U.S. patent application number 14/402574 was filed with the patent office on 2015-06-18 for target tissue-specific antigen-binding molecule.
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 Tomoyuki Igawa, Shojiro Kadono, Shun Shimizu, Shigero Tamba, Kanako Tatsumi.
Application Number | 20150166654 14/402574 |
Document ID | / |
Family ID | 49673387 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150166654 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
June 18, 2015 |
TARGET TISSUE-SPECIFIC ANTIGEN-BINDING MOLECULE
Abstract
The present inventors discovered that the above-mentioned
problems can be solved by producing antigen-binding molecules that
contain an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of a target tissue-specific
compound. Use of antigen-binding molecules of the present invention
enables various diseases that originate from a target tissue to be
treated in a manner specific to the target tissue.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Tamba; Shigero; (Shizuoka, JP) ;
Tatsumi; Kanako; (Shizuoka, JP) ; Shimizu; Shun;
(Shizuoka, JP) ; Kadono; Shojiro; (Kanagawa,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
49673387 |
Appl. No.: |
14/402574 |
Filed: |
May 30, 2013 |
PCT Filed: |
May 30, 2013 |
PCT NO: |
PCT/JP2013/064975 |
371 Date: |
November 20, 2014 |
Current U.S.
Class: |
530/387.3 ;
435/7.21; 435/7.92; 436/501; 506/9; 530/389.7 |
Current CPC
Class: |
G01N 2500/20 20130101;
C07K 16/18 20130101; G01N 2500/00 20130101; A61P 29/00 20180101;
G01N 33/6845 20130101; A61P 35/00 20180101; C07K 16/248 20130101;
C07K 16/30 20130101; C07K 2317/92 20130101; G01N 33/6869 20130101;
G01N 2333/5412 20130101; A61K 2039/505 20130101; C07K 2317/55
20130101; A61P 43/00 20180101; C07K 16/005 20130101; C07K 16/00
20130101; C07K 16/44 20130101; C07K 2317/732 20130101; C07K 2317/21
20130101; C07K 16/2866 20130101; C07K 2317/34 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; G01N 33/68 20060101 G01N033/68; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2012 |
JP |
2012-123781 |
Aug 9, 2012 |
JP |
2012-177311 |
Claims
1. An antigen-binding molecule comprising an antigen-binding domain
whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound.
2. The antigen-binding molecule of claim 1, wherein the target
tissue is a cancer tissue.
3. The antigen-binding molecule of claim 2, wherein the compound
specific to a cancer tissue is a metabolite specific to a cancer
cell, a metabolite specific to an immune cell that has infiltrated
into a cancer tissue, or a metabolite specific to a stromal cell in
a cancer tissue.
4. The antigen-binding molecule of claim 1, wherein the target
tissue is an inflamed tissue.
5. The antigen-binding molecule of claim 4, wherein the compound
specific to an inflamed tissue is a metabolite specific to an
immune cell that has infiltrated into an inflamed tissue or a
metabolite specific to a normal cell that has been damaged in an
inflamed tissue.
6. The antigen-binding molecule of claim 1, wherein the compound is
at least one compound selected from a nucleoside having a purine
ring structure, an amino acid and its metabolite, a lipid and its
metabolite, a primary metabolite of glycometabolism, and
nicotinamide and its metabolite.
7. The antigen-binding molecule of claim 6, wherein the compound is
at least one compound selected from adenosine, adenosine
triphosphate, inosine, alanine, glutamic acid, aspartic acid,
kynurenine, prostaglandin E2, succinic acid, citric acid, and
1-methylnicotinamide.
8. The antigen-binding molecule of any one of claims 1 to 7,
wherein the antigen is a membrane-type molecule.
9. The antigen-binding molecule of any one of claims 1 to 8, which
is an antigen-binding molecule that has a neutralizing
activity.
10. The antigen-binding molecule of any one of claims 1 to 9, which
is an antigen-binding molecule that has a cytotoxic activity.
11. The antigen-binding molecule of any one of claims 1 to 10,
which comprises an Fc region.
12. The antigen-binding molecule of claim 11, wherein the Fc region
is an Fc region contained in the constant region of SEQ ID NOs: 5,
6, 7, or 8.
13. The antigen-binding molecule of claim 11, wherein the Fc region
comprises an altered Fc.gamma.R-binding Fc region that has a higher
Fc.gamma. receptor-binding activity than the Fc.gamma.
receptor-binding activity of a native human IgG Fc region.
14. The antigen-binding molecule of claim 13, wherein at least one
or more amino acids selected from the group consisting of amino
acids at 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
according to EU numbering, in the amino acid sequence of the
altered Fc.gamma.R-binding Fc region are different from the amino
acids of the native human IgG Fc region.
15. The antigen-binding molecule of claim 14, which comprises at
least one or more amino acids selected from the group consisting
of: Lys or Tyr for the amino acid at position 221; Phe, Trp, Glu,
or Tyr for the amino acid at position 222; Phe, Trp, Glu, or Lys
for the amino acid at position 223; Phe, Trp, Glu, or Tyr for the
amino acid at position 224; Glu, Lys, or Trp for the amino acid at
position 225; Glu, Gly, Lys, or Tyr for the amino acid at position
227; Glu, Gly, Lys, or Tyr for the amino acid at position 228; Ala,
Glu, Gly, or Tyr for the amino acid at position 230; Glu, Gly, Lys,
Pro, or Tyr for the amino acid at position 231; Glu, Gly, Lys, or
Tyr for the amino acid at position 232; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid at 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 at 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 at 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
at position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid at position 239; Ala, Ile, Met,
or Thr for the amino acid at position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid at position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid at position 243; His for the amino
acid at position 244; Ala for the amino acid at position 245; Asp,
Glu, His, or Tyr for the amino acid at position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid at position
247; Glu, His, Gln, or Tyr for the amino acid at position 249; Glu
or Gln for the amino acid at position 250; Phe for the amino acid
at position 251; Phe, Met, or Tyr for the amino acid at position
254; Glu, Leu, or Tyr for the amino acid at position 255; Ala, Met,
or Pro for the amino acid at position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid at position 258; Asp, Glu, His, or Tyr for
the amino acid at position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid at position 262; Ala, Ile, Met, or Thr for the amino
acid at position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
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 at
position 265; Ala, Ile, Met, or Thr for the amino acid at position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid at position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid at position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid at 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 at position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid at position 272; Phe or Ile for the amino acid at
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 274;
Leu or Trp for the amino acid at position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid at position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid at
position 278; Ala for the amino acid at position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid at position
280; Asp, Lys, Pro, or Tyr for the amino acid at position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid at position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid at
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid at
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid at
position 285; Glu, Gly, Pro, or Tyr for the amino acid at position
286; Asn, Asp, Glu, or Tyr for the amino acid at position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid at
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid at position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid at position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid at position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid at position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid at position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid at 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 at position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid at position 300; Asp, Glu, His, or Tyr for the amino
acid at position 301; Ile for the amino acid at position 302; Asp,
Gly, or Tyr for the amino acid at position 303; Asp, His, Leu, Asn,
or Thr for the amino acid at position 304; Glu, Ile, Thr, or Tyr
for the amino acid at position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid at position 311; Phe for the amino acid at
position 313; Leu for the amino acid at position 315; Glu, or Gln
for the amino acid at position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid at position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid at position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid at position 322; Ile for the
amino acid at position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid at 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 at position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid at position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid at 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 at position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid at 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 at position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid at position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid at
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid at position 335; Glu, Lys,
or Tyr for the amino acid at position 336; Glu, His, or Asn for the
amino acid at position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid at position 339; Ala or Val for
the amino acid at position 376; Gly or Lys for the amino acid at
position 377; Asp for the amino acid at position 378; Asn for the
amino acid at position 379; Ala, Asn, or Ser for the amino acid at
position 380; Ala, or Ile for the amino acid at position 382; Glu
for the amino acid at position 385; Thr for the amino acid at
position 392; Leu for the amino acid at position 396; Lys for the
amino acid at position 421; Asn for the amino acid at position 427;
Phe, or Leu for the amino acid at position 428; Met for the amino
acid at position 429; Trp for the amino acid at position 434; Ile
for the amino acid at position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid at position 440 according to EU numbering in the
amino acid sequence of the altered Fc.gamma.R-binding Fc
region.
16. The antigen-binding molecule of claim 11, wherein the Fc region
is modified so that there is a higher proportion of Fc region bound
by a fucose-deficient sugar chain in a composition of sugar chain
bound at position 297, according to EU numbering, of the Fc region,
or so that there is a higher proportion of Fc region with an added
bisecting N-acetylglucosamine.
17. The antigen-binding molecule of any one of claims 11 and 13 to
16, wherein the FcRn-binding activity of the Fc region under an
acidic pH range condition is enhanced compared to the FcRn-binding
activity of the Fc region of SEQ ID NO: 5, 6, 7, or 8.
18. The antigen-binding molecule of claim 17, wherein the Fc region
is an Fc region with 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, according to EU
numbering, in the amino acid sequence of the Fc region comprised in
the constant region of SEQ ID NO: 5, 6, 7, or 8.
19. The antigen-binding molecule of claim 18, wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid at position 238; Leu for the
amino acid at position 244; Arg for the amino acid at position 245;
Pro for the amino acid at position 249; Gln or Glu for the amino
acid at position 250; Arg, Asp, Glu, or Leu for the amino acid at
position 251; Phe, Ser, Thr, or Tyr for the amino acid at position
252; Ser or Thr for the amino acid at position 254; Arg, Gly, Ile,
or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid at position 257; Asp for the
amino acid at position 258; Ser for the amino acid at position 260;
Leu for the amino acid at position 262; Lys for the amino acid at
position 270; Leu, or Arg for the amino acid at position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid at position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
position 283; Asn for the amino acid at position 285; Phe for the
amino acid at position 286; Asn or Pro for the amino acid at
position 288; Val for the amino acid at position 293, Ala, Glu,
Gln, or Met for the amino acid at position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid at position 311; Pro
for the amino acid at position 309; Ala, Asp, or Pro for the amino
acid at position 312; Ala or Leu for the amino acid at position
314; Lys for the amino acid at position 316; Pro for the amino acid
at position 317; Asn or Thr for the amino acid at position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at
position 332; Asn, Thr, or Trp for the amino acid at position 339;
Pro for the amino acid at position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid at position 343; Arg for the amino
acid at position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid at position 376; Lys for the amino acid at position 377; Asp,
Asn, or Val for the amino acid at position 378; Ala, Asn, Ser, or
Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
at position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid at position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid at position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid at position 387; Asn, Pro, or Ser for the
amino acid at position 389; Asn for the amino acid at position 423;
Asn for the amino acid at position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid at position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
at position 430; His or Asn for the amino acid at position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid at position 436; Lys, Leu, Thr, or Trp for the amino
acid at position 438; Lys for the amino acid at position 440; Lys
for the amino acid at position 442; and Ile, Pro, or Thr for the
amino acid at position 308; as indicated by EU numbering, in the
amino acid sequence of the Fc region comprised in the constant
region of SEQ ID NO: 5, 6, 7, or 8.
20. The antigen-binding molecule of any one of claims 1 to 19,
wherein the antigen-binding domain is a multispecific or a
multiparatopic antigen-binding domain.
21. The antigen-binding molecule of claim 20, wherein an antigen
bound by at least one of the antigen-binding domains is a
membrane-type molecule expressed on a cancer cell membrane, and an
antigen bound by at least one of the antigen-binding domains is a
membrane-type molecule expressed on an effector cell membrane.
22. The antigen-binding molecule of claim 21, wherein the effector
cell is an NK cell, a macrophage, or a T cell.
23. The antigen-binding molecule of claim 21 or 22, wherein the
membrane-type molecule expressed on an effector cell membrane is a
TCR-constituting polypeptide, CD2, CD3, CD28, CD44, CD16, CD32,
CD64, or NKG2D.
24. The antigen-binding molecule of claim 20, wherein an antigen
bound by at least one of the antigen-binding domains is a
membrane-type molecule expressed on a cancer cell membrane, and an
antigen bound by at least one of the antigen-binding domains is a
cytotoxic substance.
25. The antigen-binding molecule of any one of claims 20 to 24,
wherein the antigen-binding molecule is an antibody fragment.
26. The antigen-binding molecule of any one of claims 1 to 24,
wherein the antigen-binding molecule is an antibody.
27. The antigen-binding molecule of any one of claims 1 to 7,
wherein the antigen is a soluble molecule.
28. The antigen-binding molecule of claim 27, which is an
antigen-binding molecule having a neutralizing activity.
29. The antigen-binding molecule of claim 27 or 28, which comprises
an Fc region.
30. The antigen-binding molecule of claim 29, wherein the Fc region
is an Fc region comprised in the constant region of SEQ ID NO: 5,
6, 7, or 8.
31. The antigen-binding molecule of claim 29, wherein the
FcRn-binding activity of the Fc region under an acidic pH range
condition is enhanced compared to the FcRn-binding activity of the
Fc region comprised in the constant region of SEQ ID NO: 5, 6, 7,
or 8.
32. The antigen-binding molecule of claim 31, wherein the Fc region
is an Fc region with 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, according to EU
numbering, in the amino acid sequence of the Fc region comprised in
the constant region of SEQ ID NO: 5, 6, 7, or 8.
33. The antigen-binding molecule of claim 32, wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid at position 238; Leu for the
amino acid at position 244; Arg for the amino acid at position 245;
Pro for the amino acid at position 249; Gln or Glu for the amino
acid at position 250; Arg, Asp, Glu, or Leu for the amino acid at
position 251; Phe, Ser, Thr, or Tyr for the amino acid at position
252; Ser or Thr for the amino acid at position 254; Arg, Gly, Ile,
or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid at position 257; Asp for the
amino acid at position 258; Ser for the amino acid at position 260;
Leu for the amino acid at position 262; Lys for the amino acid at
position 270; Leu, or Arg for the amino acid at position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid at position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
position 283; Asn for the amino acid at position 285; Phe for the
amino acid at position 286; Asn or Pro for the amino acid at
position 288; Val for the amino acid at position 293, Ala, Glu,
Gln, or Met for the amino acid at position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid at position 311; Pro
for the amino acid at position 309; Ala, Asp, or Pro for the amino
acid at position 312; Ala or Leu for the amino acid at position
314; Lys for the amino acid at position 316; Pro for the amino acid
at position 317; Asn or Thr for the amino acid at position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at
position 332; Asn, Thr, or Trp for the amino acid at position 339;
Pro for the amino acid at position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid at position 343; Arg for the amino
acid at position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid at position 376; Lys for the amino acid at position 377; Asp,
Asn, or Val for the amino acid at position 378; Ala, Asn, Ser, or
Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
at position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid at position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid at position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid at position 387; Asn, Pro, or Ser for the
amino acid at position 389; Asn for the amino acid at position 423;
Asn for the amino acid at position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid at position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
at position 430; His or Asn for the amino acid at position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid at position 436; Lys, Leu, Thr, or Trp for the amino
acid at position 438; Lys for the amino acid at position 440; Lys
for the amino acid at position 442; and Ile, Pro, or Thr for the
amino acid at position 308; as indicated by EU numbering, in the
amino acid sequence of the Fc region comprised in the constant
region of SEQ ID NO: 5, 6, 7, or 8.
34. The antigen-binding molecule of claim 29, wherein the
FcRn-binding activity of the Fc region under a neutral pH range
condition is enhanced compared to the FcRn-binding activity of the
Fc region comprised in the constant region of SEQ ID NO: 5, 6, 7,
or 8.
35. The antigen-binding molecule of claim 34, wherein the Fc region
is an Fc region with substitution of at least one or more amino
acids selected from the group consisting of 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 according to EU numbering, in the amino acid
sequence of the Fc region comprised in the constant region of SEQ
ID NO: 5, 6, 7, or 8.
36. The antigen-binding molecule of claim 35, wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting 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 as indicated by EU
numbering, in the amino acid sequence of the Fc region of SEQ ID
NO: 5, 6, 7, or 8.
37. The antigen-binding molecule of any one of claims 29 and 31 to
36, wherein the Fc region has a higher binding activity to an
inhibitory Fc.gamma. receptor than to an activating Fc.gamma.
receptor.
38. The antigen-binding molecule of claim 37, wherein the
inhibitory Fc.gamma. receptor is human Fc.gamma.RIIb.
39. The antigen-binding molecule of claim 37 or 38, 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).
40. The antigen-binding molecule of any one of claims 37 to 39,
wherein the amino acid at position 238 or 328 (EU numbering) of the
Fc region includes an amino acid that is different from the amino
acid of the native human IgG Fc region.
41. The antigen-binding molecule of claim 40, wherein the amino
acid at position 238 indicated by EU numbering in the Fc region is
Asp or the amino acid at position 328 is Glu.
42. The antigen-binding molecule of claim 40 or 41, which comprises
at least one or more amino acids selected from the group consisting
of: Asp for the amino acid at position 233; Trp or Tyr for the
amino acid at position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp, or
Tyr for the amino acid at position 237; Asp for the amino acid at
position 239; Ala, Gln, or Val for the amino acid at position 267;
Asn, Asp, or Glu for the amino acid at position 268; Gly for the
amino acid at position 271; Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser,
or Thr for the amino acid at position 326; Arg, Lys, or Met for the
amino acid at position 330; Ile, Leu, or Met for the amino acid at
position 323; and Asp for the amino acid at position 296 according
to EU numbering, in the amino acid sequence of the Fc region.
43. The antigen-binding molecule of any one of claims 27 to 42,
wherein the antigen-binding molecule is an antibody.
44. A method for producing the antigen-binding molecule of any one
of claims 1 to 43, which comprises selecting an antigen-binding
domain whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound.
45. A method of screening for the antigen-binding molecule of any
one of claims 1 to 43, which comprises selecting an antigen-binding
domain whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound.
46. A pharmaceutical composition comprising the antigen-binding
molecule of any one of claims 1 to 43.
Description
TECHNICAL FIELD
[0001] The present invention provides antigen-binding molecules
comprising an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of a target tissue-specific
compound; production methods and screening methods for the
antigen-binding molecules; and pharmaceutical compositions
containing the antigen-binding molecules.
BACKGROUND ART
[0002] Antibodies are drawing attention as pharmaceuticals as they
are highly stable in plasma and have few side effects. In
particular, 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).
[0003] As cancer therapeutic agents using antibody pharmaceuticals,
Rituxan against a CD20 antigen, cetuximab against an EGFR antigen,
herceptin against a HER2 antigen, and such have been approved so
far (Non-Patent Document 3). These antibody molecules bind to
antigens expressed on cancer cells, and exhibit cytotoxic activity
against cancer cells by ADCC and such.
[0004] Such cytotoxic activity by ADCC and etc. are known to depend
on the number of antigens expressed on cells targeted by the
therapeutic antibodies (Non-Patent Document 4); therefore, high
expression level of the target antigen is preferable from the stand
point of the effects of the therapeutic antibodies. However, even
if the antigen expression level is high, when antigens are
expressed in normal tissues, cytotoxic activity mediated by ADCC
etc will be exerted against normal cells, and therefore
side-effects will become a major problem. Therefore, antigens
targeted by therapeutic antibodies used as therapeutic agents for
cancer are preferably antigens specifically expressed in cancer
cells. For example, antibody molecules against the EpCAM antigen
which is known as a cancer antigen have been considered to be
promising as therapeutic agents for cancer. However, the EpCAM
antigen is known to be expressed in the pancreas as well, and in
practice, administration of anti-EpCAM antibodies in clinical
trials has been reported to cause pancreatitis as a side-effect due
to cytotoxic activity towards the pancreas (Non-Patent Document
5).
[0005] Following the success of antibody pharmaceuticals that exert
cytotoxic activity by ADCC activity, a second generation of
improved antibody molecules that exert strong cytotoxic activity
through enhancement of ADCC activity by removing fucose of N-type
sugar chains in the native human IgG1 Fc region (Non-Patent
Document 6), enhancement of ADCC activity by enhancing the binding
toward Fc.gamma.RIIIa by substitution of amino acids in the native
human IgG1 Fc region (Non-Patent Document 7), and such have been
reported. As antibody pharmaceuticals that exert cytotoxic activity
against cancer cells through a mechanism other than the
above-mentioned ADCC activity mediated by NK cells, improved
antibody molecules that exert a stronger cytotoxic activity, such
as an antibody-drug conjugate (ADC) in which an antibody is
conjugated with a drug having potent cytotoxic activity (Non-Patent
Document 8), and a low molecular weight antibody that exerts toxic
activity against cancer cells by recruiting T cells to cancer
cells, have been reported as well.
[0006] Such antibody molecules exerting a stronger cytotoxic
activity can exert cytotoxic activity against cancer cells that do
not have much antigen expression, but on the other hand, they will
exert similar cytotoxic activity against normal tissues with low
antigen expression. In fact, in comparison to cetuximab which is a
natural human IgG1 against an EGFR antigen, EGFR-BiTE, which is a
bispecific antibody against CD3 and EGFR, can exert a potent
cytotoxic activity against cancer cells by recruiting T cells to
cancer cells and exert antitumor effects. On the other hand, since
EGFR is expressed also in normal tissues, when EGFR-BiTE is
administered to cynomolgus monkeys, serious side effects have
appeared (Non-Patent Document 10). Furthermore, bivatuzumab
mertansine, an ADC formed by linking mertansine to an antibody
against CD44v6 which is highly expressed in cancer cells, has been
shown to cause severe skin toxicity and liver toxicity in clinical
practice because CD44v6 is expressed also in normal tissues
(Non-Patent Document 11).
[0007] When antibodies that can exert a potent cytotoxic activity
against cancer cells having low antigen expression are used as
such, the target antigen needs to be expressed in a highly
cancer-specific manner. However, since HER2 and EGFR, which are
target antigens of herceptin and cetuximab, respectively, are also
expressed in normal tissues, the number of cancer antigens
expressed in a highly cancer-specific manner is thought to be
limited. Therefore, while it is possible to strengthen the
cytotoxic activity against cancer, the side effects occurring due
to cytotoxic actions against normal tissues may become
problematic.
[0008] Furthermore, recently, ipilimumab which enhances tumor
immunity by inhibiting CTLA4 which contributes to immunosuppression
in cancer was shown to prolong overall survival of metastatic
melanoma (Non-Patent Document 12). However, since ipulimumab
inhibits CTLA4 systemically, while tumor immunity is enhanced, the
emergence of autoimmune disease-like severe side effects due to
systemic activation of the immune system is becoming a problem
(Non-Patent Document 13).
[0009] On the other hand, as antibody pharmaceuticals against
diseases besides cancer, antibody pharmaceuticals that exert
therapeutic effects by inhibiting inflammatory cytokines in
inflammatory/autoimmune diseases are known (Non-Patent Document
14). For example, Remicade and Humira which target TNF, and Actemra
which targets IL-6R exhibit high therapeutic effects against
rheumatoid arthritis, but on the other hand, systemic
neutralization of these cytokines has led to the observation of
infection as side effects (Non-Patent Document 15).
[0010] Various techniques have been developed as techniques that
can be applied to second-generation antibody pharmaceuticals. While
techniques for improving effector functions, antigen-binding
ability, pharmacokinetics, and stability, or techniques for
reducing immunogenic risks have been reported (Non-Patent Document
16), there are hardly any reports on techniques that enable target
tissue-specific action of antibody pharmaceuticals to overcome such
side effects. For example, regarding lesions such as cancer tissues
and inflammatory tissues, pH-dependent antibodies that make use of
the acidic pH condition at these target tissues have been reported
(Patent Documents 1 and 2). However, the decrease of pH (that is,
increase in hydrogen ion concentration) in cancer tissues and
inflammatory tissues as compared to normal tissues is slight, and
since it is difficult to produce antibodies that act by detecting a
slight increase in the concentration of hydrogen ions which have an
extremely small molecular weight, and also because acidic pH
conditions may be found in normal tissues such as osteoclastic bone
resorption region or in tissues other than the lesion of interest,
use of pH conditions as a lesion-specific environmental factor was
considered to face many challenges. On the other hand, methods for
producing antibodies that exert antigen-binding activity only after
they are cleaved by a protease expressed at lesion sites such as
cancer tissues and inflammatory tissues have been reported (Patent
Document 3). However, since cleavage of antibodies by proteases is
irreversible, when the antibodies that have been cleaved at the
lesion site enter the blood stream and return to normal tissues,
they can bind to the antigens in normal tissues as well, and this
is considered to be a problem. Furthermore, cancer specificity of
such proteases is also thought to have problems that need to be
addressed. Therefore, techniques that enable reversible action at
sites of inflammation or cancer (lesion sites) without systemic
action in normal tissues and blood for exerting drug efficacy while
avoiding side effects are not known.
PRIOR ART DOCUMENTS
Patent Documents
[0011] [Patent document 1] WO 2003/105757 [0012] [Patent document
2] WO 2012/033953 [0013] [Patent document 3] WO 2010/081173
Non-Patent Documents
[0013] [0014] [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
[0015] [Non-patent document 2] The therapeutic antibodies market to
2008. Pavlou A K, Belsey M J., Eur. J. Pharm. Biopharm. (2005) 59
(3), 389-396 [0016] [Non-patent document 3] Monoclonal antibodies:
versatile platforms for cancer immunotherapy. Weiner L M, Surana R,
Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327 [0017]
[Non-patent document 4] Differential responses of human tumor cell
lines to anti-p185HER2 monoclonal antibodies. Lewis G D, Figari I,
Fendly B, Wong W L, Carter P, Gorman C, Shepard H M, Cancer
Immunol. Immunotherapy (1993) 37, 255-263 [0018] [Non-patent
document 5] ING-1, a monoclonal antibody targeting Ep-CAM in
patients with advanced adenocarcinomas. de Bono J S, Tolcher A W,
Forero A, Vanhove G F, Takimoto C, Bauer R J, Hammond L A, Patnaik
A, White M L, Shen S, Khazaeli M B, Rowinsky E K, LoBuglio A F,
Clin. Cancer Res. (2004) 10 (22), 7555-7565 [0019] [Non-patent
document 6] Non-fucosylated therapeutic antibodies as
next-generation therapeutic antibodies. Satoh M, Iida S, Shitara
K., Expert Opin. Biol. Ther. (2006) 6 (11), 1161-1173 [0020]
[Non-patent document 7] Optimizing engagement of the immune system
by anti-tumor antibodies: an engineer's perspective. Desjarlais J
R, Lazar G A, Zhukovsky E A, Chu S Y., Drug Discov. Today (2007) 12
(21-22), 898-910 [0021] [Non-patent document 8] Antibody-drug
conjugates: targeted drug delivery for cancer. Alley S C, Okeley N
M, Senter P D., Curr. Opin. Chem. Biol. (2010) 14 (4), 529-537
[0022] [Non-patent document 9] BiTE: Teaching antibodies to engage
T-cells for cancer therapy. Baeuerle P A, Kufer P, Bargou R., Curr.
Opin. Mol. Ther. (2009) 11 (1), 22-30 [0023] [Non-patent document
10] T cell-engaging BiTE antibodies specific for EGFR potently
eliminate KRAS- and BRAF-mutated colorectal cancer cells.
Lutterbuese R, Raum T, Kischel R, Hoffmann P, Mangold S, Rattel B,
Friedrich M, Thomas 0, Lorenczewski G, Rau D, Schaller E, Herrmann
I, Wolf A, Urbig T, Baeuerle P A, Kufer P., Proc. Natl. Acad. Sci.
U.S.A. (2010) 107 (28), 12605-12610 [0024] [Non-patent document 11]
Phase I trial with the C D44v6-targeting immunoconjugate
bivatuzumab mertansine in head and neck squamous cell carcinoma.
Riechelmann H, Sauter A, Golze W, Hanft G, Schroen C, Hoermann K,
Erhardt T, Gronau S., Oral Oncol. (2008) 44 (9), 823-829 [0025]
[Non-patent document 12] Ipilimumab in the treatment of melanoma.
Trinh V A, Hwu W J., Expert Opin. Biol. Ther., (2012) April 14
(doi:10.1517/14712598.2012.675325) [0026] [Non-patent document 13]
IPILIMUMAB--A NOVEL IMMUNOMODULATING THERAPY CAUSING AUTOIMMUNE
HYPOPHYSITIS: A CASE REPORT AND REVIEW. Juszczak A, Gupta A,
Karavitaki N, Middleton M R, Grossman A., Eur. J. Endocrinol.
(2012) April 10 (doi: 10.1530/EJE-12-0167) [0027] [Non-patent
document 14] The Japanese experience with biologic therapies for
rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev. Rheumatol.
(2010) 6 (11), 644-652 [0028] [Non-patent document 15] Current
evidence for the management of rheumatoid arthritis with biological
disease-modifying antirheumatic drugs: a systematic literature
review informing the EULAR recommendations for the management of R
A. Nam J L, Winthrop K L, van Vollenhoven R F, Pavelka K, Valesini
G, Hensor E M, Worthy G, Landewe R, Smolen J S, Emery P, Buch M H.,
Ann Rheum. Dis. (2010) 69 (6), 976-986 [0029] [Non-patent document
16] Antibody engineering for the development of therapeutic
antibodies. Kim S J, Park Y, Hong H J., Mol. Cells. (2005) 20 (1),
17-29
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0030] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
pharmaceutical compositions that are useful for treating diseases
originating from target tissues, and active ingredients thereof.
Another objective is to provide methods of screening for the
pharmaceutical compositions and active ingredients, as well as
their production methods.
Means for Solving the Problems
[0031] The present inventors conducted dedicated studies to achieve
the above-described objectives. As a result, they generated
antigen-binding molecules comprising an antigen-binding domain
whose antigen-binding activity varies depending on the
concentration of the target tissue-specific compound. Furthermore,
the present inventors discovered that the antigen-binding molecules
or pharmaceutical compositions comprising the antigen-binding
molecules are useful for treating diseases that originate from a
target tissue, and that they are also useful for treatment of
diseases originating from target tissues that includes
administering the antigen-binding molecules. They also discovered
that the antigen-binding molecules are useful in the production of
pharmaceuticals for treating diseases that originate from target
tissues. Furthermore, the present inventors produced screening
methods and production methods for the antigen-binding molecules,
and thereby completed the present invention.
[0032] More specifically, the present invention provides the
following:
[1] An antigen-binding molecule comprising an antigen-binding
domain whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound. [2] The
antigen-binding molecule of [1], wherein the target tissue is a
cancer tissue. [3] The antigen-binding molecule of [2], wherein the
compound specific to a cancer tissue is a metabolite specific to a
cancer cell, a metabolite specific to an immune cell that has
infiltrated into a cancer tissue, or a metabolite specific to a
stromal cell in a cancer tissue. [4] The antigen-binding molecule
of [1], wherein the target tissue is an inflamed tissue. [5] The
antigen-binding molecule of [4], wherein the compound specific to
an inflamed tissue is a metabolite specific to an immune cell that
has infiltrated into an inflamed tissue or a metabolite specific to
a normal cell that has been damaged in an inflamed tissue. [6] The
antigen-binding molecule of [1], wherein the compound is at least
one compound selected from a nucleoside having a purine ring
structure, an amino acid and its metabolite, a lipid and its
metabolite, a primary metabolite of glycometabolism, and
nicotinamide and its metabolite. [7] The antigen-binding molecule
of [6], wherein the compound is at least one compound selected from
adenosine, adenosine triphosphate, inosine, alanine, glutamic acid,
aspartic acid, kynurenine, prostaglandin E2, succinic acid, citric
acid, and 1-methylnicotinamide. [8] The antigen-binding molecule of
any one of [1] to [7], wherein the antigen is a membrane-type
molecule. [9] The antigen-binding molecule of any one of [1] to
[8], which is an antigen-binding molecule that has a neutralizing
activity. [10] The antigen-binding molecule of any one of [1] to
[9], which is an antigen-binding molecule that has a cytotoxic
activity. [11] The antigen-binding molecule of any one of [1] to
[10], which comprises an Fc region. [12] The antigen-binding
molecule of [11], wherein the Fc region is an Fc region contained
in the constant region of SEQ ID NOs: 5, 6, 7, or 8. [13] The
antigen-binding molecule of [11], wherein the Fc region comprises
an altered Fc.gamma.R-binding Fc region that has a higher Fc.gamma.
receptor-binding activity than the Fc.gamma. receptor-binding
activity of a native human IgG Fc region. [14] The antigen-binding
molecule of [13], wherein at least one or more amino acids selected
from the group consisting of amino acids at 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 according to EU numbering, in the
amino acid sequence of the altered Fc.gamma.R-binding Fc region are
different from the amino acids of the native human IgG Fc region.
[15] The antigen-binding molecule of [14], which comprises at least
one or more amino acids selected from the group consisting of: Lys
or Tyr for the amino acid at position 221; Phe, Trp, Glu, or Tyr
for the amino acid at position 222; Phe, Trp, Glu, or Lys for the
amino acid at position 223; Phe, Trp, Glu, or Tyr for the amino
acid at position 224; Glu, Lys, or Trp for the amino acid at
position 225; Glu, Gly, Lys, or Tyr for the amino acid at position
227; Glu, Gly, Lys, or Tyr for the amino acid at position 228; Ala,
Glu, Gly, or Tyr for the amino acid at position 230; Glu, Gly, Lys,
Pro, or Tyr for the amino acid at position 231; Glu, Gly, Lys, or
Tyr for the amino acid at position 232; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid at 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 at 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 at 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
at position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid at position 239; Ala, Ile, Met,
or Thr for the amino acid at position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid at position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid at position 243; His for the amino
acid at position 244; Ala for the amino acid at position 245; Asp,
Glu, His, or Tyr for the amino acid at position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid at position
247; Glu, His, Gln, or Tyr for the amino acid at position 249; Glu
or Gln for the amino acid at position 250; Phe for the amino acid
at position 251; Phe, Met, or Tyr for the amino acid at position
254; Glu, Leu, or Tyr for the amino acid at position 255; Ala, Met,
or Pro for the amino acid at position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid at position 258; Asp, Glu, His, or Tyr for
the amino acid at position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid at position 262; Ala, Ile, Met, or Thr for the amino
acid at position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
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 at
position 265; Ala, Ile, Met, or Thr for the amino acid at position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid at position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid at position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid at 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 at position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid at position 272; Phe or Ile for the amino acid at
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 274;
Leu or Trp for the amino acid at position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid at position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid at
position 278; Ala for the amino acid at position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid at position
280; Asp, Lys, Pro, or Tyr for the amino acid at position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid at position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid at
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid at
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid at
position 285; Glu, Gly, Pro, or Tyr for the amino acid at position
286; Asn, Asp, Glu, or Tyr for the amino acid at position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid at
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid at position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid at position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid at position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid at position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid at position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid at position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid at 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 at position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid at position 300; Asp, Glu, His, or Tyr for the amino
acid at position 301; Ile for the amino acid at position 302; Asp,
Gly, or Tyr for the amino acid at position 303; Asp, His, Leu, Asn,
or Thr for the amino acid at position 304; Glu, Ile, Thr, or Tyr
for the amino acid at position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid at position 311; Phe for the amino acid at
position 313; Leu for the amino acid at position 315; Glu, or Gln
for the amino acid at position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid at position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid at position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid at position 322; Ile for the
amino acid at position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid at 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 at position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid at position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid at 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 at position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid at 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 at position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid at position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid at
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid at position 335; Glu, Lys,
or Tyr for the amino acid at position 336; Glu, His, or Asn for the
amino acid at position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid at position 339; Ala or Val for
the amino acid at position 376; Gly or Lys for the amino acid at
position 377; Asp for the amino acid at position 378; Asn for the
amino acid at position 379; Ala, Asn, or Ser for the amino acid at
position 380; Ala, or Ile for the amino acid at position 382; Glu
for the amino acid at position 385; Thr for the amino acid at
position 392; Leu for the amino acid at position 396; Lys for the
amino acid at position 421; Asn for the amino acid at position 427;
Phe, or Leu for the amino acid at position 428; Met for the amino
acid at position 429; Trp for the amino acid at position 434; Ile
for the amino acid at position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid at position 440 according to EU numbering in the
amino acid sequence of the altered Fc.gamma.R-binding Fc region.
[16] The antigen-binding molecule of [11], wherein the Fc region is
modified so that there is a higher proportion of Fc region bound by
a fucose-deficient sugar chain in a composition of sugar chain
bound at position 297, according to EU numbering, of the Fc region,
or so that there is a higher proportion of Fc region with an added
bisecting N-acetylglucosamine. [17] The antigen-binding molecule of
any one of [11] and [13] to [16], wherein the FcRn-binding activity
of the Fc region under an acidic pH range condition is enhanced
compared to the FcRn-binding activity of the Fc region of SEQ ID
NO: 5, 6, 7, or 8. [18] The antigen-binding molecule of [17],
wherein the Fc region is an Fc region with 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,
according to EU numbering, in the amino acid sequence of the Fc
region comprised in the constant region of SEQ ID NO: 5, 6, 7, or
8. [19] The antigen-binding molecule of [18], wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid at position 238; Leu for the
amino acid at position 244; Arg for the amino acid at position 245;
Pro for the amino acid at position 249; Gln or Glu for the amino
acid at position 250; Arg, Asp, Glu, or Leu for the amino acid at
position 251; Phe, Ser, Thr, or Tyr for the amino acid at position
252; Ser or Thr for the amino acid at position 254; Arg, Gly, Ile,
or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid at position 257; Asp for the
amino acid at position 258; Ser for the amino acid at position 260;
Leu for the amino acid at position 262; Lys for the amino acid at
position 270; Leu, or Arg for the amino acid at position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid at position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
position 283; Asn for the amino acid at position 285; Phe for the
amino acid at position 286; Asn or Pro for the amino acid at
position 288; Val for the amino acid at position 293, Ala, Glu,
Gln, or Met for the amino acid at position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid at position 311; Pro
for the amino acid at position 309; Ala, Asp, or Pro for the amino
acid at position 312; Ala or Leu for the amino acid at position
314; Lys for the amino acid at position 316; Pro for the amino acid
at position 317; Asn or Thr for the amino acid at position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at
position 332; Asn, Thr, or Trp for the amino acid at position 339;
Pro for the amino acid at position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid at position 343; Arg for the amino
acid at position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid at position 376; Lys for the amino acid at position 377; Asp,
Asn, or Val for the amino acid at position 378; Ala, Asn, Ser, or
Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
at position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid at position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid at position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid at position 387; Asn, Pro, or Ser for the
amino acid at position 389; Asn for the amino acid at position 423;
Asn for the amino acid at position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid at position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
at position 430; His or Asn for the amino acid at position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid at position 436; Lys, Leu, Thr, or Trp for the amino
acid at position 438; Lys for the amino acid at position 440; Lys
for the amino acid at position 442; and Ile, Pro, or Thr for the
amino acid at position 308; as indicated by EU numbering, in the
amino acid sequence of the Fc region comprised in the constant
region of SEQ ID NO: 5, 6, 7, or 8. [20] The antigen-binding
molecule of any one of [1] to [19], wherein the antigen-binding
domain is a multispecific or a multiparatopic antigen-binding
domain. [21] The antigen-binding molecule of [20], wherein an
antigen bound by at least one of the antigen-binding domains is a
membrane-type molecule expressed on a cancer cell membrane, and an
antigen bound by at least one of the antigen-binding domains is a
membrane-type molecule expressed on an effector cell membrane. [22]
The antigen-binding molecule of [21], wherein the effector cell is
an NK cell, a macrophage, or a T cell. [23] The antigen-binding
molecule of [21] or [22], wherein the membrane-type molecule
expressed on an effector cell membrane is a TCR-constituting
polypeptide, CD2, CD3, CD28, CD44, CD16, CD32, CD64, or NKG2D. [24]
The antigen-binding molecule of [20], wherein an antigen bound by
at least one of the antigen-binding domains is a membrane-type
molecule expressed on a cancer cell membrane, and an antigen bound
by at least one of the
antigen-binding domains is a cytotoxic substance. [25] The
antigen-binding molecule of any one of [20] to [24], wherein the
antigen-binding molecule is an antibody fragment. [26] The
antigen-binding molecule of any one of [1] to [24], wherein the
antigen-binding molecule is an antibody. [27] The antigen-binding
molecule of any one of [1] to [7], wherein the antigen is a soluble
molecule. [28] The antigen-binding molecule of [27], which is an
antigen-binding molecule having a neutralizing activity. [29] The
antigen-binding molecule of [27] or [28], which comprises an Fc
region. [30] The antigen-binding molecule of [29], wherein the Fc
region is an Fc region comprised in the constant region of SEQ ID
NO: 5, 6, 7, or 8. [31] The antigen-binding molecule of [29],
wherein the FcRn-binding activity of the Fc region under an acidic
pH range condition is enhanced compared to the FcRn-binding
activity of the Fc region comprised in the constant region of SEQ
ID NO: 5, 6, 7, or 8. [32] The antigen-binding molecule of [31],
wherein the Fc region is an Fc region with 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,
according to EU numbering, in the amino acid sequence of the Fc
region comprised in the constant region of SEQ ID NO: 5, 6, 7, or
8. [33] The antigen-binding molecule of [32], wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid at position 238; Leu for the
amino acid at position 244; Arg for the amino acid at position 245;
Pro for the amino acid at position 249; Gln or Glu for the amino
acid at position 250; Arg, Asp, Glu, or Leu for the amino acid at
position 251; Phe, Ser, Thr, or Tyr for the amino acid at position
252; Ser or Thr for the amino acid at position 254; Arg, Gly, Ile,
or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid at position 257; Asp for the
amino acid at position 258; Ser for the amino acid at position 260;
Leu for the amino acid at position 262; Lys for the amino acid at
position 270; Leu, or Arg for the amino acid at position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid at position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at
position 283; Asn for the amino acid at position 285; Phe for the
amino acid at position 286; Asn or Pro for the amino acid at
position 288; Val for the amino acid at position 293, Ala, Glu,
Gln, or Met for the amino acid at position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid at position 311; Pro
for the amino acid at position 309; Ala, Asp, or Pro for the amino
acid at position 312; Ala or Leu for the amino acid at position
314; Lys for the amino acid at position 316; Pro for the amino acid
at position 317; Asn or Thr for the amino acid at position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at
position 332; Asn, Thr, or Trp for the amino acid at position 339;
Pro for the amino acid at position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid at position 343; Arg for the amino
acid at position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid at position 376; Lys for the amino acid at position 377; Asp,
Asn, or Val for the amino acid at position 378; Ala, Asn, Ser, or
Thr for the amino acid at position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
at position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid at position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid at position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid at position 387; Asn, Pro, or Ser for the
amino acid at position 389; Asn for the amino acid at position 423;
Asn for the amino acid at position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid at position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
at position 430; His or Asn for the amino acid at position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid at position 436; Lys, Leu, Thr, or Trp for the amino
acid at position 438; Lys for the amino acid at position 440; Lys
for the amino acid at position 442; and Ile, Pro, or Thr for the
amino acid at position 308; as indicated by EU numbering, in the
amino acid sequence of the Fc region comprised in the constant
region of SEQ ID NO: 5, 6, 7, or 8. [34] The antigen-binding
molecule of [29], wherein the FcRn-binding activity of the Fc
region under a neutral pH range condition is enhanced compared to
the FcRn-binding activity of the Fc region comprised in the
constant region of SEQ ID NO: 5, 6, 7, or 8. [35] The
antigen-binding molecule of [34], wherein the Fc region is an Fc
region with substitution of at least one or more amino acids
selected from the group consisting of 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
according to EU numbering, in the amino acid sequence of the Fc
region comprised in the constant region of SEQ ID NO: 5, 6, 7, or
8. [36] The antigen-binding molecule of [35], wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting 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 as indicated by EU
numbering, in the amino acid sequence of the Fc region of SEQ ID
NO: 5, 6, 7, or 8. [37] The antigen-binding molecule of any one of
[29] and [31] to [36], wherein the Fc region has a higher binding
activity to an inhibitory Fc.gamma. receptor than to an activating
Fc.gamma. receptor. [38] The antigen-binding molecule of [37],
wherein the inhibitory Fc.gamma. receptor is human Fc.gamma.RIIb.
[39] The antigen-binding molecule of [37] or [38], 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). [40] The antigen-binding molecule
of any one of [37] to [39], wherein the amino acid at position 238
or 328 (EU numbering) of the Fc region includes an amino acid that
is different from the amino acid of the native human IgG Fc region.
[41] The antigen-binding molecule of [40], wherein the amino acid
at position 238 indicated by EU numbering in the Fc region is Asp
or the amino acid at position 328 is Glu. [42] The antigen-binding
molecule of [40] or [41], which comprises at least one or more
amino acids selected from the group consisting of: Asp for the
amino acid at position 233; Trp or Tyr for the amino acid at
position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the
amino acid at position 237; Asp for the amino acid at position 239;
Ala, Gln, or Val for the amino acid at position 267; Asn, Asp, or
Glu for the amino acid at position 268; Gly for the amino acid at
position 271; Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for
the amino acid at position 326; Arg, Lys, or Met for the amino acid
at position 330; Ile, Leu, or Met for the amino acid at position
323; and Asp for the amino acid at position 296 according to EU
numbering, in the amino acid sequence of the Fc region. [43] The
antigen-binding molecule of any one of [27] to [42], wherein the
antigen-binding molecule is an antibody. [44] A method for
producing the antigen-binding molecule of any one of [1] to [43],
which comprises selecting an antigen-binding domain whose
antigen-binding activity varies depending on the concentration of a
target tissue-specific compound. [45] A method of screening for the
antigen-binding molecule of any one of [1] to [43], which comprises
selecting an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of a target tissue-specific
compound. [46] A pharmaceutical composition comprising the
antigen-binding molecule of any one of [1] to [43].
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows that a small-molecule-switch antibody does not
bind to antigens in a normal environment where the small molecules
are not present, but binds to the antigens in the target tissue
where the small molecules are present at a high concentration.
[0034] FIG. 2 shows that the small molecule functions as a switch
by fitting between the anti-small-molecule antibody and the
antigen. If the small molecule is absent, the antibody-antigen
interaction is insufficient and the antibody cannot bind to the
antigen, but if the small molecule is present, the antibody can
bind to the antigen by having the small molecule placed between the
antibody and the antigen.
[0035] FIG. 3 is a figure showing the result of ELISA for the
binding of the antibody to human IL-6. The vertical axis shows the
absorbance values which assess the binding activity to human IL-6
of each of the antibodies in the presence or absence of each of the
small molecules.
[0036] FIG. 4 is a sensorgram showing the interaction between 4
.mu.mol/L of human IL-6 and A11 in the presence or absence of 100
.mu.mol/L kynurenine.
[0037] FIG. 5 shows a graph that evaluates change in the response
of binding to A11 immobilized onto Sensorchip CM5, when interaction
is allowed to take place for 60 seconds with 1 .mu.mol/L of IL-6 as
the analyte. The vertical axis shows change in the response (RU)
before and after IL-6 interaction, and the horizontal axis shows
the concentration of kynurenine (.mu.mol/L) contained in the
solution at that time.
[0038] FIG. 6 shows a graph that evaluates the response to H01
which has been immobilized onto Sensorchip CM5, when interaction is
allowed to take place for 60 seconds with 1 .mu.mol/L of IL-6 as
the analyte. The vertical axis shows change in the response (RU)
before and after IL-6 interaction, and the horizontal axis shows
the concentration of kynurenine contained in the solution
(.mu.mol/L) at that time.
[0039] FIG. 7 shows a graph that evaluates the response to IL-6
which has been immobilized onto Sensorchip CM5, when interaction is
allowed to take place for 60 seconds with 0.1 .mu.mol/L of A11 as
the analyte. The vertical axis shows change in the response (RU)
before and after A11 interaction, and the horizontal axis shows the
concentration of kynurenine contained in the solution
(.mu.mol/L).
[0040] FIG. 8 shows a graph obtained by allowing A11 to interact
with IL-6 immobilized on Sensorchip CM5 in the presence of 100
.mu.mol/L kynurenine, and then observing the dissociation of A11
from IL6 in the presence of a buffer containing 100 .mu.mol/L
kynurenine or in the presence of a buffer that does not contain
kynurenine. In the figure, the vertical axis shows values
normalized by defining the amount of A11 bound in the presence of
100 .mu.mol/L kynurenine as 100, and the horizontal axis shows the
passage of time (in seconds) from the start of the interaction.
[0041] FIG. 9 shows a sensorgram obtained by allowing 800, 400,
200, 100, 50, or 25 nmol/L of kynurenine to interact with IL-6
immobilized on a sensorchip. The vertical axis shows change in the
amount of IL-6 bound by kynurenine (RU) (the response at the start
of the interaction experiment was defined as 0), and the horizontal
axis shows the passage of time from the start of the interaction
experiment.
[0042] FIG. 10 shows the structure of 2'-Adenosine-PEG-peptide
which is an adenosine analog used for immunization of rabbits.
[0043] FIG. 11 shows the structure of 5'-Adenosine-PEG-peptide
which is an adenosine analog used for immunization of rabbits.
[0044] FIG. 12 shows the structure of 2'-Adenosine-PEG-biotin
produced by substituting biotin for the peptide portion of the
adenosine analog used for immunization of rabbits.
[0045] FIG. 13 shows the structure of 5'-Adenosine-PEG-biotin
produced by substituting biotin for the peptide portion of the
adenosine analog used for immunization of rabbits.
[0046] FIG. 14 is a graph where the vertical axis shows the value
(N_binding.sub.--100) obtained by dividing the amount of binding in
the interaction of each antibody with 2'-Adenosine-PEG-biotin by
the capture level (RU) of each antibody, and the horizontal axis
shows the value (N_stability.sub.--100) obtained by dividing the
value obtained 60 seconds after dissociation of
2'-Adenosine-PEG-biotin from each antibody after interaction with
2'-Adenosine-PEG-biotin by the capture level (RU) of each
antibody.
[0047] FIG. 15A indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0002 binds to
(interacts with) adenosine. The sensorgrams show the interactions
between SMB0002 and the antigen at 7.81, 31.3, 125, and 500 nM in
order from the bottom.
[0048] FIG. 15B indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0002 binds to
(interacts with) ATP. The sensorgrams show the interactions between
SMB0002 and the antigen at 78.1, 313, 1250, and 5000 nM in order
from the bottom.
[0049] FIG. 15C indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0089 binds to
(interacts with) adenosine. The sensorgrams show the interactions
between SMB0089 and the antigen at 7.81, 31.3, 125, and 500 nM in
order from the bottom.
[0050] FIG. 15D indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0089 binds to
(interacts with) ATP. The sensorgrams show the interactions between
SMB00089 and the antigen at 78.1, 313, 1250, and 5000 nM in order
from the bottom.
[0051] FIG. 15E indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0104 binds to
(interacts with) adenosine. The sensorgrams show the interactions
between SMB0104 and the antigen at 7.81, 31.3, and 500 nM in order
from the bottom.
[0052] FIG. 15F indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0104 binds to
(interacts with) ATP. The sensorgrams show the interactions between
SMB0104 and the antigen at 78.1, 313, 1250, and 5000 nM in order
from the bottom.
[0053] FIG. 16 indicates sensorgrams of surface plasmon
resonance-based analyses which show that clone SMB0171 binds to
(interacts with) ATP. The sensorgrams show the interactions between
SMB0171 and the antigen at 5 and 50 .mu.M in order from the
bottom.
[0054] FIG. 17 indicates the results of competitive ELISA which
shows that clone SMB0002 binds to adenosine and ATP.
[0055] FIG. 18 shows a graph that assesses the inhibitive ability
of ATP towards binding of biotin-labeled antigens (a mixture of
5'-Adenosine-PEG-biotin and ATP-PEG-biotin) by ATNLSA1-4_D12.
[0056] FIG. 19 shows the concept of a rationally designed antibody
library that can obtain adenosine/ATP-switch antibodies against any
antigen, wherein the library is made from antibody variable region
portions that contact with the antigens such that adenosine or ATP
is positioned between the antibody and antigen.
[0057] FIG. 20 shows the concept of an adenosine-immunized rabbit
antibody library which yields adenosine/ATP-switch antibodies
against any antigen and in which adenosine or ATP is sandwiched
between the antibody and the antigen.
[0058] FIG. 21 is a figure showing the result of ELISA for binding
of the antibody to human IL-6. The vertical axis shows the binding
activity of each antibody to human IL-6 depending on the presence
or absence of amino acids or amino acid metabolites (kynurenine,
tryptophan, phenylalanine, anthranilic acid, 3-hydroxykynurenine,
and kynurenic acid), presented as absorbance values at wavelength
of 450 nm.
[0059] FIG. 22 is a figure showing the result of ELISA for binding
of the antibody to human IL-6. The vertical axis shows the binding
activity of the I6NMSC1-3.sub.--#03 antibody to human IL-6
depending on the presence or absence of each small molecule (ATP,
adenosine, inosine, PGE2, succinic acid, lactic acid, kynurenine,
and a small-molecule cocktail), presented as specific activity
values calculated from absorbance values at wavelength of 450
nm.
[0060] FIG. 23 is a figure showing the result of ELISA for binding
of the antibody to human IL-6. The vertical axis shows the binding
activity of the I6NMSC1-3.sub.--#17 antibody to human IL-6
depending on the presence or absence of each small molecule (ATP,
adenosine, inosine, PGE2, succinic acid, lactic acid, kynurenine,
and a small-molecule cocktail), presented as specific activity
values calculated from absorbance values at wavelength of 450
nm.
[0061] FIG. 24 is a figure showing the result of ELISA for binding
of the antibody to HSA. The vertical axis shows the binding
activity of the HSNMSC1-4.sub.--#22 antibody to HSA depending on
the presence or absence of each small molecule (ATP, adenosine,
inosine, PGE2, succinic acid, lactic acid, kynurenine, and a
small-molecule cocktail), presented as absorbance values at
wavelength of 450 nm.
[0062] FIG. 25 is a figure showing the result of ELISA performed on
clone I6DL2C5-4.sub.--076, which was obtained from the rationally
designed antibody library against human IL-6 in the presence or
absence of ATP and/or adenosine at 1 mM. The vertical axis shows
the absorbance value which evaluates binding activity of the
antibody to human IL-6. Results obtained when using M13KO7 Helper
Phage are presented as the negative control.
[0063] FIG. 26 is a figure showing the result of ELISA performed on
clone HSDL3C5-4.sub.--015, which was obtained from the rationally
designed antibody library against human serum albumin in the
presence or absence of ATP and/or adenosine at 1 mM. The vertical
axis shows the absorbance value which assesses binding activity of
the antibody to human serum albumin. Results obtained when using
M13KO7 Helper Phage are presented as the negative control.
[0064] FIG. 27 is a figure showing the result of ELISA performed on
clone 6RAD2C1-4.sub.--011 and 6RAD2C1-4.sub.--076, which were
obtained from the rationally designed antibody library against
human IL-6 receptor in the presence or absence of ATP and/or
adenosine (written as ADO) at 1 mM, and in the presence or absence
of a small-molecule cocktail (SC). The vertical axis shows
absorbance values which assess the binding activity of the antibody
to the human IL-6 receptor. Results obtained when using M13KO7
Helper Phage are presented as the negative control.
[0065] FIG. 28 is a figure showing the result of ELISA for binding
of clone 6RNMSC1-2_F02 to human IL-6R. The vertical axis shows the
absorbance values which assess the binding activity of the antibody
to human IL-6R in the presence or absence of each small
molecule.
[0066] FIG. 29 is a figure showing the result of ELISA for binding
of clone 6RNMSC1-3_G02 to human IL-6R. The vertical axis shows the
absorbance values which assess the binding activity of the antibody
to human IL-6R in the presence or absence of each small
molecule.
[0067] FIG. 30 is a figure showing the result of ELISA for binding
of an antibody to human IL-6R. The vertical axis shows the
absorbance values which assess the binding activity of the antibody
to human IL-6R in the presence or absence of each amino acid or
amino acid metabolite.
[0068] FIG. 31 presents sensorgrams showing the interaction between
6RNMSC1-2_F02 and 1 .mu.mol/L IL-6R in the presence of 100
.mu.mol/L kynurenine, in the presence of 10 mmol/L ATP, and in the
absence of kynurenine and ATP. The solid line indicates the
interaction in the presence of kynurenine, the dotted line
indicates the interaction in the presence of ATP, and the dashed
line indicates the interaction in their absence.
[0069] FIG. 32 is a graph obtained by allowing 6RNMSC1-2_F02 to
interact with IL-6R immobilized on Sensorchip CM5 in the presence
of 100 .mu.mol/L kynurenine, and then observing the dissociation of
6RNMSC1-2_F02 from IL-6R in the presence of a buffer containing 100
.mu.mol/L kynurenine or in the presence of a buffer that does not
contain kynurenine. In the figure, the vertical axis shows values
normalized by defining the amount of 6RNMSC1-2_F02 bound in the
presence of 100 .mu.mol/L kynurenine as 100, and the horizontal
axis shows the passage of time (in seconds) from the start of the
interaction. The solid line shows the dissociation of 6RNMSC1-2_F02
from IL-6R in the presence of kynurenine, and the dotted line shows
the dissociation of 6RNMSC1-2_F02 from IL-6R in the absence of
kynurenine.
[0070] FIG. 33 is a graph produced by allowing 5 .mu.g/L of
6RNMSC1-2_F02 to interact as an analyte for 180 seconds, and
assessing the response to IL-6R immobilized onto Sensorchip CM5.
The vertical axis shows change in the response (RU) before and
after 6RNMSC1-2_F02 interaction, and the horizontal axis shows the
concentration (.mu.mol/L) of kynurenine contained in the
solution.
[0071] FIG. 34 is a figure showing assessment of the binding of
antibodies to membrane-type human IL-6R by FCM. The top panel shows
results obtained in the presence of Kynurenine, and the bottom
panel shows results obtained in the absence of Kynurenine. The
horizontal axis shows the fluorescence intensity and the vertical
axis shows the cell count.
[0072] FIG. 35A shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells expressing
the antigens. It shows the ADCC activity of clone 6RNMSC1-2_F02,
which binds to hIL-6R in the presence of kynurenine, toward BaF
cells expressing hIL-6R in the presence (triangles) or absence
(circles) of kynurenine. The open triangles and circles show the
measured values, and the filled triangles and circles show the mean
values.
[0073] FIG. 35B shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells that
express the antigen. It shows the ADCC activity of MRA, which binds
to hIL-6R regardless of the presence of kynurenine, toward BaF
cells expressing hIL-6R in the presence (triangles) or absence
(circles) of kynurenine. The open triangles and circles show the
measured values, and the filled triangles and circles show the mean
values.
[0074] FIG. 36 shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells expressing
the antigen. It shows the ADCC activity of clone 6RNMSC1-2_F02
toward BaF cells expressing hIL-6R in the presence (triangles) or
absence (circles) of clone 6RNMSC1-2_F02 which binds to hIL-6R in
the presence of kynurenine. The horizontal axis shows the
kynurenine concentration and the vertical axis shows the ADCC
activity (%). The mean values and standard deviations of ADCC
activity are shown.
[0075] FIG. 37 is a figure showing the result of ELISA for the
binding of clone 6RNMSC1-2_F02 in mouse serum to human IL-6R. The
vertical axis shows the absorbance values which evaluate the
binding activities of the antibody to human IL-6R in the presence
or absence of kynurenine.
[0076] FIG. 38 is a figure showing the result of ELISA performed
with clone I6RLSA1-6.sub.--011, which was obtained from the
rationally designed antibody library, against human IL-6 in the
presence or absence of ATP and adenosine at 10 mM. The vertical
axis shows the absorbance value which evaluates binding activity of
the antibody to human IL-6. Results obtained when using a clone
obtained from the rationally designed antibody library and showing
binding activity toward human IL-6 regardless of the presence of
small molecules are presented as the positive control. Results
obtained when using the M13KO7 Helper Phage are presented as the
negative control.
[0077] FIG. 39 is a figure showing the result of ELISA performed
with clone 6RRLSA1-6.sub.--037 and 6RRLSA1-6.sub.--045, which were
obtained from the rationally designed antibody library, against the
human IL-6 receptor in the presence or absence of ATP and adenosine
at 10 mM. The vertical axis shows the absorbance value which
evaluates the binding activity of the antibodies to the human IL-6
receptor. Results obtained when using the M13KO7 Helper Phage are
presented as the negative control.
[0078] FIG. 40 is a figure showing the result of ELISA performed on
96 clones obtained by panning the rationally designed antibody
library four times against human IgA-Fc using a multivalent
antibody phage display. The absorbance values which evaluate the
binding activity of the antibodies to human IgA-Fc in the absence
of ATP and adenosine are shown on the vertical axis, and absorbance
values which evaluate the binding activity of the antibodies to
human IgA-Fc in the presence of ATP and adenosine are shown on the
horizontal axis.
[0079] FIG. 41 is a figure showing the result of ELISA performed on
96 clones obtained by panning the rationally designed antibody
library four times against human IgA-Fc using a monovalent antibody
phage display. The absorbance values which evaluate the binding
activity of the antibodies to human IgA-Fc in the absence of ATP
and adenosine are shown on the vertical axis, and the absorbance
values which evaluate the binding activity of the antibodies to
human IgA-Fc in the presence of ATP and adenosine are shown on the
horizontal axis.
[0080] FIG. 42 is a figure showing the result of ELISA performed on
clone IADL3C5-4.sub.--048 obtained from the rationally designed
antibody library against human IgA-Fc in the presence or absence of
ATP and adenosine at 1 mM. The vertical axis shows the absorbance
value which evaluates binding activity of the antibody to human
IgA-Fc. Results obtained when using a clone obtained from the
rationally designed antibody library and showing binding activity
toward human IgA-Fc regardless of the presence of small molecules
are presented as the positive control. Results obtained when using
the M13KO7 Helper Phage are presented as the negative control.
[0081] FIG. 43 is a graph showing the binding level (binding
response (RU)) when each clone at 1 .mu.M was made to interact for
120 seconds with IL-6R immobilized on Sensorchip CM5 in the
presence or absence of each of the small molecules at 1 mM.
[0082] FIG. 44A shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells expressing
the antigen. It is a figure showing the ADCC activity of clone
6RAD2C1-4.sub.--030, which binds to hIL-6R in the presence of ATP,
toward CHO cells expressing hIL-6R in the presence (triangles) or
absence (circles) of ATP. The open triangles and circles show the
measured values, and the filled triangles and circles show the mean
values.
[0083] FIG. 44B shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells expressing
the antigen. It is a figure showing the ADCC activity of clone
6RAD2C1-4.sub.--011, which binds to hIL-6R in the presence of ATP,
toward CHO cells expressing hIL-6R in the presence (triangles) or
absence (circles) of ATP. The open triangles and circles show the
measured values, and the filled triangles and circles show the mean
values.
[0084] FIG. 44C shows the ADCC activity of antibodies that bind to
antigens in the presence of small molecules toward cells expressing
the antigen. It is a figure showing the ADCC activity of MRA, which
binds to hIL-6R regardless of the presence or absence of ATP,
toward CHO cells expressing in the presence (triangles) or absence
(circles) of ATP. The open triangles and circles show the measured
values, and the filled triangles and circles show the mean
values.
[0085] FIG. 45 is a figure showing the result of ELISA performed on
clone HSADSA1-6.sub.--020 obtained from the rationally designed
antibody library against HSA in the presence or absence of ATP and
adenosine at 10 mM. The vertical axis shows the absorbance value
which evaluates binding activity of the antibody to HSA. Results
obtained when using a clone obtained from the rationally designed
antibody library and showing binding activity toward HSA regardless
of the presence of small molecules are presented as the positive
control. Results obtained when using the M13KO7 Helper Phage are
presented as the negative control.
MODE FOR CARRYING OUT THE INVENTION
[0086] The definitions and detailed description below are provided
to facilitate understanding of the present invention illustrated
herein.
Amino Acids
[0087] Herein, amino acids are described by 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.
Alteration of Amino Acids
[0088] For amino acid alteration 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
employed. Furthermore, several known methods may also be employed
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 which has a
non-natural amino acid bound to a complementary amber suppressor
tRNA of one of the stop codons, the UAG codon (amber codon).
[0089] 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 alterations: 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.
[0090] Furthermore, herein, as an expression showing alteration of
amino acids, an expression that shows before and after a number
indicating a specific position, one-letter or three-letter codes
for amino acids before and after alteration, respectively, may be
used appropriately. For example, the alteration N100bL or
Asn100bLeu used when substituting an amino acid contained in an
antibody variable region indicates substitution of Asn at position
100b (according to Kabat numbering) with Leu. That is, the number
shows the amino acid position according to Kabat numbering, the
one-letter or three-letter amino-acid code written before the
number shows the amino acid before substitution, and the one-letter
or three-letter amino-acid code written after the number shows the
amino acid after substitution. Similarly the alteration P238D or
Pro238Asp used when substituting an amino acid of the Fc region
contained in an antibody constant region indicates substitution of
Pro at position 238 (according to EU numbering) with Asp. That is,
the number shows the amino acid position according to EU numbering,
the one-letter or three-letter amino-acid code written before the
number shows the amino acid before substitution, and the one-letter
or three-letter amino-acid code written after the number shows the
amino acid after substitution.
Antigens
[0091] 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 RIAALK-2, activin RIB ALK-4, activin RIIA, activin RIIB,
ADAM, ADAM10, ADAM12, ADAM15, 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, Ax1, 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, PD1, PDL1,
LAG3, TIM3, galectin-9, 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 gp120 V3 loop, HLA, HLA-DR,
HM 1.24, 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-21, IL-23,
IL-27, 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 (Muc1), 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), P1GF, 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, 5100, 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-betaRI (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 Rill, 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-.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), TP-1, t-PA, Tpo,
TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor,
TRF, Trk, TROP-2, TLR1 (Toll-like receptor 1), TLR2, TLR3, TLR4,
TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, 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
(fit-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, WNT10B, 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 S1P; and receptors for hormone and growth
factors. Preferred antigens are antigens that are expressed in
cancer cells, immune cells, stromal cells, or such present in
cancer tissues or inflammatory tissues.
[0092] While receptors are recited as examples of the
above-mentioned antigens, when these receptors exist in soluble
forms in biological fluids, they may be used as antigens that bind
to the antigen-binding molecule of the present invention, which
contains an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of the target tissue-specific
compound. An example of a non-limiting embodiment of such a soluble
receptor is the 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).
[0093] Membrane-type molecules expressed on cell membranes and
soluble molecules secreted from cells to the outside of the cells
are included in the examples of the above-mentioned antigens. When
the antigen-binding molecule of the present invention, which
contains an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of the target tissue-specific
compound, binds to a soluble molecule secreted from cells, it is
preferable that the antigen-binding molecule has neutralizing
activity as described later.
[0094] The fluids in which the soluble molecules exist are not
limited, and the soluble molecules 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 soluble molecules to which antigen-binding
molecules of the present invention bind may be present in the
extracellular fluid. 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 effusion, 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).
[0095] When an antigen-binding molecule of the present invention
comprising an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of a target tissue-specific
compound binds to a membrane-type molecule expressed on a cell
membrane, suitable examples of the antigen-binding molecule include
antigen-binding molecules which have cytotoxic activity, bind to a
cytotoxic substance, or have the ability to bind to a cytotoxic
substance, as described later. Furthermore, antigen-binding
molecules having a neutralizing activity instead of the properties
of having a cytotoxic activity, binding to a cytotoxic substance,
or having the ability to bind to a cytotoxic substance; or in
addition to these properties are also suitable examples of a
non-limiting embodiment.
Epitopes
[0096] "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.
[0097] 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 about 10 or 6 to 20 amino
acids in a specific sequence.
[0098] 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.).
[0099] 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 antigens and a plurality of
antigen-binding molecules bind is referred to as avidity. When an
antibody comprising a plurality of antigen-binding domains (i.e.,
multivalent antibody) or such binds to a plurality of epitopes, the
affinity acts synergistically, and therefore avidity becomes higher
than affinity.
Binding Activity
[0100] Examples of a method for assessing the epitope binding by a
test antigen-binding molecule containing an IL-6R antigen-binding
domain are described below. According to the examples below,
methods for assessing the epitope binding by a test antigen-binding
molecule containing an antigen-binding domain for an antigen other
than IL-6R, can also be appropriately conducted.
[0101] For example, whether a test antigen-binding molecule
containing an IL-6R antigen-binding domain recognizes a linear
epitope in the IL-6R molecule can be confirmed for example as
mentioned below. A linear peptide comprising an amino acid sequence
forming the extracellular domain of IL-6R 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 extracellular domain
in an IL-6R cDNA. Then, a test antigen-binding molecule containing
an IL-6R antigen-binding domain is assessed for its binding
activity towards a linear peptide comprising the amino acid
sequence forming the extracellular domain. For example, an
immobilized linear peptide can be used as an antigen by ELISA 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 that the linear
peptide inhibits the binding of the antigen-binding molecule to
IL-6R-expressing cells. These tests can demonstrate the binding
activity of the antigen-binding molecule towards the linear
peptide.
[0102] Whether a test antigen-binding molecule containing an IL-6R
antigen-binding domain recognizes a conformational epitope can be
assessed as follows. IL-6R-expressing cells are prepared for the
above purpose. A test antigen-binding molecule containing an IL-6R
antigen-binding domain can be determined to recognize a
conformational epitope when it strongly binds to IL-6R-expressing
cells upon contact, but does not substantially bind to an
immobilized linear peptide comprising an amino acid sequence
forming the extracellular domain of IL-6R. Herein, "not
substantially bind" means that the binding activity is 80% or less,
generally 50% or less, preferably 30% or less, and particularly
preferably 15% or less compared to the binding activity towards
cells expressing human IL-6R.
[0103] Methods for assaying the binding activity of a test
antigen-binding molecule containing an IL-6R antigen-binding domain
towards IL-6R-expressing cells 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
fluorescence activated cell sorting (FACS) using IL-6R-expressing
cells as antigen.
[0104] In the ELISA format, the binding activity of a test
antigen-binding molecule containing an IL-6R antigen-binding domain
towards IL-6R-expressing cells can be assessed quantitatively by
comparing the levels of signal generated by enzymatic reaction.
Specifically, a test polypeptide complex is added to an ELISA plate
onto which IL-6R-expressing cells are immobilized. Then, the test
antigen-binding molecule bound to the cells is detected using an
enzyme-labeled antibody that recognizes the test antigen-binding
molecule. Alternatively, when FACS is used, a dilution series of a
test antigen-binding molecule is prepared, and the antibody binding
titer for IL-6R-expressing cells can be determined to compare the
binding activity of the test antigen-binding molecule towards
IL-6R-expressing cells.
[0105] 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
[0106] FACSCalibur.TM. (all are trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
[0107] Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of
Beckman Coulter).
[0108] Preferable methods for assaying the binding activity of a
test antigen-binding molecule containing an IL-6R antigen-binding
domain towards an antigen include, for example, the following
method. First, IL-6R-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.
[0109] Whether a test antigen-binding molecule containing an IL-6R
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.
[0110] Specifically, in cross-blocking assay, the IL-6R 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
IL-6R 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 IL-6R protein-coated
wells.
[0111] The quantity of the test antigen-binding molecule bound to
the wells via the IL-6R 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.
[0112] When the candidate competitor antigen-binding molecule can
block the binding by a test antigen-binding molecule containing an
IL-6R 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.
[0113] When the structure of an epitope bound by a test
antigen-binding molecule containing an IL-6R 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.
[0114] 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.
[0115] Alternatively, when the identified epitope is a
conformational epitope, whether test and control antigen-binding
molecules share a common epitope can be assessed by the following
method. First, IL-6R-expressing cells and cells expressing IL-6R
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.
[0116] In the above method, whether an antigen-binding molecule
does "not substantially bind to cells expressing mutant IL-6R" can
be assessed, for example, by the following method. First, the test
and control antigen-binding molecules bound to cells expressing
mutant IL-6R 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 Formula 1 below 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)
Formula 1
[0117] The Geometric Mean comparison value (.DELTA.Geo-Mean value
for the mutant IL-6R molecule) determined by the above analysis,
which reflects the quantity of a test antigen-binding molecule
bound to cells expressing mutant IL-6R, is compared to the
.DELTA.Geo-Mean comparison value that reflects the quantity of the
test antigen-binding molecule bound to IL-6R-expressing cells. In
this case, the concentrations of the test antigen-binding molecule
used to determine the .DELTA.Geo-Mean comparison values for
IL-6R-expressing cells and cells expressing mutant IL-6R are
particularly preferably adjusted to be equal or substantially
equal. An antigen-binding molecule that has been confirmed to
recognize an epitope in IL-6R is used as a control antigen-binding
molecule.
[0118] If the .DELTA.Geo-Mean comparison value of a test
antigen-binding molecule for cells expressing mutant IL-6R is
smaller than the .DELTA.Geo-Mean comparison value of the test
antigen-binding molecule for IL-6R-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 IL-6R". 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.
Target Tissue
[0119] The term "target tissue" as used herein refers to a tissue
containing cells carrying antigens to which the antigen-binding
molecules of the present invention bind in a manner dependent on
compounds. It is a tissue that yields positive pharmacological
effects for the organism carrying the tissue, when the
antigen-binding molecules bind to a membrane-type molecule
expressed on the cells or bind to a soluble molecule present in the
tissue. In this case, the phrase "positive pharmacological effects"
refers to effects that relieve, alleviate, ameliorate, or cure
symptoms brought about by pathological sites containing the target
tissue for the organism carrying the tissue. When the symptoms are
brought about by malignant tumors such as cancer, a non-limiting
embodiment of a mechanism that yields such a pharmacological effect
is, for example, cytotoxic activity and growth inhibition against
cancer cells, and immunostimulation in cancer tissues. In the case
of inflammatory diseases, examples of such a non-limiting
embodiment of the mechanism include immunosuppression and activity
to block actions of inflammatory cytokines in inflammatory
tissues.
Cancer Tissue-Specific Compounds
[0120] The term "compound specific to a cancer tissue (cancer
tissue-specific compound)" as used herein refers to a compound
differentially present in cancer tissues as compared to
non-cancerous tissues. Herein, the term "cancer" is generally used
to describe malignant neoplasms, which may be metastatic or
non-metastatic. Non-limiting examples of carcinomas developed from
epithelial tissues such as skin or digestive tract include brain
tumor, skin cancer, head and neck cancer, esophageal cancer, lung
cancer, stomach cancer, duodenal cancer, breast cancer, prostate
cancer, cervical cancer, endometrial cancer, pancreatic cancer,
liver cancer, colorectal cancer, colon cancer, bladder cancer, and
ovarian cancer. Non-limiting examples of sarcomas developed from
non-epithelial (interstitial) tissues such as muscles include
osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma,
liposarcoma, and angiosarcoma. Non-limiting examples of
hematological cancer derived from hematopoietic organs include
malignant lymphomas including Hodgkin's lymphoma and non Hodgkin's
lymphoma; leukemia including acute myelocytic leukemia or chronic
myelocytic leukemia, and acute lymphatic leukemia or chronic
lymphatic leukemia; and multiple myeloma. The term "neoplasm"
widely used herein refers to any newly formed diseased tissue
tumor. In the present invention, neoplasms cause formation of
tumors, which are partly characterized by angiogenesis. Neoplasms
may be benign such as hemangioma, glioma, or teratoma, or malignant
such as carcinoma, sarcoma, glioma, astrocytoma, neuroblastoma, or
retinoblastoma.
[0121] The term "cancer tissue" refers to a tissue containing at
least one cancer cell. Therefore, as cancer tissues contain cancer
cells and blood vessels, it refers to all cell types contributing
to the formation of a tumor mass containing cancer cells and
endothelial cells. Herein, "tumor mass" refers to a foci of tumor
tissue. The term "tumor" is generally used to mean a benign
neoplasm or a malignant neoplasm.
[0122] For example, in several embodiments, cancer tissue-specific
compounds may be compounds defined by qualitative properties of
cancer tissues such as being present in cancer tissues but absent
in non-cancer tissues, or being absent in cancer tissues but
present in non-cancer tissues. In other embodiments, cancer
tissue-specific compounds may be compounds defined by quantitative
properties of cancer tissues such as being present in cancer
tissues at a concentration different (for example, higher
concentration or lower concentration) from that in non-cancer
tissues. For example, cancer tissue-specific compounds are present
differentially at arbitrary concentrations. Generally, cancer
tissue-specific compounds can be present at a concentration
increased by at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 100%, at least 110%, at least 120%, at least
130%, at least 140%, at least 150%, at least 2-fold, at least
5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at
least 10.sup.3-fold, at least 10.sup.4-fold, at least
10.sup.5-fold, at least 10.sup.6-fold, or more, or up to infinity
(i.e., when the compound is absent in non-cancerous tissues).
Alternatively, they can generally be present at a concentration
decreased by at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 100% (i.e., absent). Preferably, cancer tissue-specific
compounds are differentially present at statistically significant
concentrations (that is, as determined using either Welch's t-test
or Wilcoxon rank sum test, the p value is less than 0.05 and/or the
q value is less than 0.10). Examples of a non-limiting embodiment
of a cancer tissue-specific compound include compounds which are
cancer tissue-specific metabolites produced by metabolic activities
characteristic of cancer cells, immune cells, or stromal cells
contained in cancer tissues, such as those described below (cancer
tissue-specific metabolites, cancer cell-specific metabolites,
metabolites specific to immune cells that infiltrated into cancer
tissues, and cancer stromal cell-specific metabolites).
Cancer Tissue-Specific Metabolites
[0123] The term "metabolism" refers to chemical changes that take
place in biological tissues and includes "anabolism" and
"catabolism". Anabolism refers to biosynthesis or accumulation of
molecules, and catabolism refers to degradation of molecules.
"Metabolites" are intermediates or products that arise from
metabolism. "Primary metabolites" refers to metabolites directly
involved in the process of growth or proliferation of cells or
organisms. "Secondary metabolites" refer to products that are not
directly involved in such process of growth or proliferation, and
are products such as pigments or antibiotics that are produced as a
result of metabolism which biosynthesizs substances that are not
directly involved in biological phenomena common to cells and
organisms. The metabolites may be metabolites of "biopolymers", or
they may be metabolites of "small molecules". "Biopolymers" are
polymers comprising one or more types of repeating units.
Biopolymers are generally found in biological systems, and examples
include cells forming the organism and intercellular matrices that
adhere to them, molecules having a molecular weight of
approximately 5000 or more which form structures such as
interstitial matrices, particularly polysaccharides (carbohydrates
and such), peptides (this term is used so as to include
polypeptides and proteins), and polynucleotides, and similarly
their analogs such as compounds composed of or including amino acid
analogs or non-amino acid groups. "Small molecules" refers to
natural chemical substances other than "biopolymers" that exist in
vivo. Suitable examples of a non-limiting embodiment of a cancer
tissue-specific metabolite described herein include cancer
cell-specific small-molecule metabolites (Eva Gottfried, Katrin
Peter and Marina P. Kreutz, From Molecular to Modular Tumor Therapy
(2010) 3 (2), 111-132). In addition, metabolites that are highly
produced by immune cells that infiltrate into cancer tissues, and
metabolites that are highly produced by stromal cells that support
the survival and/or growth of cancer cells (cancer stromal cells or
cancer associated stromal fibroblasts (CAF)) are also included.
Infiltrating immune cells are, for example, dendritic cells,
inhibitory dendritic cells, inhibitory T cells, exhausted T cells,
and myeloma derived suppressor cells (MDSC). Furthermore,
metabolites of the present invention include compounds released
from inside the cells to outside the cells when cells present in
cancer tissues (cancer cells, immune cells, or stromal cells) die
due to apoptosis, necrosis, or such.
[0124] To identify cancer cell-specific metabolites, metabolomic
analyses focused on metabolic profiling can be suitably used, in
addition to transcriptome-level analyses (for example, Dhanasekaran
et al. (Nature (2001) 412, 822-826), Lapointe et al. (Proc. Natl.
Acad. Sci. U.S.A. (2004) 101, 811-816) or Perou et al. (Nature
(2000) 406, 747-752)) and proteome-level analyses (for example,
Ahram et al. (Mol. Carcinog. (2002) 33, 9-15), Hood et al. (Mol.
Cell. Proteomics (2005) 4, 1741-1753)). More specifically, to
identify metabolites in test samples, metabolic profiling that uses
high-pressure liquid chromatography (HPLC), nuclear magnetic
resonance (NMR) (Brindle et al. (J. Mol. Recognit. (1997) 10,
182-187), mass spectrometry (Gates and Sweeley (Clin. Chem. (1978)
24, 1663-1673) (GC/MS and LC/MS)), and ELISA or such individually
and/or in combination may be used appropriately.
[0125] These studies elucidated heterogeneity within the
constituted tumors which results from changing the concentration
gradient of growth factors and metabolites (glucose, oxygen, or
such) that enable cancer cell growth under low oxygen pressure
conditions (Dang and Semenza (Trends Biochem. Sci. (1999) 24,
68-72)). In these studies, cell line models are also used to
understand the change in energy utilization pathway depending on
the different malignancy levels of tumors (Vizan et al. (Cancer
Res. (2005) 65, 5512-5515)). Examples of a non-limiting embodiment
of the technical components of the metabolomics platform include
sample extraction, separation, detection, spectroscopic analysis,
data normalization, description of class-specific metabolites,
pathway mapping, confirmation, and functional characterization of
candidate metabolites described by Lawton et al. (Pharmacogenomics
(2008) 9, 383). These methods enable identification of cancer
cell-specific metabolites in desired cancer tissues.
[0126] Examples of a non-limiting embodiment of cancer
tissue-specific compounds or cancer tissue-specific metabolites
used in the present invention preferably include at least one
compound selected from the compounds below. At least one compound
means that in addition to cases where the antigen-binding activity
of a same antigen-binding domain described below depends on one
type of cancer tissue-specific compound or metabolite, cases where
it depends on several types of cancer tissue-specific compounds or
metabolites are included.
(1) Primary Metabolites of the Krebs Cycle or of the Glycolytic
System Such as Lactic Acid, Succinic Acid, and Citric Acid
[0127] Preferable examples of a non-limiting embodiment of a cancer
tissue-specific compound, particularly a cancer cell-specific
metabolite, used in the present invention include primary
metabolites such as lactic acid, succinic acid, and citric acid,
which are produced as a result of glucose metabolism, and are
present at higher concentrations in cancer tissues as compared to
in the surrounding non-cancerous tissues. The glycolytic system
phenotype, which is characterized as an up-regulation of enzymes of
the glycolytic system (Embden-Meyerhof pathway) such as pyruvate
kinase, hexokinase, and lactic acid dehydrogenase (LDH), has been
conventionally known to be a characteristic of solid tumors as
Warburg effect.
[0128] That is, in tumor cells, high expression of the pyruvate
kinase isoform M2 which is necessary for anaerobic glycolysis, and
not isoform Ml, is considered to be working advantageously for the
growth of tumor cells in vivo (Christofk et al. (Nature (2008) 452,
230-233). Pyruvic acid produced by pyruvate kinase is subjected to
feedback inhibition by lactic acid produced as a result of
equilibrium reaction by lactic acid dehydrogenase (LDH) under
anaerobic conditions. Since the feedback inhibition causes
promotion of respiration in mitochondria (Krebs cycle) and cell
growth inhibition, up regulation of LDH, hexokinase, and glucose
transporter (GLUT) is said to play an important role in the
proliferation of cancer cells (Fantin et al. (Cancer Cell (2006) 9,
425-434)). Glucose is metabolized by the glycolytic system, and the
final metabolite lactic acid is transported together with protons
to the tumor surrounding, and as a result, the pH of the tissues
surrounding the tumor is said to become acidic. Lactic acid, which
is the final product of the glycolytic pathway, as well as succinic
acid and citric acid produced by promotion of respiration in
mitochondria are known to be accumulated in cancer tissues (Teresa
et al. (Mol. Cancer (2009) 8, 41-59)). Examples of a non-limiting
embodiment of cancer tissue-specific compounds, particularly cancer
cell-specific metabolites, used in the present invention preferably
include such primary metabolites such as lactic acid, succinic
acid, and citric acid produced by metabolism by the glycolytic
pathway. Furthermore, succinic acid which is present at high
concentration in cells is known to leak out to the outside of the
cells upon cell death (Nature Immunology, (2008) 9, 1261-1269).
Therefore, succinic acid concentration is thought to be increased
in cancer tissues in which cell death occurs frequently.
(2) Amino Acids Such as Alanine, Glutamic Acid, and Aspartic
Acid
[0129] Besides the above-mentioned glucose metabolism, the amino
acid metabolism is also known to be altered in tumor cells which
require continuous supply of essential amino acids and
non-essential amino acids that are necessary for the biosynthesis
of biopolymers under anaerobic conditions. Glutamine which contains
two nitrogens in its side chain acts as a nitrogen transporter, and
is an amino acid that is most widely distributed in an organism.
Tumor cells, in which the rate of glutamine uptake into cells is
increased, is said to be functioning as a glutamine trap. Such
increase in the uptake of glutamine and activity of converting into
glutamic acid and lactic acid is called "glutaminolysis", and is
considered to be a characteristic of transformed (tumor) cells
(Mazurek and Eigenbrodt (Anticancer Res. (2003) 23, 1149-1154); and
Mazurek et al. (J. Cell. Physiol. (1999) 181, 136-146)). As a
result, cancer patients show an increase in glutamic acid
concentration while showing a decrease in plasma glutamine level
(Droge et al. (Immunobiology (1987) 174, 473-479)). Furthermore,
correlation was observed between concentrations of .sup.13C-labeled
succinic acid, .sup.13C-labeled alanine, .sup.13C-labeled glutamic
acid, and .sup.13C-labeled citric acid in studies on
.sup.13C-radiolabeled glucose metabolism in lung cancer tissues.
Suitable examples of a non-limiting embodiment of cancer
tissue-specific compounds used in this invention include alanine,
glutamic acid, and aspartic acid which accumulate at high
concentrations in cancer tissues through such glutaminolysis and
the like.
(3) Amino Acid Metabolite Such as Kynurenine
[0130] Indolamine 2,3-dioxygenase (IDO) is a
tryptophan-metabolizing enzyme which is highly expressed in many
cancers such as melanoma, colon cancer, and kidney cancer
(Uyttenhove et al. (Nat. Med. (2003) 9, 1269-127)); and it is known
to have two isoforms (Lob et al. (Cancer Immunol. Immunother.
(2009) 58, 153-157)). IDO catalyzes the conversion of tryptophan to
kynurenine (shown as Compound 1), and is the first enzyme in the
nicotinamide nucleotide (NAD) de novo pathway. Furthermore, in
glioma which does not express IDO, kynurenine is produced from
tryptophan by tryptophan 2,3-dioxygenase (TDO) in the liver (Opitz
et al. (Nature (2011) 478, 7368, 197-203)). IDO is also expressed
in dendritic cells infiltrated into cancer tissues, and dendritic
cells also produce kynurenine (J. Immunol. (2008) 181, 5396-5404).
IDO is also expressed in myeloid-derived suppressor cells (MDSC) in
cancer tissues, and MDSC also produces kynurenine (Yu et al. (J.
Immunol. (2013) 190, 3783-3797)).
##STR00001##
[0131] Kynurenine is known to suppress the same type of T cell
response (Frumento et al. (J. Exp. Med. (2002) 196, 459-468); and a
mechanism has been suggested, in which tumor cells evade antitumor
immune responses through such inhibition, and proliferation of
glioma cells is promoted through an autocrine proliferation
mechanism in which kynurenine acts as an endogenous ligand for the
aryl hydrocarbon receptor expressed on gliomas (Optiz et al.
(mentioned above)). Kynurenine is converted to anthranilic acid
(shown as Compound 2) by kynurenidase, and to 3-hydroxykynurenine
(shown as Compound 3) by kynurenine 3-hydroxylase. Anthranilic acid
and 3-hydroxykynurenine are both converted to 3-hydroxyanthranilic
acid, the precursor of NAD.
##STR00002##
[0132] Kynurenine is converted to kynurenic acid (shown as Compound
4) by kynurenine aminotransferase. Examples of a non-limiting
embodiment of cancer tissue-specific compounds, particularly cancer
cell-specific metabolites, used in the present invention preferably
include such amino acid metabolites such as kynurenine and its
metabolites such as anthranilic acid, 3-hydroxykynurenine, and
kynurenic acid.
##STR00003##
(4) Arachidonic Acid Metabolites Such as Prostaglandin E2
[0133] Prostaglandin E2 (PGE2) (Compound 5) is an arachidonic acid
metabolite called a prostanoid, which includes thromboxane and
prostaglandin synthesized by cyclooxygenase (COX)-1/2 (Warner and
Mitchell (FASEB J. (2004) 18, 790-804)). PGE2 promotes the
proliferation of colon cancer cells and suppresses their apoptosis
(Sheng et al. (Cancer Res. (1998) 58, 362-366)). Cyclooxygenase
expression is known to be altered in many cancer cells. More
specifically, while COX-1 is expressed constitutively in almost all
tissues, COX-2 has been found to be mainly induced by certain types
of inflammatory cytokines and cancer genes in tumors (Warner and
Mitchell (mentioned above)). In addition, COX-2 overexpression has
been reported to be related to bad prognosis for breast cancer
(Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)), and rapid
disease progression for ovarian cancer (Denker et al. (Mod. Pathol.
(2006) 19, 1261-1269)). Inhibitory T cells that have infiltrated
into cancer tissues also produce prostaglandin E2 (Curr. Med. Chem.
(2011) 18, 5217-5223). Small molecules such as the arachidonic acid
metabolites prostaglandin and leukotriene are known to act as a
stimulating factor that regulates autocrine and/or paracrine growth
of cancer (Nat. Rev. Cancer (2012) 12 (11) 782-792). Examples of a
non-limiting embodiment of cancer tissue-specific compounds used in
the present invention, particularly cancer cell-specific
metabolites and immune cell-specific metabolites that have
infiltrated into cancer tissues, preferably include such
arachidonic acid metabolites such as prostaglandin E2. Besides
prostaglandin E2, production of thromboxane A2 (TXA2) is enhanced
in cancer tissues such as colorectal cancer tissues (J. Lab. Clin.
Med. (1993) 122, 518-523), and thromboxane A2 can be suitably
presented as a non-limiting embodiment of an arachidonic acid
metabolite of the present invention.
##STR00004##
(5) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,
Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and
Adenosine Monophosphate (AMP)
[0134] When cancer cells undergo cell death, a large amount of ATP
in the cell is known to leak out to the outside of the cells.
Therefore, the ATP concentration is remarkably higher in cancer
tissues than in normal tissues (PLoS One. (2008) 3, e2599).
Multiple types of cells release adenine nucleotides in the form of
ATP, ADP, and AMP. Metabolism takes place through an extracellular
enzyme on the cell surface such as extracellular 5'-nucleotidase
(ecto-5'-nucleotidase) (CD73) (Resta and Thompson (Immunol. Rev.
(1998) 161, 95-109) and Sadej et al. (Melanoma Res. (2006) 16,
213-222)). Adenosine is a purine nucleoside that exists
constitutively at low concentration in the extracellular
environment, but in hypoxic tissues found in solid cancers, a
remarkable increase in the extracellular adenosine concentration
has been reported (Blay and Hoskin (Cancer Res. (1997) 57,
2602-2605). CD73 is expressed on the surface of immune cells and
tumors (Kobie et al. (J. Immunol. (2006) 177, 6780-6786)), and its
activity has been found to be increased in breast cancer (Canbolat
et al. (Breast Cancer Res. Treat. (1996) 37, 189-193)), stomach
cancer (Durak et al. (Cancer Lett. (1994) 84, 199-202)), pancreatic
cancer (Flocke and Mannherz (Biochim. Biophys. Acta (1991) 1076,
273-281), and glioblastoma (Bardot et al. (Br. J. Cancer (1994) 70,
212-218)). It has been proposed that the accumulation of adenosine
in cancer tissues may be caused by an increase in the intracellular
adenosine production through dephosphorylation of AMP by
5'-nucleotidase in the cytoplasm (Headrick and Willis (Biochem. J.
(1989) 261, 541-550)). Furthermore, inhibitory T cells and such
that have infiltrated into cancer tissues also express ATPase and
produce adenosine (Proc. Natl. Acad. Sci. (2006) 103 (35),
13132-13137; Curr. Med. Chem. (2011) 18, 5217-5223). The produced
adenosine is considered to be rendering the cancer tissue an
immunosuppressive environment through adenosine receptors such as
the A2A receptor (Curr. Med. Chem. (2011), 18, 5217-23). Examples
of a non-limiting embodiment of the cancer tissue-specific compound
used in the present invention preferably include ATP, ADP, AMP, and
adenosine which accumulate at high concentration in cancer tissues
through such metabolism of purine nucleotides such as ATP.
Furthermore, since adenosine is degraded to inosine by adenosine
deaminase, inosine accumulates at high concentration.
(6) Uric Acid
[0135] Uric acid is a product of the metabolic pathway of purine
nucleosides in vivo, and is released to the outside of cells such
as the interstitial space and blood. In recent years, it has been
found to be released from dead cells that are present at sites of
lesions such as cancer tissues (Nat. Med. (2007) 13, 851-856).
Examples of a non-limiting embodiment of cancer tissue-specific
compounds used in the present invention preferably include such
uric acid which accumulates at high concentration in cancer tissues
due to metabolism of purine nucleotides such as ATP.
(7) 1-Methyl Nicotinamide
[0136] The enzyme nicotinamide N-methyl transferase is known to be
highly expressed in several human cancer tissues. When this enzyme
produces the stable metabolite 1-methylnicotinamide from
nicotinamide, the methyl group of S-adenosylmethionine (SAM) which
serves as a methyl donor is consumed; therefore, the high
expression of nicotinamide N-methyltransferase has been suggested
to contribute to tumorigenesis through a mechanism that impairs the
DNA methylation ability accompanying a decrease in the SAM
concentration in cancer cells (Ulanovskaya et al. (Nat. Chem. Biol.
(2013) 9 (5) 300-306)). The stable metabolite of this enzyme,
1-methylnicotinamide is known to be secreted to the outside of
cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol. (2010) 56,
83-86)), and preferable examples of a non-limiting embodiment of
cancer tissue-specific compounds used in the present invention
include 1-methylnicotinamide and such which accumulate at high
concentration in cancer tissues through nicotinamide
metabolism.
Inflammatory Tissue-Specific Compounds
[0137] The term "compound specific to inflammatory tissue
(inflammatory tissue-specific compound)" as used herein refers to a
compound that is present differentially in inflammatory tissues as
compared to non-inflammatory tissues. Herein, suitable examples of
"inflammatory tissues" include:
joints with rheumatoid arthritis or osteoarthritis; lungs (alveoli)
with bronchial asthma or COPD; digestive organs of inflammatory
bowel disease, Crohn's disease, or ulcerative colitis; fibrotic
tissues of fibrosis of the liver, kidney, or lung; tissues
undergoing rejection reaction in organ transplantation; blood
vessels and heart (myocardium) in arteriosclerosis or heart
failure; visceral fat in metabolic syndrome; skin tissues in atopic
detrmatitis or other dermatitis; and spinal nerves in disk
herniation or chronic low back pain.
Inflammatory Tissue-Specific Metabolites
[0138] "Inflammatory tissue-specific metabolite" refers to
metabolites highly produced by immune cells that have infiltrated
into inflammatory tissues, and metabolites highly produced by
specifically normal cells that have been damaged in inflammatory
tissues. Examples of infiltrating immune cells include effector T
cells, mature dendritic cells, neutrophils, granule cells (mast
cells), and basophils. Furthermore, metabolites in the present
invention include compounds that are released from inside the cells
to the outside of the cells when the cells that are present in
inflammatory tissues (immune cells and normal cells) die by
apoptosis, necrosis, or such.
[0139] Examples of a non-limiting embodiment of the inflammatory
tissue-specific compounds or inflammatory tissue-specific
metabolites used in the present invention preferably include at
least one compound selected from the compounds below. At least one
compound means including cases where the antigen-binding activity
of a same antigen-binding domain described below depends on one
type of inflammatory tissue-specific compound or metabolite, as
well as cases where it depends on several types of inflammatory
tissue-specific compounds or metabolites.
(1) Arachidonic Acid Metabolites Such as Prostaglandin E2
[0140] The PGE2 concentration has been known to be high in
rheumatoid arthritis and osteoarthritis (Eur. J. Clin. Pharmacol.
(1994) 46, 3-7.; Clin. Exp. Rheumatol. (1999) 17, 151-160; Am. J.
Vet. Res. (2004) 65, 1269-1275). Examples of a non-limiting
embodiment of inflamatory tissue-specific compounds, particularly
inflammatory tissue-specific metabolites and metabolites specific
to immune cells that infiltrate into inflammatory tissues used in
the present invention preferably include such arachidonic acid
metabolites such as prostaglandin E2.
(2) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,
Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and
Adenosine Monophosphate (AMP)
[0141] ATP concentration is known to be high in pulmonary alveoli
where inflammation caused by bronchial asthma is taking place (Nat.
Med. (2007) 13, 913-919). ATP concentration is also known to be
high in pulmonary alveoli where inflammation caused by COPD is
taking place (Am. J. Respir. Crit. Care Med. (2010) 181, 928-934).
Furthermore, adenosine concentration has been observed to be high
in the joint fluid of rheumatoid arthritis patients (Journal of
Pharmaceutical and Biomedical Analysis (2004) 36, 877-882).
Furthermore, ATP concentration is known to be high in tissues where
a rejection reaction is taking place due to GVHD (Nat. Med. (2010)
16, 1434-1438). Adenosine concentration is known to be enhanced in
fibrotic tissues of the liver, kidney, and lung (FASEB J. (2008)
22, 2263-2272; J. Immunol. (2006) 176, 4449-4458; J. Am. Soc.
Nephrol. (2011) 22 (5), 890-901; PLoS ONE J. (2010) 5 (2), e9242).
Furthermore, ATP concentration has been observed to be increased in
fibrotic tissues of pulmonary fibrosis patients (Am. J. Respir.
Crit. Care Med. (2010) 182, 774-783). Examples of a non-limiting
embodiment of an inflammatory tissue-specific compound used in the
present invention suitably include ATP, ADP, AMP, adenosine and
such which accumulate at high concentration in inflammatory tissues
by metabolism of such purine nucleotides such as ATP. In addition,
inosine accumulates at a high concentration due to degradation of
adenosine by adenosine deaminase to produce inosine.
(3) Uric Acid
[0142] Uric acid is a product of the metabolic pathway of purine
nucleosides in vivo, and is released to the outside of cells such
as the interstitial space and blood. In recent years, uric acid
released from cells undergoing necrosis has been found to promote
inflammatory response (J. Clin. Invest. (2010) 120 (6), 1939-1949).
Examples of a non-limiting embodiment of inflammatory
tissue-specific compounds to be used in the present invention
suitably include such uric acid which accumulates at high
concentration in inflammatory tissues due to metabolism of purine
nucleotides such as ATP.
Antigen-Binding Domain
[0143] 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 1 OFn3 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".
[0144] 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: 1. 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: 1.
Specificity
[0145] "Specific" means that one of the molecules that specifically
bind does not substantially bind 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 substantially bind" is determined
according to the method described in the above-mentioned section on
binding activity, and refers to the binding activity of a molecule
that specifically binds to a molecule other than the partner
molecule, where the binding activity is not more than 80%, normally
not more than 50%, preferably not more than 30%, or particularly
preferably not more than 15% of the binding activity to its partner
molecule.
Cytotoxic Activity
[0146] In a non-limiting embodiment, the present invention provides
antigen-binding molecules that comprise an antigen-binding domain
whose antigen-binding activity varies depending on the
concentration of a cancer-tissue specific compound, and which have
cytotoxic activity against cells expressing a membrane-type
molecule on their cell membrane; and pharmaceutical compositions
comprising these antigen-binding molecules as an active ingredient.
In the present invention, cytotoxic activity includes, for example,
antibody-dependent cell-mediated cytotoxicity (ADCC) activity,
complement-dependent cytotoxicity (CDC) activity, and cytotoxic
activity by T cells. In the present invention, CDC activity refers
to cytotoxic activity by the complement system. On the other hand,
ADCC activity refers to the activity of immune cells to damage
target cells when the immune cells and such bind to the Fc region
of antigen-binding molecules comprising an antigen-binding domain
that binds to a membrane-type molecule expressed on the cell
membrane of target cells via an Fc.gamma. receptor expressed on the
immune cells. Whether an antigen-binding molecule of interest has
an ADCC activity or whether it has a CDC activity can be determined
using known methods (for example, Current Protocols in Immunology,
Chapter 7. Immunologic studies in humans, Editor, Coligan et al.,
(1993)).
[0147] Specifically, effector cells, complement solution, and
target cells are first prepared.
(1) Preparation of Effector Cells
[0148] Spleen is removed from a CBA/N mouse or the like, and spleen
cells are dispersed in an RPMI1640 medium (Invitrogen). After the
cells are washed in the same medium containing 10% fetal bovine
serum (FBS, HyClone), effector cells are prepared by adjusting the
spleen cell concentration to 5.times.10.sup.6/mL.
(2) Preparation of Complement Solution
[0149] Baby Rabbit Complement (CEDARLANE) is diluted 10-fold in a
culture medium (Invitrogen) containing 10% FBS to prepare a
complement solution.
(3) Preparation of Target Cells
[0150] The target cells can be radioactively labeled by culturing
cells expressing the antigen with 0.2 mCi of .sup.51Cr-sodium
chromate-(GE Healthcare Bio-Sciences) in a DMEM medium containing
10% FBS for one hour at 37.degree. C. After radioactive labeling,
cells are washed three times in an RPMI1640 medium containing 10%
FBS, and the target cells can be prepared by adjusting the cell
concentration to 2.times.10.sup.5/mL.
[0151] ADCC activity or CDC activity can be measured by the method
described below. In the case of ADCC activity measurement, 50 .mu.L
each of the target cell and antigen-binding molecule are added to a
96-well U-bottom plate (Becton Dickinson), and allowed to react for
15 minutes at room temperature. Then, 100 .mu.L of effector cells
are added to the plate and this plate is placed in a carbon dioxide
incubator for four hours. The final concentration of the
antigen-binding molecule may be set, for example, to 0 .mu.g/mL or
10 .mu.g/mL. After incubation, 100 .mu.L of the supernatant is
collected from each well, and the radioactivity is measured with a
gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument
Company). The cytotoxic activity (%) can be calculated using the
measured values according to the equation: (A-C)/(B-C).times.100. A
represents the radioactivity (cpm) in each sample, B represents the
radioactivity (cpm) in a sample to which 1% NP-40 (Nacalai Tesque)
has been added, and C represents the radioactivity (cpm) of a
sample containing the target cells alone.
[0152] Meanwhile, in the case of CDC activity measurement, 50 .mu.L
of target cell and 50 .mu.L of an antigen-binding molecule are
added to a 96-well flat-bottomed plate (Becton Dickinson), and
allowed to react for 15 minutes on ice. Then, 100 .mu.L of a
complement solution is added to the plate, and this plate is placed
in a carbon dioxide incubator for four hours. The final
concentration of the antigen-binding molecule may be set, for
example, to 0 .mu.g/mL or 3 .mu.g/mL. After incubation, 100 .mu.L
of supernatant is collected from each well, and the radioactivity
is measured with a gamma counter. The cytotoxic activity can be
calculated in the same way as in the determination of ADCC
activity.
[0153] The later-described modified antigen-binding molecules to
which cytotoxic substances such as chemotherapeutic agents, toxic
peptides, or radioactive chemical substances have been ligated can
also be suitably used as the antigen-binding molecules of the
present invention having cytotoxic activity. Such modified
antigen-binding molecules (hereinafter referred to as
"antigen-binding molecule-drug conjugate") can be obtained by
chemically modifying the obtained antigen-binding molecules.
Methods that have been already established in the field of
antibody-drug conjugates and such may be used appropriately as a
method for modifying antigen-binding molecules. Furthermore, a
modified antigen-binding molecule with a linked toxic peptide can
be obtained by expressing in an appropriate host cell a fusion gene
produced by linking a gene encoding the toxic peptide in frame with
a gene encoding an antigen-binding molecule of the present
invention, and then isolating the molecule from the culture
solution of the cells.
Neutralizing Activity
[0154] The present invention provides in a non-limiting embodiment
a pharmaceutical composition that induces an immune response,
comprising as an active ingredient an antigen-binding molecule that
contains an antigen-binding domain whose antigen-binding activity
varies depending on the concentration of a cancer tissue-specific
compound and has a neutralizing activity against a membrane-type
molecule. In another non-limiting embodiment, the present invention
provides a pharmaceutical composition that induces an immune
response, comprising as an active ingredient an antigen-binding
molecule that contains an antigen-binding domain whose
antigen-binding activity varies depending on the concentration of a
cancer tissue-specific compound and has a neutralizing activity
against a membrane-type molecule in addition to a cytotoxic
activity against cells expressing the membrane-type molecule on
their cell membrane. Generally, a neutralizing activity refers to
an activity of inhibiting the biological activity of a ligand which
has a biological activity towards cells, such as viruses and
toxins. Thus, a substance having a neutralizing activity refers to
a substance that binds to a ligand or a receptor to which the
ligand binds and inhibits the binding between the ligand and the
receptor. A receptor whose binding to the ligand has been blocked
by the neutralizing activity will not be able to exhibit the
biological activity through the receptor. When the antigen-binding
molecule is an antibody, the antibody having such a neutralizing
activity is generally called a neutralizing antibody. The
neutralizing activity of a test substance may be measured by
comparing the biological activities in the presence of a ligand
between conditions when the test substance is present or
absent.
[0155] A suitable example of a major ligand for the IL-6 receptor
is IL-6, which is shown in SEQ ID NO: 27. The IL-6 receptor, which
is an I-type membrane protein whose amino terminus forms the
extracellular domain, forms a hetero-tetramer with the gp130
receptor which was induced by IL-6 to dimerize (Heinrich et al.
(Biochem. J. (1998) 334, 297-314)). Formation of the heterotetramer
activates Jak associated with the gp130 receptor. Jak carries out
autophosphorylation and receptor phosphorylation. The
phosphorylation sites of the receptor and of Jak serve as binding
sites for molecules belonging to the Stat family having SH2 such as
Stat3, and for the MAP kinases, PI3/Akt, and other proteins and
adapters having SH2. Next, Stat that bound to the gp130 receptor is
phosphorylated by Jak. The phosphorylated Stat dimerizes and
translocates to the nucleus, and regulates transcription of target
genes. Jak and Stat can also be involved in the signaling cascade
through receptors of other classes. A deregulated IL-6 signaling
cascade is observed in inflammation and pathological conditions of
autoimmune diseases, and cancers such as prostate cancer and
multiple myeloma. Stat3 which may act as an oncogene is
constitutively activated in many cancers. In prostate cancer and
multiple myeloma, there is a crosstalk between the signaling
cascade from the IL-6 receptor and the signaling cascade from
members of the epidermal growth factor receptor (EGFR) family
(Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2),
55-66)).
[0156] Such intracellular signaling cascades are different for each
cell type; therefore, an appropriate target molecule can be set
according to each of the target cells of interest, and the target
molecule is not limited to the above-mentioned factors. The
neutralization activity can be evaluated by measuring the in vivo
signal activation. Furthermore, activation of in vivo signals can
also be detected by using as an indicator the
transcription-inducing action on a target gene that exists
downstream of the in vivo signaling cascade. A change in the
transcription activity of a target gene can be detected by the
principle of a reporter assay. Specifically, a reporter gene such
as the green fluorescence protein (GFP) or luciferase is placed
downstream of a transcription factor or a promoter region of the
target gene; and a change in transcription activity can be measured
in terms of reporter activity by measuring the reporter activity.
Commercially available kits for measuring in vivo signal activation
can be suitably used (for example, the Mercury Pathway Profiling
Luciferase System (Clontech)).
[0157] Furthermore, as a method for measuring the neutralization
activity on a receptor ligand in the EGF receptor family and such
which acts on a signaling cascade that typically works toward
enhancing cell proliferation, neutralization activity of an
antigen-binding molecule can be evaluated by measuring the
proliferation activity of the target cells. For example, the
following method is suitably used as a method for measuring or
evaluating inhibitory effects based on the neutralization activity
of an anti-HB-EGF antibody against the proliferation of cells whose
proliferation is promoted by EGF family growth factors such as
HB-EGF. As a method for evaluating or measuring the activity of
inhibiting cell proliferation in a test tube, a method that
measures the incorporation by living cells of [.sup.3H]-labeled
thymidine added to the culture medium as an index of the DNA
replication ability is used. As a more convenient method, a dye
exclusion method that measures under a microscope the ability of a
cell to release a dye such as trypan blue to the outside of the
cell, or the MTT method is used. The latter makes use of the
ability of living cells to convert
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), which is a tetrazolium salt, to a blue formazan product.
More specifically, a test antibody is added along with a ligand to
the culture solution of a test cell; and after a certain period of
time has elapsed, an MTT solution is added to the culture, and this
is left to stand for a certain amount of time to let the cell
incorporate MTT. As a result, MTT which is a yellow compound is
converted to a blue compound by succinate dehydrogenase in the
mitochondria of the cell. After this blue product is dissolved for
coloration, its absorbance is measured and used as an indicator of
the number of viable cells. Besides MTT, reagents such as MTS, XTT,
WST-1, and WST-8 are also commercially available (Nacalai Tesque,
and such), and can be suitably used. For measurement of the
activity, a binding antibody that has the same isotype as the
anti-HB-EGF antibody but does not have the cell
proliferation-inhibiting activity can be used as a control antibody
in the same manner as the anti-HB-EGF antibody, and the anti-HB-EGF
antibody is judged to have the activity when it shows a stronger
cell proliferation-inhibiting activity than the control
antibody.
[0158] As cells for evaluating activity, for example, cells showing
HB-EGF-promoted proliferation such as the RMG-1 cell line which is
an ovarian cancer cell line may be suitably used; and mouse Ba/F3
cells transformed with a vector in which a gene encoding
hEGFR/mG-CSFR, which is a fusion protein of the extracellular
domain of human EGFR fused in frame with the intracellular domain
of the mouse G-CSF receptor, is linked so as to allow expression,
may also be suitably used. This way, those skilled in the art may
appropriately select cells for evaluating activity to measure the
cell proliferation activity mentioned above.
Antibody
[0159] 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 IgG1sequence, 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.
[0160] 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
IL-6R (anti-IL-6R antibody). Antibodies that bind to an antigen
other than IL-6R can also be produced according to the example
described below.
[0161] Anti-IL-6R antibodies can be obtained as polyclonal or
monoclonal antibodies using known methods. The anti-IL-6R
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. "Humanized antibodies" or "chimeric antibodies" are
included in the monoclonal antibodies of the present invention.
[0162] 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 IL-6R protein as a sensitizing antigen. Resulting
immune cells are fused with known parental cells by conventional
cell fusion methods. Then, hybridomas producing an anti-IL-6R
antibody can be selected by screening for monoclonal
antibody-producing cells using conventional screening methods.
[0163] Specifically, monoclonal antibodies are prepared as
mentioned below. First, the IL-6R gene whose nucleotide sequence is
disclosed in SEQ ID NO: 2 can be expressed to produce an IL-6R
protein shown in SEQ ID NO: 1, which will be used as a sensitizing
antigen for antibody preparation. That is, a gene sequence encoding
IL-6R is inserted into a known expression vector, and appropriate
host cells are transformed with this vector. The desired human
IL-6R protein is purified from the host cells or their culture
supernatants by known methods. In order to obtain soluble IL-6R
from culture supernatants, for example, a protein consisting of the
amino acids at positions 1 to 357 in the IL-6R polypeptide sequence
of SEQ ID NO: 1, such as described in Mullberg et al. (J. Immunol.
(1994) 152 (10), 4958-4968), is expressed as a soluble IL-6R,
instead of the IL-6R protein of SEQ ID NO: 1. Purified natural
IL-6R protein can also be used as a sensitizing antigen.
[0164] The purified IL-6R protein can be used as a sensitizing
antigen for immunization of mammals. A partial IL-6R 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 IL-6R, or by inserting a partial IL-6R gene
into an expression vector for expression. Alternatively, a partial
peptide can be produced by degrading an IL-6R protein with a
protease. The length and region of the partial IL-6R peptide are
not limited to particular embodiments. A preferred region can be
arbitrarily selected from the amino acid sequence at amino acid
positions 20 to 357 in the amino acid sequence of SEQ ID NO: 1. 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.
[0165] For sensitizing antigen, alternatively it is possible to use
a fusion protein prepared by fusing a desired partial polypeptide
or peptide of the IL-6R 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 IL-6R to be used as a sensitizing
antigen, and immunization methods using IL-6R are specifically
described in WO 2003/000883, WO 2004/022754, WO 2006/006693, and
such.
[0166] 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.
[0167] The above animals are immunized with a sensitizing antigen
by known methods. Generally performed immunization methods include,
for example, intraperitoneal or subcutaneous injection
administration 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.
[0168] 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: [0169]
immunostimulation can be provided while retaining the structure of
a membrane protein such as IL-6R; and [0170] there is no need to
purify the antigen for immunization.
[0171] In order to prepare a monoclonal antibody of the present
invention using DNA immunization, first, a DNA expressing an IL-6R
protein is administered to an animal to be immunized. The
IL-6R-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 recognized IL-6R
can also be produced by the methods described in WO
2003/104453.
[0172] After immunizing a mammal as described above, an increase in
the titer of an IL-6R-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.
[0173] 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.
[0174] 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.
[0175] 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);
[0176] 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.
[0177] 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).
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] Desired antibodies can be preferably selected and singly
cloned by screening methods based on known antigen/antibody
reaction. For example, an IL-6R-binding monoclonal antibody can
bind to IL-6R expressed on the cell surface. Such a monoclonal
antibody can be screened by fluorescence activated cell sorting
(FACS). FACS is a system that assesses the binding of an antibody
to cell surface by analyzing cells contacted with a fluorescent
antibody using laser beam, and measuring the fluorescence emitted
from individual cells.
[0184] To screen for hybridomas that produce a monoclonal antibody
of the present invention by FACS, IL-6R-expressing cells are first
prepared. Cells preferably used for screening are mammalian cells
in which IL-6R is forcedly expressed. As control, the activity of
an antibody to bind to cell-surface IL-6R can be selectively
detected using non-transformed mammalian cells as host cells.
Specifically, hybridomas producing an anti-IL-6R monoclonal
antibody can be isolated by selecting hybridomas that produce an
antibody which binds to cells forced to express IL-6R, but not to
host cells.
[0185] Alternatively, the activity of an antibody to bind to
immobilized IL-6R-expressing cells can be assessed based on the
principle of ELISA. For example, IL-6R-expressing cells are
immobilized to the wells of an ELISA plate. Culture supernatants of
hybridomas are contacted with the immobilized cells in the wells,
and antibodies that bind to the immobilized cells 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.
[0186] Monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen for a long period.
[0187] 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.
[0188] 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.
[0189] For example, a cDNA encoding the variable region (V region)
of an anti-IL-6R antibody is prepared from hybridoma cells
expressing the anti-IL-6R antibody. For this purpose, total RNA is
first extracted from hybridomas. Methods used for extracting mRNAs
from cells include, for example: [0190] the guanidine
ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299),
and [0191] the AGPC method (Anal. Biochem. (1987) 162(1),
156-159)
[0192] 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. U.S.A. (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.
[0193] 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.
[0194] 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.
[0195] 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).
[0196] 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.
[0197] 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 IL-6R-binding
activity of a reshaped immunoglobulin as an indicator. For example,
when the objective is to isolate an antibody against IL-6R, it is
more preferred that the binding of the antibody to IL-6R is
specific. An IL-6R-binding antibody can be screened, for example,
by the following steps:
[0198] (1) contacting an IL-6R-expressing cell with an antibody
comprising the V region encoded by a cDNA isolated from a
hybridoma;
[0199] (2) detecting the binding of the antibody to the
IL-6R-expressing cell; and
[0200] (3) selecting an antibody that binds to the IL-6R-expressing
cell.
[0201] Methods for detecting the binding of an antibody to
IL-6R-expressing cells are known. Specifically, the binding of an
antibody to IL-6R-expressing cells can be detected by the
above-described techniques such as FACS. Immobilized samples of
IL-6R-expressing cells are appropriately used to assess the binding
activity of an antibody.
[0202] 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.
[0203] After isolation of the cDNA encoding the V region of the
anti-IL-6R 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-IL-6R 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. A chimeric antibody
expression vector is constructed by fusing in frame the two genes
digested with the same combination of restriction enzymes.
[0204] To produce an anti-IL-6R 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 below, a peptide having the amino acid sequence
MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is 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-IL-6R antibody-encoding DNA are
obtained.
[0205] 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 WO 1994/011523).
[0206] 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 domains
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 (Sp2/0, NSO, etc.), BHK (baby
hamster kidney cell line), HeLa, Vero, 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) E1A and E1B genes) and 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.
[0207] 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.
[0208] Furthermore, the following cells can be used as fungal
cells: [0209] yeasts: the Saccharomyces genus such as Saccharomyces
serevisiae, and the Pichia genus such as Pichia pastoris; and
[0210] filamentous fungi: the Aspergillus genus such as Aspergillus
niger.
[0211] 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.
[0212] 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).
[0213] 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 altered
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 altered
antibodies are appropriately produced by known methods.
[0214] 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 (FRs).
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.
[0215] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by grafting
the CDR 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.
[0216] 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.
[0217] 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).
[0218] 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).
[0219] Alternatively, desired human antibodies can be obtained by
immunizing transgenic animals having the entire repertoire of human
antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602;
WO 1994/025585; WO 1996/034096; WO 1996/033735) by DNA
immunization.
[0220] 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 WO
1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO
1993/019172; WO 1995/001438; WO 1995/015388).
[0221] 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 WO 2008/081008 can be
appropriately used to isolate antibody genes.
EU Numbering and Kabat Numbering
[0222] 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 by Kabat numbering, while constant region amino
acids are indicated by EU numbering based on Kabat's amino acid
positions.
Antigen-Binding Domains Dependent on a Target Tissue-Specific
Compound
[0223] To obtain an antigen-binding domain (or an antigen-binding
molecule containing the domain) whose antigen-binding activity
varies depending on the concentration of a target tissue-specific
compound, or more specifically, an antigen-binding domain (or an
antigen-binding molecule containing the domain) dependent on a
target tissue-specific compound, the methods indicated in the above
section on binding activity may be appropriately applied. As a
non-limiting embodiment, some specific examples of the methods are
presented below. For example, to confirm that the antigen-binding
activity of an antigen-binding domain (or an antigen-binding
molecule containing the domain) in the presence of a target
tissue-specific compound becomes higher than the antigen-binding
activity of an antigen-binding domain (or an antigen-binding
molecule containing the domain) in the absence of the compound, the
antigen-binding activities of the antigen-binding domain (or the
antigen-binding molecule containing the domain) in the presence and
absence of the target tissue-specific compound or in the presence
of high and low concentrations of the compound are compared. In
another non-limiting embodiment, for example, to confirm that the
antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) in the presence of
a high concentration of a target tissue-specific compound becomes
higher than the antigen-binding activity of an antigen-binding
domain (or an antigen-binding molecule containing the domain) in
the presence of a low concentration of the compound, the
antigen-binding activities of the antigen-binding domain (or the
antigen-binding molecule containing the domain) in the presence of
high and low concentrations of the target tissue-specific compound
are compared.
[0224] Furthermore, in the present invention, the phrase "the
antigen-binding activity in the presence of a target
tissue-specific compound is higher than the antigen-binding
activity in the absence of the compound" can be alternatively
expressed as "the antigen-binding activity of an antigen-binding
domain (or an antigen-binding molecule containing the domain) in
the absence of a target tissue-specific compound is lower than the
antigen-binding activity in the presence of the compound".
Furthermore, in the present invention, "the antigen-binding
activity of an antigen-binding domain (or an antigen-binding
molecule containing the domain) in the absence of a target
tissue-specific compound is lower than the antigen-binding activity
in the presence of the compound" may be alternatively described as
"the antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) in the absence of a
target tissue-specific compound is weaker than the antigen-binding
activity in the presence of the compound".
[0225] Furthermore, in the present invention, the phrase "the
antigen-binding activity in the presence of a high concentration of
a target tissue-specific compound is higher than the
antigen-binding activity in the presence of a low concentration of
the compound" can be alternatively expressed as "the
antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) in the presence of
a low concentration of a target tissue-specific compound is lower
than the antigen-binding activity in the presence of a high
concentration of the compound". In the present invention, "the
antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) in the presence of
a low concentration of a target tissue-specific compound is lower
than the antigen-binding activity in the presence of a high
concentration of the compound" may be alternatively described as
"the antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) in the presence of
a low concentration of a target tissue-specific compound is weaker
than the antigen-binding activity in the presence of a high
concentration of the compound".
[0226] Conditions when measuring antigen-binding activity other
than the concentration of a target tissue-specific compound are not
particularly limited, and can be selected appropriately by those
skilled in the art. For example, it is possible to measure under
conditions of HEPES buffer and 37.degree. C. For example, Biacore
(GE Healthcare) or such can be used for measurement. When the
antigen is a soluble molecule, the activity of an antigen-binding
domain (or an antigen-binding molecule containing the domain) to
bind to the soluble molecule can be determined by loading the
antigen as an analyte onto a chip immobilized with the
antigen-binding domain (or an antigen-binding molecule containing
the domain). Alternatively, when the antigen is a membrane-type
molecule, the binding activity towards the membrane-type molecule
can be determined by loading the antigen-binding domain (or an
antigen-binding molecule containing the domain) as an analyte onto
a chip immobilized with the antigen.
[0227] As long as the antigen-binding activity of an
antigen-binding domain (or an antigen-binding molecule containing
the domain) contained in antigen-binding molecules of the present
invention in the absence of a target tissue-specific compound is
weaker than the antigen-binding activity in the presence of the
target tissue-specific compound, the ratio between the
antigen-binding activity in the absence of the compound and the
antigen-binding activity in the presence of the compound is not
particularly limited. However, the value of KD (in the absence of
the compound)/KD (in the presence of the compound), which is a
ratio of dissociation constant (KD) against an antigen in the
absence of the target tissue-specific compound to KD in the
presence of the compound, is preferably 2 or greater, more
preferably 10 or greater, and still more preferably 40 or greater.
The upper limit of the value of KD (in the absence of the
compound)/KD (in the presence of the compound) is not particularly
limited, and may be any value, for example, 400, 1,000, or 10,000,
as long as it can be provided by the technologies of those skilled
in the art. When antigen-binding activity is not observed in the
absence of the target tissue-specific compound, the value of the
upper limit is infinity.
[0228] As long as the antigen-binding activity of an
antigen-binding domain (or an antigen-binding molecule containing
the domain) contained in antigen-binding molecules of the present
invention in the presence of a low concentration of a target
tissue-specific is weaker than the antigen-binding activity in the
presence of a high concentration of the target tissue-specific
compound, the ratio between the antigen-binding activity in the
presence of a low concentration of the compound and the
antigen-binding activity in the presence of a high concentration of
the compound is not particularly limited. However, the value of KD
(in the presence of a low concentration of the compound)/KD (in the
presence of a high concentration of the compound), which is a ratio
of dissociation constant (KD) against an antigen in the presence of
a low concentration of the target tissue-specific compound to KD in
the presence of a high concentration of the compound, is preferably
2 or greater, more preferably 10 or greater, and still more
preferably 40 or greater. The upper limit of the value of KD (in
the presence of a low concentration of the compound)/KD (in the
presence of a high concentration of the compound) is not
particularly limited, and may be any value, for example, 400,
1,000, or 10,000, as long as it can be provided by the technologies
of those skilled in the art. When antigen-binding activity is not
observed in the presence of a low concentration of the target
tissue-specific compound, the value of the upper limit is
infinity.
[0229] For the value of antigen-binding activity, if the antigen is
a soluble molecule, dissociation constant (KD) can be used; and if
the antigen is a membrane-type molecule, apparent dissociation
constant (apparent KD) can be used. The dissociation constant (KD)
and apparent dissociation constant (apparent KD) can be determined
by methods known to those skilled in the art, for example, using
Biacore (GE Healthcare), a Scatchard plot, a flow cytometer, or
such.
[0230] As another indicator that shows the ratio between the
antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) of the present
invention in the absence of a target tissue-specific compound and
the antigen-binding activity in the presence of the compound, for
example, dissociation rate constant kd can be suitably used. When
the dissociation rate constant (kd) is used instead of the
dissociation constant (KD) as an indicator that shows the binding
activity ratio, the value of kd (in the absence of the compound)/kd
(in the presence of the compound), which is a ratio between kd
(dissociation rate constant) for an antigen in the absence of a
target tissue-specific compound and kd in the presence of the
compound, is preferably 2 or greater, more preferably 5 or greater,
even more preferably 10 or greater, and still more preferably 30 or
greater. The upper limit of the value of kd (in the absence of the
compound)/kd (in the presence of the compound) is not particularly
limited, and may be any value, for example, 50, 100, or 200, as
long as it can be provided by the common technical knowledge of
those skilled in the art. When antigen-binding activity is not
observed in the absence of the tissue-specific compound, there is
no dissociation and the value of the upper limit becomes
infinity.
[0231] As another indicator that shows the ratio between the
antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) of the present
invention in the presence of a low concentration of a target
tissue-specific compound and the antigen-binding activity in the
presence of a high concentration of the compound, for example,
dissociation rate constant kd can be suitably used. When the
dissociation rate constant (kd) is used instead of the dissociation
constant (KD) as an indicator showing the binding activity ratio,
the value of kd (in the presence of a low concentration of the
compound)/kd (in the presence of a high concentration of the
compound), which is a ratio between kd (dissociation rate constant)
for an antigen in the presence of a low concentration of a target
tissue-specific compound and kd in the presence of a high
concentration of the compound, is preferably 2 or greater, more
preferably 5 or greater, even more preferably 10 or greater, and
still more preferably 30 or greater. The upper limit of the value
of kd (in the presence of a low concentration of the compound)/kd
(in the presence of a high concentration of the compound) is not
particularly limited, and may be any value, for example, 50, 100,
or 200, as long as it can be provided by the common technical
knowledge of those skilled in the art. When antigen-binding
activity is not observed in the presence of a low concentration of
the target tissue-specific compound, there is no dissociation and
the value of the upper limit becomes infinity.
[0232] For the value of antigen-binding activity, if the antigen is
a soluble molecule, dissociation rate constant (kd) can be used;
and if the antigen is a membrane-type molecule, apparent
dissociation rate constant (apparent kd) can be used. The
dissociation rate constant (kd) and apparent dissociation rate
constant (apparent kd) can be determined by methods known to those
skilled in the art, for example, using Biacore (GE Healthcare), a
flow cytometer, or such. In the present invention, when measuring
the antigen-binding activity of an antigen-binding domain (or an
antigen-binding molecule containing the domain) at a certain
concentration of the target tissue-specific compound, conditions
other than the concentration of the compound concentration are
preferably the same.
[0233] For example, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may be obtained by screening of
antigen-binding domains (or antigen-binding molecules) that
comprises the steps of:
[0234] (a) determining antigen-binding activity of antigen-binding
domains (or antigen-binding molecules) in the absence of a target
tissue-specific compound;
[0235] (b) determining antigen-binding activity of the
antigen-binding domains (or antigen-binding molecules) in the
presence of the target tissue-specific compound; and
[0236] (c) selecting an antigen-binding domain (or an
antigen-binding molecule) with lower antigen-binding activity in
the absence of the target tissue-specific compound than in the
presence of the compound.
[0237] For example, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may be obtained by screening of antigen-binding domains
(or antigen-binding molecules) that comprises the steps of:
[0238] (a) determining antigen-binding activity of antigen-binding
domains (or antigen-binding molecules) in the presence of a low
concentration of a target tissue-specific compound;
[0239] (b) determining antigen-binding activity of the
antigen-binding domains (or antigen-binding molecules) in the
presence of a high concentration of the target tissue-specific
compound; and
[0240] (c) selecting an antigen-binding domain (or an
antigen-binding molecule) with lower antigen-binding activity in
the presence of a low concentration of the target tissue-specific
compound than in the presence of a high concentration of the
compound.
[0241] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may be obtained by screening of
antigen-binding domains (or antigen-binding molecules) or a library
thereof that comprises the steps of:
[0242] (a) contacting antigen-binding domains (or antigen-binding
molecules) or a library thereof with an antigen in the presence of
a target tissue-specific compound;
[0243] (b) placing antigen-binding domains (or antigen-binding
molecules) that bind to the antigen in said step (a) in the absence
of the compound;
[0244] (c) isolating an antigen-binding domain (or an
antigen-binding molecule) that dissociated in said step (b).
[0245] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may be obtained by screening of antigen-binding domains
(or antigen-binding molecules) or a library thereof that comprises
the steps of:
[0246] (a) contacting antigen-binding domains (or antigen-binding
molecules) or a library thereof with an antigen in the presence of
a high concentration of a target tissue-specific compound;
[0247] (b) placing antigen-binding domains (or antigen-binding
molecules) that bind to the antigen in said step (a) in the
presence of a low concentration of the compound;
[0248] (c) isolating an antigen-binding domain (or an
antigen-binding molecule) that dissociates in said step (b).
[0249] Alternatively, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may be obtained by screening of
antigen-binding domains (or antigen-binding molecules) or a library
thereof that comprises the steps of:
[0250] (a) contacting a library of antigen-binding domains (or
antigen-binding molecules) with an antigen in the absence of a
target tissue-specific compound;
[0251] (b) selecting antigen-binding domains (or antigen-binding
molecules) that do not bind to the antigen in said step (a);
[0252] (c) allowing the antigen-binding domains (or antigen-binding
molecules) selected in said step (b) to bind to the antigen in the
presence of the compound; and
[0253] (d) isolating an antigen-binding domain (or an
antigen-binding molecule) that binds to the antigen in said step
(c).
[0254] Alternatively, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may be obtained by screening of antigen-binding domains
(or antigen-binding molecules) or a library thereof that comprises
the steps of:
[0255] (a) contacting a library of antigen-binding domains (or
antigen-binding molecules) with an antigen in the presence of a low
concentration of a target tissue-specific compound;
[0256] (b) selecting antigen-binding domains (or antigen-binding
molecules) that do not bind to the antigen in said step (a);
[0257] (c) allowing the antigen-binding domains (or antigen-binding
molecules) selected in said step
[0258] (b) to bind to the antigen in the presence of a high
concentration the compound; and
[0259] (d) isolating an antigen-binding domain (or an
antigen-binding molecule) that binds to the antigen in said step
(c).
[0260] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may be obtained by a screening method
comprising the steps of:
[0261] (a) contacting a library of antigen-binding domains (or
antigen-binding molecules) with an antigen-immobilized column in
the presence of a target tissue-specific compound;
[0262] (b) eluting an antigen-binding domain (or antigen-binding
molecule) that binds to the column in said step (a) from the column
in the absence of the compound; and
[0263] (c) isolating the antigen-binding domain (or antigen-binding
molecule) eluted in said step (b).
[0264] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may be obtained by a screening method comprising the
steps of:
[0265] (a) contacting a library of antigen-binding domains (or
antigen-binding molecules) with an antigen-immobilized column in
the presence of a high concentration of a target tissue-specific
compound;
[0266] (b) eluting an antigen-binding domain (or antigen-binding
molecule) that binds to the column in said step (a) from the column
in the presence of a low concentration of the compound; and
[0267] (c) isolating the antigen-binding domain (or antigen-binding
molecule) eluted in said step (b).
[0268] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may be obtained by a screening method
comprising the steps of:
[0269] (a) allowing a library of antigen-binding domains (or
antigen-binding molecules) to pass through an antigen-immobilized
column in the absence of a target tissue-specific compound;
[0270] (b) collecting an antigen-binding domain (or antigen-binding
molecule) eluted without binding to the column in said step
(a);
[0271] (c) allowing the antigen-binding domain (or antigen-binding
molecule) collected in said step (b) to bind to the antigen in the
presence of the compound; and
[0272] (d) isolating an antigen-binding domain (or antigen-binding
molecule) that binds to the antigen in said step (c).
[0273] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may be obtained by a screening method comprising the
steps of:
[0274] (a) allowing a library of antigen-binding domains (or
antigen-binding molecules) to pass through an antigen-immobilized
column in the presence of a low concentration of a target
tissue-specific compound;
[0275] (b) collecting an antigen-binding domain (or antigen-binding
molecule) eluted without binding to the column in said step
(a);
[0276] (c) allowing the antigen-binding domain (or antigen-binding
molecule) collected in said step (b) to bind to the antigen in the
presence of a high concentration of the compound; and
[0277] (d) isolating an antigen-binding domain (or antigen-binding
molecule) that binds to the antigen in said step (c).
[0278] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the absence of a target tissue-specific compound than in the
presence of the compound, may 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) in the presence of a target
tissue-specific compound; (b) obtaining an antigen-binding domain
(or antigen-binding molecule) that binds to the antigen in said
step (a); (c) placing the antigen-binding domain (or
antigen-binding molecule) obtained in said step (b) in the absence
of the compound; and (d) isolating an antigen-binding domain (or
antigen-binding molecule) whose antigen-binding activity in said
step (c) is weaker than that of the reference selected in said step
(b).
[0279] Furthermore, in an embodiment provided by the present
invention, an antigen-binding domain (or an antigen-binding
molecule containing the domain) with lower antigen-binding activity
in the presence of a low concentration of a target tissue-specific
compound than in the presence of a high concentration of the
compound, may 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) in the presence of a high
concentration of a target tissue-specific compound; (b) obtaining
an antigen-binding domain (or antigen-binding molecule) that binds
to the antigen in said step (a); (c) placing the antigen-binding
domain (or antigen-binding molecule) obtained in said step (b) in
the presence of a low concentration of the compound; and (d)
isolating an antigen-binding domain (or antigen-binding molecule)
whose antigen-binding activity in said step (c) is weaker than that
of the reference selected in said step (b).
[0280] The above-mentioned steps may be repeated two or more times.
Thus, the present invention provides an antigen-binding domain (or
an antigen-binding molecule containing the domain) with lower
antigen-binding activity in the absence of a target tissue-specific
compound than in the presence of the compound, or an
antigen-binding domain (or an antigen-binding molecule containing
the domain) with lower antigen-binding activity in the presence of
a low concentration of a target tissue-specific compound than in
the presence of a high concentration of the compound, obtained by
screening methods that further comprise the step of repeating steps
(a) to (c) or (a) to (d) two or more times in the above-mentioned
screening methods. The number of repeats of steps (a) to (c) or (a)
to (d) is not particularly limited, and it is generally ten or
less.
[0281] In the screening methods of the present invention, a target
tissue-specific compound may be a compound defined by quantitative
target tissue specificity such as presence in the target tissue at
a concentration (for example, high concentration or low
concentration) different from the concentration in non-target
tissues. For example, a target tissue-specific compound is
differentially present at any concentrations. However, generally, a
target tissue-specific compound can be present at a concentration
increased by at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 100%, at least 110%, at least 120%, at least
130%, at least 140%, at least 150%, at least 2-fold, at least
5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at
least 10.sup.3-fold, at least 10.sup.4-fold, at least
10.sup.5-fold, at least 10.sup.6-fold, or more, or up to infinity
(when the compound is absent in non-target tissues).
[0282] The threshold differentiating low and high concentrations
can be set appropriately according to the compound. For example, in
a non-limiting embodiment of the threshold of ATP or adenosine, the
threshold for a low-concentration condition may be selected
appropriately from the values of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM,
and 0 M. Depending on the predetermined threshold, the
high-concentration condition may be set appropriately at a value
selected from at least 110%, at least 120%, at least 130%, at least
140%, at least 150%, at least twice, at least five-fold, at least
10-fold, at least 50-fold, at least 100-fold, at least
10.sup.3-fold, at least 10.sup.4-fold, at least 10.sup.5-fold, and
at least 10.sup.6-fold the value of each threshold. Furthermore, in
a non-limiting embodiment of PGE2, the threshold for a
low-concentration condition may be selected appropriately from the
values of 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, and 0 M. Depending on
the predetermined threshold, the high-concentration condition may
be set appropriately at a value selected from at least 110%, at
least 120%, at least 130%, at least 140%, at least 150%, at least
twofold, at least five-fold, at least 10-fold, at least 50-fold, at
least 100-fold, at least 10.sup.3-fold, at least 10.sup.4-fold, at
least 10.sup.5-fold, and at least 10.sup.6-fold the value of each
threshold. Furthermore, in a non-limiting embodiment of Kynurenine,
the threshold for a low-concentration condition may be selected
appropriately from the values of 10 .mu.M, 1 .mu.M, 100 nM, 10 nM,
and 1 nM, and 0 M. Depending on the predetermined threshold, the
high-concentration condition may be set appropriately at a value
selected from at least 110%, at least 120%, at least 130%, at least
140%, at least 150%, at least twofold, at least five-fold, at least
10-fold, at least 50-fold, at least 100-fold, at least
10.sup.3-fold, at least 10.sup.4-fold, at least 10.sup.5-fold, and
at least 10.sup.6-fold the value of each threshold.
[0283] The antigen-binding activity of an antigen-binding domain
(or an antigen-binding molecule) may be measured by a method known
to those skilled in the art, and conditions other than the
concentration of a target tissue-specific compound can be set
appropriately by one skilled in the art. The antigen-binding
activity of an antigen-binding domain (or an antigen-binding
molecule) can be assessed as dissociation constant (KD), apparent
dissociation constant (apparent KD), dissociation rate constant
(kd), apparent dissociation rate constant (apparent kd), etc. They
can be determined by methods known to those skilled in the art, for
example, using Biacore (GE Healthcare), the Scatchard plot, FACS,
or such.
[0284] In the present invention, the step of selecting an antibody
or an antigen-binding domain with higher antigen-binding activity
in the presence of a target tissue-specific compound than in the
absence of the compound has the same meaning as the step of
selecting an antibody or an antigen-binding domain with lower
antigen-binding activity in the absence of a target tissue-specific
compound than in the presence of the compound.
[0285] In the present invention, the step of selecting an antibody
or an antigen-binding domain with higher antigen-binding activity
in the presence of a high concentration of a target tissue-specific
compound than in the presence of a low concentration of the
compound has the same meaning as the step of selecting an antibody
or an antigen-binding domain with lower antigen-binding activity in
the absence of a target tissue-specific compound than in the
presence of the compound.
[0286] As long as antigen-binding activity in the absence of a
target tissue-specific compound is lower than the antigen-binding
activity in the presence of the compound, the difference between
antigen-binding activity in the presence of the compound and
antigen-binding activity in the absence of the compound is not
particularly limited, but preferably, the antigen-binding activity
in the presence of the compound relative to the antigen-binding
activity in the absence of the compound is twofold or more, more
preferably 10-fold or more, and even more preferably 40-fold or
more. The upper limit of the difference between the antigen-binding
activities is not particularly limited, and as long as it can be
produced by the techniques of those skilled in the art, any value
such as 400-fold, 1000-fold, or 10000-fold is possible. In the
absence of a target tissue-specific compound, when antigen-binding
activity is not observed, this upper limit becomes infinity.
[0287] The antigen-binding domains (or antigen-binding molecules
containing the domains) of the present invention which are to be
screened by the aforementioned screening methods may be any
antigen-binding domains (or antigen-binding molecules); and for
example, the above-mentioned antigen-binding domains (or
antigen-binding molecules) can be screened. For example,
antigen-binding domains (or antigen-binding molecules) having
naturally-occurring sequences can be screened, and antigen-binding
domains (or antigen-binding molecules) with substituted amino acid
sequences may be screened.
Library
[0288] According to a certain embodiment, the antigen-binding
domain (or an antigen-binding molecule containing this domain) of
the present invention can be obtained from a library mainly
comprising a plurality of antigen-binding molecules having
different sequences from one another, in which at least one amino
acid residue that changes the binding activity of the
antigen-binding molecule toward an antigen dependent on a target
tissue-specific compound is contained in the antigen-binding
domain. Examples of the compound include (1) primary metabolites of
the Krebs cycle or the glycolytic pathway such as lactose, succinic
acid, or citric acid, (2) amino acids such as alanine, glutamic
acid, or asparagine, (3) kynurenine and amino acid metabolites
thereof such as anthranilic acid, 3-hudroxykynurenine, and
kynurenic acid, (4) arachidonic acid metabolites such as
prostaglandin E2, and (5) nucleosides carrying a purine ring
structure such as adenosine, adenosine triphosphate (ATP),
adenosine diphosphate (ADP), and adenosine monophosphate (AMP).
Below are examples of such a library mainly comprising a plurality
of antigen-binding molecules having different sequences from one
another, in which at least one amino acid residue that changes the
binding activity of the antigen-binding molecule toward adenosine-
and/or ATP-dependent antigens which are target tissue-specific
compounds is contained in the antigen-binding domain.
[0289] 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.
[0290] 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 to 10,000 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.
[0291] 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.
[0292] 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 the
concentration of a target tissue-specific compound, 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 in which antigen-binding
activity of the antigen-binding domain varies depending on the
presence or absence of adenosine and/or ATP 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 Change the Antigen-Binding Activity of the
Antigen-Binding Domain Depending on the Presence or Absence of
Adenosine and/or ATP
[0293] Antigen-binding domains or antibodies of the present
invention screened by the above-described screening methods may be
prepared in any manner. It is possible to use preexisting
antibodies, preexisting libraries (phage libraries, etc.),
antibodies or libraries prepared from hybridomas obtained by
immunizing animals or from B cells of immunized animals, and
antibodies or libraries prepared from immune cells such as B cells
of animals immunized by a conjugate in which adenosine or ATP is
suitably linked to an adjuvant agent such as a highly immunogenic T
cell epitope peptide. A non-limiting example of the T cell epitope
peptide suitably includes Tetanus toxin-derived p30 helper peptide
(shown in SEQ ID NO: 4, and also referred to as Fragment C
(FrC)).
[0294] Examples of amino acids that change the antigen-binding
activity of the antigen-binding molecule depending on the presence
or absence of adenosine and/or ATP as described above include amino
acids that form an adenosine- and/or ATP-binding motif. The amino
acid positions where the above-mentioned amino acids are contained
in the antigen-binding domain are not limited to any specific
position. As long as the antigen-binding activity of the
antigen-binding domain is changed depending on the presence or
absence of adenosine and/or ATP, any position in the heavy chain
variable region or light chain variable region forming the
antigen-binding domain is possible. More specifically, the
antigen-binding domains of the present invention may be obtained
from a library mainly comprising antigen-binding molecules having
different sequences from one another, in which the amino acids that
change the antigen-binding activity of the antigen-binding molecule
depending on the presence or absence of adenosine and/or ATP are
contained in the antigen-binding domain of the heavy chain. In a
non-limiting embodiment, antigen-binding domains of the present
invention may be obtained from a library mainly comprising
antigen-binding molecules having different sequences from one
another, in which the amino acids that change the antigen-binding
activity of the antigen-binding molecule depending on the presence
or absence of adenosine and/or ATP are contained in CDR1, CDR2,
and/or CDR3 of the heavy chain. In another non-limiting embodiment,
antigen-binding domains of the present invention may be obtained
from a library mainly comprising antigen-binding molecules having
different sequences from one another, in which the amino acids that
change the antigen-binding activity of the antigen-binding molecule
depending on the presence or absence of adenosine and/or ATP are
contained in FR1, FR2, FR3 and/or FR4 of the heavy chain.
[0295] Furthermore, in an embodiment of the present invention,
antigen-binding domains of the present invention may be obtained
from a library mainly comprising antigen-binding molecules having
different sequences from one another, in which the amino acids that
change the antigen-binding activity of the antigen-binding molecule
depending on the presence or absence of adenosine and/or ATP are
contained in the antigen-binding domain of the heavy chain and/or
light chain. In a non-limiting embodiment, antigen-binding domains
of the present invention may be obtained from a library mainly
comprising antigen-binding molecules having different sequences
from one another, in which the amino acids that change the
antigen-binding activity of the antigen-binding molecule depending
on the presence or absence of adenosine and/or ATP are contained in
CDR1, CDR2, and/or CDR3 of the heavy chain and/or light chain. In
another non-limiting embodiment, antigen-binding domains of the
present invention may be obtained from a library mainly comprising
antigen-binding molecules having different sequences from one
another, in which the amino acids that change the antigen-binding
activity of the antigen-binding molecule depending on the presence
or absence of adenosine and/or ATP are contained in FR1, FR2, FR3
and/or FR4 of the heavy chain and/or light chain.
[0296] In a non-limiting embodiment, examples of such amino acids
include any one or more amino acids selected from amino acids at
positions 52, 52a, 53, 96, 100a, and 100c contained in the heavy
chain variable region. Also, in a non-limiting embodiment, examples
of such amino acids include one or more amino acids selected from
amino acids including Ser at position 52, Ser at position 52a, Arg
at position 53, Gly at position 96, Leu at position 100a, and Trp
at position 100c contained in the heavy chain variable region.
[0297] Any framework sequence can be used as the framework sequence
of the light-chain and/or heavy-chain variable regions of an
antigen-binding molecule as long as the amino acids that change the
antigen-binding activity of the antigen-binding molecule depending
on the presence or absence of adenosine and/or ATP are contained in
the antigen-binding domain of the heavy chain and/or light chain.
The origin of the framework sequences 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 a particularly
preferred embodiment, the framework sequences of the light chain
and/or heavy chain variable region of an antigen-binding molecule
preferably have human germ-line framework sequences. Thus, in an
embodiment of the present invention, if the entire framework
sequences are human sequences, it is thought that an
antigen-binding molecule of the present invention induces little or
no immunogenic response when it is administered to humans (for
example, to treat diseases). 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 "containing a
germ line sequence" in the present invention. Even when the
framework sequences of antigen-binding molecules of the present
invention are sequences with substitutions, they are
antigen-binding molecules "containing a germ line sequence" of the
present invention. Examples of such sequences with substitutions
include, in particular, sequences in which amino acids of part of
human germ line framework sequences have been substituted with
amino acids that change the antigen-binding activity of the
antigen-binding molecule depending on the presence or absence of
adenosine and/or ATP.
[0298] 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
V.kappa., which is grouped into seven subgroups; V.lamda., which is
grouped into ten subgroups; and VH, which is grouped into seven
subgroups.
[0299] 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.
[0300] 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. Hoppe Seyler
(1993) 374, 1001-1022); Brensing-Kuppers et al. (Gene (1997) 191,
173-181)).
[0301] 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)).
[0302] 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 domain depending on the presence or absence of
adenosine and/or ATP" 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 domain depending on the presence or absence of
adenosine and/or ATP" 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)).
[0303] 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.
[0304] 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 domain depending on the presence or absence of
adenosine and/or ATP.
[0305] 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 CDR sequence and/or framework sequence originally containing
at least one amino acid residue that alters the antigen-binding
activity of the antigen-binding domain depending on the presence or
absence of adenosine and/or ATP.
[0306] Alternatively, a heavy chain and/or light chain variable
region sequence selected as a CDR sequence and/or a framework
sequence originally containing at least one amino acid residue that
changes the antigen-binding activity of an antigen-binding domain
depending on the presence or absence of adenosine and/or ATP as
mentioned above, can be designed to contain various amino acid
residues other than the above amino acid residue(s). 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 the concentration of a
tissue-specific compound. Specifically, the CDR sequences and/or FR
sequences of the heavy chain and/or light chain may contain one or
more flexible residues. One can identify the flexible residues and
those residues that can be substituted into other amino acids for
library production by introducing mutations or by crystal structure
analysis of complexes formed between an antibody and adenosine
and/or ATP. For example, from crystal structure analysis of
complexes formed between an antibody and adenosine and/or ATP, one
can identify residues in the antibody that are not involved in
binding to adenosine and/or ATP. One can select amino acids that
can maintain binding to the compounds at an appropriate level even
when the residues that have been identified as not being involved
in binding to adenosine and/or ATP are substituted into other amino
acids. Accordingly, it is possible to design a library that has the
selected amino acids for the selected residues. In this case, one
can design a library mainly comprising multiple antigen-binding
molecules to be an assembly of antigen-binding molecules in which
residues identified as not being involved in binding to adenosine
and/or ATP have been substituted with amino acids that are
different from one another. That is, the combination of individual
flexible residues substituted with amino acids that are different
from one another can provide sequence diversity in antigen-binding
molecules containing the flexible residues.
[0307] Antigen-binding molecules can be designed to include
residues wherein at least one of the residues identified to be
involved in binding to adenosine and/or ATP binding becomes any
residue selected from the residue and other residues that are
different from the residue. In a non-limiting embodiment, examples
of amino acids identified as being involved in binding to adenosine
and/or ATP may include one or more amino acids selected from amino
acids at positions 52, 52a, 53, 96, 100a, and 100c in the heavy
chain variable region. In a non-limiting embodiment, examples of
such amino acids include one or more amino acids selected from
amino acids including Ser at position 52, Ser at position 52a, Arg
at position 53, Gly at position 96, Leu at position 100a, and Trp
at position 100c contained in the heavy chain variable region. For
example, when Leu at position 100a mentioned above is identified to
be involved in binding to adenosine and/or ATP, the amino acid
residue at position 100a in the antigen-binding molecules included
in the library may be any amino acid residue selected from the
flexible residues of His, Met, Leu, Arg, Trp, or Tyr, in addition
to Leu.
[0308] In a non-limiting embodiment, examples of the flexible
residues may include amino acids at positions 31, 32, 33, 35, 50,
55, 56, 57, 58, 59, 95, 96, 97, 98, 99, 100, 100a, and 100b
contained in the heavy chain variable region. In another
non-limiting embodiment, examples of such amino acids may include
amino acids at positions 26, 27, 27a, 27b, 27c, 28, 29, 31, 32, 50,
51, 52, 53, 54, 55, 89, 90, 91, 92, 93, 94, 95a, 96, and 97
contained in the light chain variable region.
[0309] In a non-limiting embodiment, examples of the aforementioned
flexible residues may include the following amino acids contained
in the heavy chain variable region:
Asp, Gly, Asn, Ser, Arg, or Thr for the amino acid at position 31;
Ala, Phe, His, Asn, Ser, or Tyr for the amino acid at position 32;
Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro,
Ser, Arg, Trp, Val, Tyr, or Thr for the amino acid at position 33;
His, Ser, Thr, Tyr, or Asn for the amino acid at position 35; Ala,
Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Arg,
Thr, Trp, Val, Tyr, or Ser for the amino acid at position 50; Ala,
Glu, Asp, Gly, Leu, Thr, Ser, Arg, or Asn for the amino acid at
position 55; Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln,
Pro, Ser, Thr, Trp, Val, or Tyr for the amino acid at position 56;
Ala, Lys, Arg, Thr, or Ile for the amino acid at position 57; Asp,
Gly, Phe, His, Ser, Thr, Tyr, or Asn for the amino acid at position
58; Leu, or Tyr for the amino acid at position 59; Ala, Ile, Lys,
Met, Leu, Arg, Trp, Val, Tyr, or Phe for the amino acid at position
95; Ala, Asp, Asn, or Ser for the amino acid at position 96; Ala,
Asp, Gly, Ile, His, Lys, Met, Leu, Asn, Ser, Val, Tyr, or Arg for
the amino acid at position 97; Ala, Glu, Asp, Gly, Phe, Ile, His,
Met, Leu, Asn, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Lys for
the amino acid at position 98; Ala, Glu, Asp, Phe, His, Lys, Asn,
Gln, Ser, Arg, Trp, Val, Tyr, or Gly for the amino acid at position
99; Ala, Glu, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro,
Ser, Arg, Thr, Trp, Val, Tyr, or Asp for the amino acid at position
100; Ala, Phe, Ile, His, Lys, Met, Arg, Trp, Val, or Tyr for the
amino acid at position 100a; or Ala, Glu, Asp, Gly, Phe, Ile, His,
Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or Asn for
the amino acid at position 100b.
[0310] In a non-limiting embodiment, examples of the aforementioned
flexible residues may include the following amino acids contained
in the light chain variable region:
Ala, Ser, or Thr for the amino acid at position 26; Thr or Ser for
the amino acid at position 27; Gly, Asn, Thr, or Ser for the amino
acid at position 27a; Asn or Asp for the amino acid at position
27b; Ile or Val for the amino acid at position 27c; Asp or Gly for
the amino acid at position 28; Ala, Asp, Phe, Ser, Arg, Thr, Tyr,
or Gly for the amino acid at position 29; Glu, Asp, Lys, or Asn for
the amino acid at position 31; Ala, Asp, Ser, Thr, or Tyr for the
amino acid at position 32; Asp, Gly, Lys, Asn, Gln, Ser, Arg, Tyr,
or Glu for the amino acid at position 50; Asp, Gly, Lys, Asn, Thr,
or Val for the amino acid at position 51; Ala, Asp, Asn, Thr, or
Ser for the amino acid at position 52; Glu, Asp, His, Asn, Gln,
Ser, Tyr, or Lys for the amino acid at position 53; Lys or Arg for
the amino acid at position 54; Leu or Pro for the amino acid at
position 55; Ala, Gly, Phe, Leu, Asn, Gln, Thr, Val, Tyr, or Ser
for the amino acid at position 89; Ala, Leu, Thr, Val, or Ser for
the amino acid at position 90; Ala, Asp, Phe, His, Lys, Asn, Ser,
Arg, Thr, Trp, Val, or Tyr for the amino acid at position 91; Glu,
Asp, Ser, Arg, Thr, Val, Tyr, or Ala for the amino acid at position
92; Ala, Asp, Ile, Asn, Ser, Arg, Thr, Val, Tyr, or Gly for the
amino acid at position 93; Ala, Asp, Gly, Ile, Asn, Arg, Thr, or
Ser for the amino acid at position 94; Ala, Glu, Asp, Gly, Phe,
Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg, Thr, Trp, Val, Tyr, or
Asn for the amino acid at position 95; Ala, Glu, Asp, Gly, Ile,
His, Lys, Leu, Gln, Pro, Ser, Arg, Thr, Tyr, or Asn for the amino
acid at position 95a; Ala, Asp, Gly, Phe, His, Lys, Leu, Asn, Gln,
Pro, Ser, Thr, Trp, Tyr, or Val for the amino acid at position 96;
or Ala, Gly, Ile, Met, Leu, Ser, or Val for the amino acid at
position 97.
[0311] Herein, "flexible residue" refers to amino acid residue
variations present at hypervariable amino acid positions of
light-chain and heavy-chain variable regions at which several
different amino acids exist, when the amino acid sequences of known
and/or native antibodies or antigen-binding domains are compared.
The 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 for determining the
hypervariable positions in known and/or native antibodies.
Furthermore, databases on the Internet
(http://vbase.mrc-cpe.cam.ac.uk/, and
http://www.bioinf.org.uk/abs/index.html) provide many collected
sequences of human light chains and heavy chains, and their
locations. The information on the sequences and locations is useful
for determining the hypervariable positions in the present
invention. According to the present invention, when a certain amino
acid position has preferably about 2 to about 20, 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 can be said to be 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 possible amino acid
residue variations.
[0312] A library of the present invention that contains a plurality
of antigen-binding molecules having different sequences from one
another can be constructed by combining heavy chain variable
regions produced as a randomized variable region sequence library
with the aforementioned light chain variable regions introduced
with at least one amino acid residue that changes the
antigen-binding activity of the antigen-binding domains depending
on the presence or absence of adenosine and/or ATP. Similarly, a
library of the present invention that contains a plurality of
antigen-binding molecules having different sequences from one
another can also be produced by combining the heavy-chain variable
regions introduced with at least one amino acid residue that
changes the antigen-binding activity of the antigen-binding domains
depending on the presence or absence of adenosine and/or ATP, and
having the other amino acid residues designed as flexible
residues.
[0313] 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 the concentration of the target tissue-specific compound 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 the presence or absence of adenosine and/or ATP.
Specifically, the CDR sequences and/or FR sequences of heavy chain
and/or light chain can contain one or more flexible residues.
[0314] 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.
[0315] 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).
[0316] Furthermore, in a non-limiting embodiment of the present
invention, amino acids of the variable region including the CDR
region and/or the framework region may be altered appropriately to
improve antibody stability. In a non-limiting embodiment, examples
of such amino acids may include the amino acids of positions 1, 5,
10, 30, 48, and 58. More specifically, examples may include Gln at
position 1, Gln at position 5, Asp at position 10, Asn at position
30, Leu at position 48, and Asn at position 58. For the improvement
of antibody stability, these amino acids can be substituted for
corresponding amino acids contained in a germ-line sequence. In a
non-limiting embodiment, an example of such a germ line sequence
may be the VH3-21 sequence. In this case, Gln of position 1 may be
substituted with Glu, Gln of position 5 may be substituted with
Val, Asp of position 10 may be substituted with Gly, Asn of
position 30 may be substituted with Ser, Leu of position 48 may be
substituted with Val, and Asn of position 58 may be substituted
with Tyr.
[0317] In another non-limiting embodiment of the present invention,
a naive library which is constructed from antibody genes derived
from lymphocytes of healthy individuals and consists of naive
sequences which are antibody sequences that do not have bias in
their repertoire, 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.
Fc Region
[0318] An Fc region contains an amino acid sequence derived from
the heavy chain constant region of an antibody. An Fc region is a
portion of the antibody heavy chain constant region that includes
the N terminal end of the hinge region, which is the papain
cleavage site, at an amino acid around position 216 (indicated by
EU numbering), and the hinge, CH2, and CH3 domains. Fc regions can
be obtained from human IgG1; however, they are not limited to any
specific IgG subclass. Preferred examples of the Fc regions include
Fc regions having FcRn-binding activity in an acidic pH range as
described below. Preferred examples of the Fc regions include Fc
regions having Fc.gamma. receptor-binding activity as described
below. In a non-limiting embodiment, examples of such Fc regions
include the Fc regions of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID
NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).
Fc.gamma. Receptor (Fc.gamma.R)
[0319] "Fc.gamma. receptor" (also called "Fc.gamma.R") refers to a
receptor capable of binding to the Fc region of monoclonal IgG1,
IgG2, IgG3, or IgG4 antibodies; and means all members belonging to
the family of proteins substantially encoded by Fc.gamma. receptor
genes. In humans, 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 allotype 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 isoform
Fc.gamma.RIIIa (including allotype V158 and F158, i.e.,
Fc.gamma.RIIIa(V) and Fc.gamma.RIIIa(F)) and Fc.gamma.RIIIb
(including allotype 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; but the family is not limited to these
examples. Without being limited thereto, Fc.gamma.Rs include those
derived from humans, mice, rats, rabbits, and monkeys. Fc.gamma.Rs
may be derived from any organism. Mouse Fc.gamma.Rs include
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, but
they are not limited to these examples. Preferred examples of such
Fc.gamma. receptors include, 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 human Fc.gamma.RI are shown in SEQ ID NOs: 9 (NM
000566.3) and 10 (NP 000557.1), respectively; the polynucleotide
sequence and amino acid sequence of human Fc.gamma.RIIa (allotype
H131) are shown in SEQ ID NOs: 11 (BCO20823.1) and 12 (AAH20823.1),
respectively (allotype R131 is a sequence in which the amino acid
at position 166 of SEQ ID NO: 12 is substituted with Arg); the
polynucleotide sequence and amino acid sequence of Fc.gamma.IIb are
shown in SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1),
respectively; the polynucleotide sequence and amino acid sequence
of Fc.gamma.RIIIa are shown in SEQ ID NOs: 15 (BCO33678.1) and 16
(AAH33678.1), respectively; and the polynucleotide sequence and
amino acid sequence of Fc.gamma.RIIIb are shown in SEQ ID NOs: 17
(BC128562.1) and 18 (AAI28563.1), respectively (RefSeq accession
number or such is shown in parentheses). 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
(Amplified Luminescent Proximity Homogeneous Assay) screen, surface
plasmon resonance (SPR)-based BIACORE methods, and others (Proc.
Natl. Acad. Sci. USA (2006) 103(11), 4005-4010), in addition to the
above-described FACS and ELISA formats.
[0320] 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 region 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 region 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 herein referred to as
activating Fc.gamma. receptors.
[0321] 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 herein referred to as inhibitory Fc.gamma.
receptor.
Fc.gamma.R-Binding Activity of Fc Region
[0322] 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: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO:
7), or IgG4 (SEQ ID NO: 8). 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.
U.S.A. (2006) 103(11), 4005-4010), in addition to the
above-described FACS and ELISA formats.
[0323] 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.
[0324] 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 an antigen-binding
molecule comprising a native 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).
[0325] 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 constants.
Inhibition assay is preferably used in the BIACORE methods.
Examples of such inhibition assay are described in Proc. Natl.
Acad. Sci. U.S.A. (2006) 103(11), 4005-4010.
Fc Regions with Altered Fc.gamma. Receptor (Fc.gamma.R) Binding
[0326] In addition to the Fc region of human IgG1 (SEQ ID NO: 5),
IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8),
an Fc region with altered 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: 5, 6, 7, or 8 is a fucose-containing sugar
chain. Such Fc regions with altered Fc.gamma.R binding may be
produced by altering amino acids of the Fc region of a native human
IgG. Whether the Fc.gamma.R-binding activity of an Fc region with
altered 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.
[0327] In the present invention, "alteration of amino acids" or
"amino acid alteration" of an Fc region includes alteration 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
altered from a starting Fc region which had been already altered
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 native
IgG variants or altered 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.
[0328] 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 altered Fc region of the present
invention and its starting Fc region. Amino acid difference between
an Fc.gamma.R-binding altered 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 by EU numbering. Examples of methods
of preparing such variants are shown in the section "Alteration of
amino acids".
[0329] Included in the antigen-binding molecules of the present
invention, an Fc region with altered 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-altered Fc
region) may be obtained by any method. Specifically, the Fc region
with altered Fc.gamma.R binding may be obtained by altering amino
acids of an IgG-type human immunoglobulin used as a starting Fc
region. Preferred Fc regions of the IgG-type immunoglobulins for
alteration include, for example, those of human IgGs shown in SEQ
ID NOs: 5, 6, 7, or 8 (IgG1, IgG2, IgG3, or IgG4, respectively, and
variants thereof).
[0330] Amino acids of any positions may be altered 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 an alteration
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 alterations 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.
[0331] Examples of such amino acids that may be altered 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 altered 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
altering these amino acids.
[0332] Examples of particularly preferable alterations for use in
the present invention include at least one or more amino acid
alterations 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 in
the Fc region. The number of amino acids to be altered is not
particularly limited; and amino acid may be altered at only one
site or amino acids may be altered at two or more sites. Examples
of combinations for amino acid alterations at two or more sites
include those described in Table 1 (Tables 1-1 to 1-3).
TABLE-US-00001 TABLE 1-1 Combination of amino acids Combination of
amino acids K370E/P396L/D270E S239Q/I332Q Q419H/P396L/D270E
S267D/I332E V240A/P396L/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 F243L/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/N297E/I332E S298A/E333A/K334A
D265Y/N297D/I332E E380A/T307A F243L/V262I/V264W K326M/E333S
N297D/A330Y/I332E K326A/E333A N297D/T299E/I332E S3I7A/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 E272I/N276D
P244H/P245A/P247V E272R/I332E S239D/A330L/I332E E283H/I332E
S239D/A330Y/I332E E293R/I332E S239D/H268E/A330Y F241L/V262I
S239D/I332E/A327A F241W/F243W S239D/I332E/A330I
Table 1-2 is a continuation of Table 1-1.
TABLE-US-00002 TABLE 1-2 F243L/V264I S239D/N297D/I332E H268D/A330Y
S239D/S298A/I332E H268E/A330Y S239D/V264I/I332E K246H/I332E
S239E/N297D/I332E L234D/I332E S239E/V264I/I332E L234E/I332E
S239N/A330L/I232E L234G/I332E S239N/A330Y/I332E L234I/I332E
S239N/S298A/I332E L234I/L235D S239Q/V2641/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 F241L/F243L/V262I/V264I
L328M/I332D F241R/F243Q/V262T/V264R L328M/I332E
F241S/F243H/V262T/V264T I328N/I332D F241W/F243W/V262A/V264A
L328N/I332E F241Y/F243Y/V626T/V264T L328Q/I332D
I332E/A330Y/H268E/A327A L328Q/I332E N297D/I332E/S239D/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 1-3 is a continuation of Table 1-2.
TABLE-US-00003 TABLE 1-3 L328Y/I332E S239D/A330Y/I332E/V240I
N297D/I332E S239D/A330Y/I332E/V264T N297E/I332E
S239D/A330Y/I332E/V266I N297S/I332E S239D/D265F/N297D/I322E
P227G/I332E S239D/D265H/N297D/I332E P230A/E233D
S239D/D265I/N297D/I332E Q295E/I332E S239D/D265L/N297D/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/V264I/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/A330Y/I332E S239Q/I332N
F241Y/F243Y/V262T/V264T/N297D/I332E S267E/L328F G236D/S267E
S239D/S267E
[0333] For the pH conditions to measure the binding activity of the
Fc.gamma. receptor binding domain 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 acidic pH range or neutral pH range, as a
condition to measure the binding activity of the Fc.gamma. receptor
binding domain and the Fc.gamma. receptor contained in the
antigen-binding molecule of the present invention, generally
indicates pH 5.8 to pH 8.0. Preferably, it is a range indicated
with arbitrary pH values between pH 6.0 and pH 7.4; and preferably,
it is selected from pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5,
pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, and
pH 7.4; and particularly preferably, it is pH 6.15 to 7.4, which is
close to the pH of cancer tissues (Vaupel et al., Cancer Res.
(1989) 49, 6449-6665). With regard to the temperature used as a
measurement condition, the binding affinity between an Fc.gamma.
receptor binding domain and a human 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 a human
Fc.gamma. receptor binding domain and 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.gamma. receptor
binding domain and an Fc.gamma. receptor. A temperature of
25.degree. C. is a non-limiting example in an embodiment of the
present invention.
[0334] Herein, "Fe region with altered 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 altered 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 altered
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.
[0335] 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 (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-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.
[0336] As an antigen-binding molecule containing a native Fc region
of an 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: 5 (A is added to the N
terminus of Database Accession No. AAC82527.1), SEQ ID NO: 6 (A is
added to the N terminus of Database Accession No. AAB59393.1), SEQ
ID NO: 7 (Database Accession No. CAA27268.1), and SEQ ID NO: 8 (A
is added to the N terminus of Database 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 Toward an Fc.gamma.
Receptor
[0337] Examples of Fc.gamma. receptor binding domains suitable for
use in the present invention include Fc.gamma. receptor binding
domains having a higher binding activity to a particular Fc.gamma.
receptor than to other Fc.gamma. receptors (Fc.gamma. receptor
binding domains having a selective binding activity to an Fc.gamma.
receptor). When an antibody is used as the antigen-binding molecule
(when an Fc region is used as the Fc.gamma. receptor binding
domain), 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.
Fc Regions with a Higher Binding Activity Toward an Activating
Fc.gamma. Receptor than the Binding Activity Toward an Inhibitory
Fc.gamma. Receptor
[0338] As described above, preferable activating Fc.gamma.
receptors include Fc.gamma.RI (CD64) including Fc.gamma.RIa,
Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RIIa; and Fc.gamma.RIII
(CD16) including Fc.gamma.RIIIa (including allotypes V158 and F158)
and Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2). Meanwhile, preferred examples of inhibitory
Fc.gamma. receptors include Fc.gamma.RIIb (including
Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2).
[0339] Herein, an example of a case where the binding activity
toward a certain Fc.gamma. receptor is higher than the binding
activity toward another Fc.gamma. receptor is the case where the
binding activity toward an activating Fc.gamma. receptor is higher
than the binding activity toward an inhibitory Fc.gamma. receptor.
In this case, the binding activity of the Fc region toward any of
the human Fc.gamma. receptors of Fc.gamma.RIa, Fc.gamma.RIIa,
Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb is said to be higher than the
binding activity toward Fc.gamma.RIIb. For example, this means
that, based on an above-described analytical method, the binding
activity of an antigen-binding molecule containing the Fc region
toward any of the human Fc.gamma. receptors, Fc.gamma.RIa,
Fc.gamma.RIIa, Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb, is 105% or
more, preferably 110% or more, 120% or more, 130% or more, 140% or
more, particularly preferably 150% or more, 160% or more, 170% or
more, 180% or more, 190% or more, 200% or more, 250% or more, 300%
or more, 350% or more, 400% or more, 450% or more, 500% or more,
750% or more, 10-fold or more, 20-fold or more, 30-fold or more,
40-fold or more, 50-fold or more, 60-fold, 70-fold, 80-fold,
90-fold, or 100-fold or more as compared with the binding activity
toward Fc.gamma.RIIb. The Fc region with a higher binding activity
toward activating Fc.gamma. receptors than to inhibitory Fc.gamma.
receptors may be favorably included in antigen-binding molecules of
the present invention whose antigen-binding domain binds to a
membrane-type molecule. IgG1 antibodies containing such Fc regions
are known to enhance the ADCC activity mentioned below. Therefore,
antigen-binding molecules containing the Fc-region are also useful
as antigen-binding molecules to be included in the pharmaceutical
compositions of the present invention.
[0340] In a non-limiting embodiment of the present invention,
examples of the Fc region with a higher binding activity toward
activating Fc.gamma. receptors than to inhibitory Fc.gamma.
receptors (or having a selective binding activity toward inhibitory
Fc.gamma. receptors) preferably include Fc regions in which at
least one or more amino acids selected from the group consisting of
amino acids at 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
indicated by EU numbering mentioned above, have been altered to
amino acids different from those of the native Fc region.
[0341] In a non-limiting embodiment of the present invention,
examples of the Fc region with a higher binding activity toward
activating Fc.gamma. receptors than to inhibitory Fc.gamma.
receptors (or having a selective binding activity toward inhibitory
Fc.gamma. receptors) preferably include Fc regions in which
multiple amino acids indicated in Tables 1-1 to 1-3 have been
altered to amino acids different from those of the native Fc
region.
Fc Regions Whose Binding Activity Toward an Inhibitory Fc.gamma.
Receptor is Higher than the Binding Activity Toward an Activating
Fc.gamma. Receptor
[0342] Herein, an example of a case where the binding activity
toward a certain Fc.gamma. receptor is higher than the binding
activity toward another Fc.gamma. receptor is the case where the
binding activity toward an inhibitory Fc.gamma. receptor is higher
than the binding activity toward an activating Fc.gamma. receptor.
In this case, the binding activity of the Fc region toward
Fc.gamma.RIIb is said to be higher than the binding activity toward
any of the human Fc.gamma. receptors of Fc.gamma.RIa,
Fc.gamma.RIIa, Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb. For example,
this means that, based on an above-described analytical method, the
binding activity of an antigen-binding molecule containing the Fc
region toward Fc.gamma.RIIb is 105% or more, preferably 110% or
more, 120% or more, 130% or more, 140% or more, particularly
preferably 150% or more, 160% or more, 170% or more, 180% or more,
190% or more, 200% or more, 250% or more, 300% or more, 350% or
more, 400% or more, 450% or more, 500% or more, 750% or more,
10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more,
50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more as
compared with the binding activity toward any of the human
Fc.gamma. receptors of Fc.gamma.RIa, Fc.gamma.RIIa, Fc.gamma.RIIIa,
and/or Fc.gamma.RIIIb. The Fc region with a higher binding activity
toward inhibitory Fc.gamma. receptors than to activating Fc.gamma.
receptors may be favorably included in antigen-binding molecules of
the present invention whose antigen-binding domain binds to a
soluble molecule.
[0343] In a non-limiting embodiment of the present invention,
examples of the Fc region with a higher binding activity toward
inhibitory Fc.gamma. receptors than to activating Fc.gamma.
receptors (or having a selective binding activity toward inhibitory
Fc.gamma. receptors) preferably include Fc regions in which, of the
amino acids of the above Fc region, the amino acids at 238 and 328
indicated by EU numbering are altered to amino acids different from
those of the native Fc region.
[0344] In a non-limiting embodiment of the present invention,
examples of the Fc region with a higher binding activity toward
inhibitory Fc.gamma. receptors than to activating Fc.gamma.
receptors (or having a selective binding activity toward inhibitory
Fc.gamma. receptors) preferably include Fc regions altered at any
one or more of the amino acids in the above Fc region as indicated
by EU numbering: the amino acid at position 238 (indicated by EU
numbering) is altered into Asp; and the amino acid at position 328
(indicated by EU numbering) is altered into Glu. Furthermore, as
the Fc regions having a selective binding activity toward
inhibitory Fc.gamma. receptors, the Fc regions or alterations
described in US 2009/0136485 can be suitably selected.
[0345] In another non-limiting embodiment of the present invention,
preferred examples include Fc regions altered at any one or more of
the amino acids in the above Fc region as indicated by EU
numbering: the amino acid at position 238 (indicated by EU
numbering) to Asp; and the amino acid at position 328 (indicated by
EU numbering) to Glu.
[0346] In still another non-limiting embodiment of the present
invention, preferred examples include Fc regions that have one or
more of the alterations exemplified in PCT/JP2012/054624:
substitution of Pro at position 238 (indicated by EU numbering)
with Asp; alteration of the amino acid at position 237 (indicated
by EU numbering) to Trp; alteration of the amino acid at position
237 (indicated by EU numbering) to Phe; alteration of the amino
acid at position 267 (indicated by EU numbering) to Val; alteration
of the amino acid at position 267 (indicated by EU numbering) to
Gln; alteration of the amino acid at position 268 (indicated by EU
numbering) to Asn; alteration of the amino acid at position 271
(indicated by EU numbering) to Gly; alteration of the amino acid at
position 326 (indicated by EU numbering) to Leu; alteration of the
amino acid at position 326 (indicated by EU numbering) to Gln;
alteration of the amino acid at position 326 (indicated by EU
numbering) to Glu; alteration of the amino acid at position 326
(indicated by EU numbering) to Met; alteration of the amino acid at
position 239 (indicated by EU numbering) to Asp; alteration of the
amino acid at position 267 (indicated by EU numbering) to Ala;
alteration of the amino acid at position 234 (indicated by EU
numbering) to Trp; alteration of the amino acid at position 234
(indicated by EU numbering) to Tyr; alteration of the amino acid at
position 237 (indicated by EU numbering) to Ala; alteration of the
amino acid at position 237 (indicated by EU numbering) to Asp;
alteration of the amino acid at position 237 (indicated by EU
numbering) to Glu; alteration of the amino acid at position 237
(indicated by EU numbering) to Leu; alteration of the amino acid at
position 237 (indicated by EU numbering) to Met; alteration of the
amino acid at position 237 (indicated by EU numbering) to Tyr;
alteration of the amino acid at position 330 (indicated by EU
numbering) to Lys; alteration of the amino acid at position 330
(indicated by EU numbering) to Arg, alteration of the amino acid at
position 233 (indicated by EU numbering) to Asp, alteration of the
amino acid at position 268 (indicated by EU numbering) to Asp,
alteration of the amino acid at position 268 (indicated by EU
numbering) to Glu, alteration of the amino acid at position 326
(indicated by EU numbering) to Asp, alteration of the amino acid at
position 326 (indicated by EU numbering) to Ser, alteration of the
amino acid at position 326 (indicated by EU numbering) to Thr,
alteration of the amino acid at position 323 (indicated by EU
numbering) to Ile, alteration of the amino acid at position 323
(indicated by EU numbering) to Leu, alteration of the amino acid at
position 323 (indicated by EU numbering) to Met, alteration of the
amino acid at position 296 (indicated by EU numbering) to Asp,
alteration of the amino acid at position 326 (indicated by EU
numbering) to Ala, alteration of the amino acid at position 326
(indicated by EU numbering) to Asn, and alteration of the amino
acid at position 330 (indicated by EU numbering) to Met.
Fc Regions with Modified Sugar Chains
[0347] Fc regions contained in the antigen-binding molecules
provided by the present invention may include Fc regions that have
been modified so that the composition of the sugar-chain-attached
Fc regions has a high percentage of fucose-deficient
sugar-chain-attached Fc regions, or a high percentage of bisecting
N-acetylglucosamine-added Fc regions. Removal of fucose residue
from N-acetylglucosamine at the reducing end of N-glycoside linkage
complex sugar chains bonded to the antibody Fc region is known to
enhance the affinity to Fc.gamma.RIIIa (Non-Patent Document 6). It
is known that for IgG1 antibodies containing such Fc regions, the
ADCC activity mentioned below is enhanced; therefore,
antigen-binding molecules containing such Fc regions are also
useful as antigen-binding molecules to be contained in
pharmaceutical compositions of the present invention. Examples of
antibodies with fucose residue removed from N-acetylglucosamine at
the reducing end of N-glycoside linkage complex sugar chains bonded
to the antibody Fc regions are antibodies such as:
antibodies modified by glycosylation (for example, WO 1999/054342);
and antibodies deficient in fucose attached to sugar chains (for
example, WO 2000/061739, WO 2002/031140, and WO 2006/067913).
[0348] More specifically, to produce antibodies deficient in fucose
attached to sugar chains (for example, WO 2000/061739, WO
2002/031140, and WO 2006/067913) as another non-limiting embodiment
of antibodies with fucose residue removed from N-acetylglucosamine
at the reducing end of N-glycoside linkage complex sugar chains
bonded to the antibody Fc regions, host cells having a low ability
to add fucose to sugar chains are produced by altering the activity
of forming the sugar chain structure of the polypeptide to be
glycosylated. Antibodies that lack fucose in their sugar chains can
be collected from culture of the host cells by expressing a desired
antibody gene in the host cells. Non-limiting suitable examples of
the activity to form the sugar chain structure of a polypeptide
include the activity of a transporter or an enzyme selected from
the group consisting of fucosyltransferase (EC 2.4.1.152), fucose
transporter (SLC35C1), GMD (GDP-mannose-4,6-dehydratase) (EC
4.2.1.47), Fx (GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase)
(EC 1.1.1.271), and GFPP (GDP-.beta.-L-fucose pyrophosphorylase (EC
2.7.7.30). As long as these enzymes or transporters can exhibit
their activities, their structures are not necessarily specified.
Herein, proteins that can exhibit these activities are referred to
as "functional proteins". In a non-limiting embodiment, methods for
altering these activities include deletion of these activities. To
produce host cells deficient in these activities, known methods
such as a method for destroying the genes of these functional
proteins to make them unable to function may be appropriately
employed (for example, WO2000/061739, WO2002/031140, and
WO2006/067913). Host cells deficient in such activities can be
produced, for example, by a method that destroys the genes of these
functional proteins endogenous to CHO cells, BHK cells, NSO cells,
SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER
cells, PER.C6 cells, HEK293 cells, hybridoma cells, or such, so
that the genes are unable to function.
[0349] Antibodies that have a sugar chain containing bisecting
GlcNAc (WO2002/079255, etc.) are known. In a non-limiting
embodiment, host cells for expressing a gene that encodes a
functional protein having GnTIII (.beta.-1,4-mannosyl-glycoprotein
4-.beta.-N-acetylglucosaminyltransferase) (EC 2.4.1.144) activity
or Ga1T (.beta.-1,4-galactosyltransferase) (EC 2.4.1.38) activity
are produced to prepare antibodies that have bisecting
GlcNAc-containing sugar chains. In another suitable non-limiting
embodiment, host cells that co-express, in addition to the
aforementioned functional proteins, a gene encoding a functional
protein having human ManII (manosidase II) (3.2.1.114) activity, a
gene encoding a functional protein having GnTI
(.beta.-1,2-acetylglucosaminyltransferase I) (EC 2.4.1.94)
activity, a gene encoding a functional protein having GnTII
(.beta.-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143)
activity, a gene encoding a functional protein having ManI
(mannosidase) (EC 3.2.1.113) activity, and .alpha.-1,6-fucosyl
transferase (EC 2.4.1.68), are produced (WO2004/065540).
[0350] Antibodies with fucose residue removed from
N-acetylglucosamine at the reducing end of N-glycoside linkage
complex sugar chains bonded to the antibody Fc regions and
antibodies having sugar chains containing bisecting GlcNAc can be
produced, respectively, by transfecting an expression vector
containing the antibody gene into host cells with a low ability to
add fucose to sugar chains, and into host cells having the activity
to form bisecting GlcNAc structure-containing sugar chains. Methods
for producing these antibodies can be applied to methods for
producing antigen-binding molecules containing altered Fc regions
that have been modified so that the composition of the
sugar-chain-attached Fc regions of the present invention has a high
percentage of fucose-deficient sugar chain-attached Fc regions or a
high percentage of bisecting N-acetylglucosamine-added Fc regions.
The composition of the sugar-chain-attached Fc regions contained in
the antigen-binding molecules of the present invention produced by
such production methods can be assessed by the method described in
"Fc regions with altered Fc.gamma. receptor (Fc.gamma.R) binding"
above.
Multispecific Antigen-Binding Molecules or Multiparatopic
Antigen-Binding Molecules
[0351] An antigen-binding molecule comprising at least two
antigen-binding domains in which 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
"multispecific antigen-binding molecule" from the viewpoint of its
reaction specificity. When two types of antigen-binding domains
contained in a single antigen-binding molecule allow binding to two
different epitopes by the antigen-binding molecule, this molecule
is called "bispecific antigen-binding molecule". When three types
of antigen-binding domains contained in a single antigen-binding
molecule allow binding to three different epitopes by the
antigen-binding molecule, this antigen-binding molecule is called
"trispecific antigen-binding molecule".
[0352] A paratope in the antigen-binding domain that binds to the
first epitope in the antigen molecule and a paratope in the
antigen-binding domain that binds to the second epitope which is
structurally different from the first epitope have different
structures. Therefore, an antigen-binding molecule comprising at
least two antigen-binding domains in which 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
"multiparatopic antigen-binding molecule" from the viewpoint of the
specificity of its structure. When two types of antigen-binding
domains contained in a single antigen-binding molecule allow
binding to two different epitopes by the antigen-binding molecule,
this molecule is called "biparatopic antigen-binding molecule".
When three types of antigen-binding domains contained in a single
antigen-binding molecule allow binding to three different epitopes
by the antigen-binding molecule, this molecule is called
"triparatopic antigen-binding molecule".
[0353] Multivalent multispecific or multiparatopic antigen-binding
molecules comprising one or more antigen-binding domains and
methods for preparing them are 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 WO1996/034103 and WO1999/023221. Antigen-binding
molecules of the present invention can be produced using
multispecific or multiparatopic antigen-binding molecules, and
their preparation methods described in these documents.
Bispecific Antibodies and Methods for Producing them
[0354] In an embodiment, bispecific antibodies and methods for
producing them are mentioned below as examples of the
aforementioned multispecific or multiparatopic antigen-binding
molecules and methods for preparing them. 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).
[0355] 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.
[0356] 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).
[0357] 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.
[0358] In a non-limiting embodiment of the present invention, two
polypeptides constituting an Fc region derived from a bispecific
antibody described above can be suitably used as an Fc region to be
included in the antigen-binding molecule. More specifically, it is
preferable to use two polypeptides that constitute an Fc region,
and which comprise Cys for the amino acid at position 349 and Trp
for the amino acid at position 366 according to EU numbering in the
amino acid sequence of one of the polypeptides; and Cys for the
amino acid at position 356, Ser for the amino acid at position 366,
Ala for the amino acid at position 368, and Val for the amino acid
at position 407 as indicated by EU numbering in the amino acid
sequence of the other polypeptide.
[0359] In another non-limiting embodiment of the present invention,
two polypeptides that constitute an Fc region and which comprise
Asp for the amino acid at position 409 according to EU numbering in
the amino acid sequence of one of the polypeptides, and Lys for the
amino acid at position 399 according to EU numbering in the amino
acid sequence of the other polypeptide, 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 be suitably be added as the amino acid at
position 360 or Asp may suitably be added as the amino acid at
position 392.
[0360] In still another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region, and which
comprise Glu for the amino acid at position 370 according to EU
numbering in the amino acid sequence of one of the polypeptides,
and Lys for the amino acid at position 357 according to EU
numbering in the amino acid sequence of the other polypeptide, may
be suitably used as the Fc region.
[0361] In yet another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region, and which
comprise Glu for the amino acid at position 439 according to EU
numbering in the amino acid sequence of one of the polypeptides,
and Lys for the amino acid at position 356 according to EU
numbering in the amino acid sequence of the other polypeptide, may
be suitably used as the Fc region.
[0362] In still yet another non-limiting embodiment of the present
invention, any of the embodiments indicated below of combinations
from the above may be suitably used as the Fc region:
[0363] (i) two polypeptides that constitute an Fc region, and which
comprise Asp for the amino acid at position 409 and Glu for the
amino acid at position 370 according to EU numbering in the amino
acid sequence of one of the polypeptides, and Lys for the amino
acid at position 399 and Lys for the amino acid at position 357
according to EU numbering in the amino acid sequence of the other
polypeptide (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 may be used instead of the amino acid Glu
at position 370 according to EU numbering);
[0364] (ii) two polypeptides that constitute an Fc region, and
which comprise Asp for the amino acid at position 409 and Glu for
the amino acid at position 439 according to EU numbering of the
amino acid sequence of one of the polypeptides; and Lys for the
amino acid at position 399 and Lys for the amino acid at position
356 according to EU numbering in the amino acid sequence of the
other polypeptide (in this embodiment, the amino acid Asp at
position 360, the amino acid Asp at position 392, or the amino acid
Asp at position 439 may be used instead of the amino acid Glu at
position 439 according to EU numbering);
[0365] (iii) two polypeptides that constitute an Fc region, and
which comprise Glu for the amino acid at position 370 and Glu for
the amino acid at position 439 according to EU numbering in the
amino acid sequence of one of the polypeptides, and Lys for the
amino acid at position 357 and Lys for the amino acid at position
356 according to EU numbering in the amino acid sequence of the
other polypeptide; or
two polypeptides that constitute an Fc region, and which comprise
Asp the amino acid at position 409, Glu for the amino acid at
position 370, and Glu for the amino acid at position 439 according
to EU numbering in the amino acid sequence of one of the
polypeptides; and Lys for the amino acid at position 399, Lys for
the amino acid at position 357, and Lys for the amino acid at
position 356 according to EU numbering in the amino acid sequence
of the other polypeptide (in this embodiment, the amino acid at
position 370 may not be substituted with Glu, and furthermore, when
the amino acid at position 370 is not substituted with 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, according to EU numbering).
[0366] Further, in another non-limiting embodiment of the present
invention, it may also be suitable to use two polypeptides that
constitute an Fc region, and which comprise Lys for the amino acid
at position 356 according to EU numbering in the amino acid
sequence of one of the polypeptides, and Arg for the amino acid at
position 435 and Glu for the amino acid at position 439 according
to EU numbering in the amino acid sequence of the other
polypeptide.
[0367] In still another non-limiting embodiment of the present
invention, it may also be suitable to use two polypeptides that
constitute an Fc region and which comprise Lys for the amino acid
at position 356 and Lys for the amino acid at position 357
according to EU numbering in the amino acid sequence of one of the
polypeptides, and Glu for the amino acid at position 370, Arg for
the amino acid at position 435, and Glu for the amino acid at
position 439 according to EU numbering in the amino acid sequence
of the other polypeptide.
[0368] Furthermore, in addition to the above-mentioned technologies
of associating heterologous heavy chains, 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. Furthermore, Fab-Arm Exchange which is known as a
technology for associating heterologous heavy chains, in which a
heavy chain forming a variable region that binds to a first epitope
and a heavy chain forming a variable region that binds to a second
epitope by utilizing that heterologous IgG4 heavy chains exchange
each other (Labrijn et al. (Proc. Natl. Acad. Sci. U.S.A. (2013)
110, 5145-5150), WO2008119353), may also be used to produce the
multispecific or multiparatopic antigen-binding molecules provided
by the present invention.
Effector Cells
[0369] In the present invention, the term "effector cells" may be
used in the broadest sense including T cells (CD4.sup.+ (helper
lymphocyte) T cells and/or CD8.sup.+ (cytotoxic) T cells),
multinuclear leucocytes (neutrophils, eosinophils, basophils, mast
cells), monocytes, macrophages, histiocytes, or leukocytes such as
natural killer cells (NK cells), NK-like T cells, Kupffer cells,
Langerhans cells, or lymphokine-activated killer cells (LAK cells),
B-lymphocytes, or antigen-presenting cells such as dendritic cells
or macrophages. Preferred examples of effector cells include
CD8.sup.+ (cytotoxic) T cells, NK cells, or macrophages.
Membrane-type molecules expressed on the cell membrane of effector
cells may be used as antigens to which at least one antigen-binding
domain contained in the antigen-binding molecule of the present
invention binds. Non-limiting examples of a preferred membrane-type
molecule may be CD3, CD2, CD28, CD44, CD16, CD32, CD64, or NKG2D,
NK cell-activating ligands, or polypeptides constituting TCR.
Cytotoxic Substances
[0370] In order for antigen-binding molecules of the present
invention to bind to cancer cells and exhibit cytotoxic activity,
cytotoxic substances may be linked to antigen-binding molecules.
The cytotoxic substances may be chemotherapeutic agents exemplified
below, or compounds disclosed in Curr Opin Chem Biol (2010) 14,
529-37 and WO 2009/140242; and these compounds are linked to
antigen-binding molecules by appropriate linkers and such. When
antigen-binding molecules of the present invention are used as
pharmaceutical compositions, these cytotoxic substances may be
linked to the antigen-binding molecules prior to administration, or
they may be administered before, after, or at the same time when
the antigen-binding molecules are administered to subjects (test
individuals, patients, and such).
[0371] The later-described modified antigen-binding molecules to
which cytotoxic substances such as chemotherapeutic agents, toxic
peptides, or radioactive chemical substances have been linked may
also be used preferably as antigen-binding molecules of the present
invention having cytotoxic activity. Such modified antigen-binding
molecules (hereinafter referred to as antigen-binding molecule-drug
conjugate) can be obtained by chemically modifying the obtained
antigen-binding molecules. Methods that have been already
established in the field of antibody-drug conjugates and such may
be used appropriately as methods for modifying antigen-binding
molecules. Furthermore, a modified antigen-binding molecule to
which a toxic peptide is linked can be obtained by expressing in
appropriate host cells a fused gene produced by linking a gene
encoding the toxic peptide in frame with a gene encoding an
antigen-binding molecule of the present invention, and then
isolating it from the cell culture.
[0372] Examples of chemotherapeutic agents linked to the
antigen-binding molecules of the present invention may include:
azaribine, anastrozole, azacytidine, bleomycin, bortezomib,
bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin,
carmustine, celebrex, chlorambucil, cisplatin, irinotecan,
carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,
docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,
dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin
glucuronide, epirubicin, ethinyl estradiol, estramustine,
etoposide, etoposide glucuronide, floxuridine, fludarabine,
flutamide, fluorouracil, fluoxymesterone, gemcitabine,
hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,
leucovorin, lomustine, maytansinoid, mechlorethamine,
medroxyprogesterone acetate, megestrol acetate, melphalan,
mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,
mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel,
pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa,
teniposide, topotecan, uracil mustard, vinblastine, vinorelbine,
and vincristine.
[0373] In the present invention, preferred chemotherapeutic agents
are low-molecular-weight chemotherapeutic agents.
Low-molecular-weight chemotherapeutic agents are unlikely to
interfere with the function of antigen-binding molecules even after
they bind to antigen-binding molecules of the present invention. In
the present invention, low-molecular-weight chemotherapeutic agents
usually have a molecular weight of 100 to 2000, preferably 200 to
1000. The chemotherapeutic agents exemplified herein are all
low-molecular-weight chemotherapeutic agents. The chemotherapeutic
agents of the present invention include prodrugs that are converted
into active chemotherapeutic agents in vivo. Prodrug activation may
be enzymatic conversion or non-enzymatic conversion.
[0374] Moreover, cytotoxic substances that are linked to
antigen-binding molecules of the present invention include, for
example, toxic peptides (toxins) such as Pseudomonas exotoxin A,
Saporin-s6, Diphtheria toxin, Cnidarian toxin; radioiodine; and
photosensitizers. Suitable examples of the toxic peptides include
the following:
Diphtheria toxin A Chain (Langone et al. (Methods in Enzymology
(1983) 93, 307-308)); Pseudomonas Exotoxin (Nature Medicine (1996)
2, 350-353); Ricin Chain (Ricin A Chain) (Fulton et al. (J. Biol.
Chem. (1986) 261, 5314-5319), Sivam et al. (Cancer Res. (1987) 47,
3169-3173), Cumber et al. (J. Immunol. Methods (1990) 135, 15-24),
Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562), and Gheeite
et al. (J. Immunol. Methods (1991) 142, 223-230));
Deglicosylated Ricin A Chain (Thorpe et al. (Cancer Res. (1987) 47,
5924-5931));
[0375] Abrin A Chain (Wawrzynczak et al. (Br. J. Cancer (1992) 66,
361-366), Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562),
Sivam et al. (Cancer Res. (1987) 47, 3169-3173), and Thorpe et al.
(Cancer Res. (1987) 47, 5924-5931)); Gelonin (Sivam et al. (Cancer
Res. (1987) 47, 3169-3173), Cumber et al. (J. Immunol. Methods
(1990) 135, 15-24), Wawrzynczak et al. (Cancer Res., (1990) 50,
7519-7562), and Bolognesi et al. (Clin. exp. Immunol. (1992) 89,
341-346)); PAP-s; Pokeweed anti-viral protein from seeds (Bolognesi
et al. (Clin. exp. Immunol. (1992) 89, 341-346)); Briodin
(Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));
Saporin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89,
341-346)); Momordin (Cumber et al. (J. Immunol. Methods (1990) 135,
15-24); Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562); and
Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));
Momorcochin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89,
341-346)); Dianthin 32 (Bolognesi et al. (Clin. exp. Immunol.
(1992) 89, 341-346)); Dianthin 30 (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Modeccin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Viscumin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Volkesin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Dodecandrin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Tritin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); Luffin (Stirpe F., Barbieri L. (FEBS
letter (1986) 195, 1-8)); and Trichokirin (Casellas et al. (Eur. J.
Biochem. (1988) 176, 581-588), and Bolognesi et al. (Clin. exp.
Immunol., (1992) 89, 341-346)).
Antigen-Binding Molecule
[0376] In the present invention, "an antigen-binding molecule
comprising an antigen-binding domain whose antigen-binding activity
in the presence of a target tissue-specific compound is higher than
in the absence of the target tissue-specific compound" is used in
the broadest sense; and specifically, it includes various types of
molecules as long as they show 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.
[0377] An antigen-binding molecule of the present invention may
contain at least some portions of an Fc region that mediates the
binding to Fc.gamma. receptor and/or FcRn. 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 a polypeptide comprising 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 Fc.gamma.
receptor, and/or a portion of an Fc region responsible for the
binding to FcRn). 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.
[0378] Respective domains 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, Holliger et al., 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.
[0379] For example, such peptide linkers preferably include:
TABLE-US-00004 Ser Gly.cndot.Ser Gly.cndot.Gly.cndot.Ser
Ser.cndot.Gly.cndot.Gly (SEQ ID NO: 19)
Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 20)
Se.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 21)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 22)
Se.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 23)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO:
24) Se.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID
NO: 25)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser
(SEQ ID NO: 26)
Se.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly
(Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 21))n
(Se.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 22))n
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.
[0380] Synthetic linkers (chemical crosslinking agents) is
routinely used to crosslink peptides, and for example:
N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS),
bis(sulfosuccinimidyl) suberate (BS.sup.3), dithiobis(succinimidyl
propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),
ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).
These crosslinking agents are commercially available.
[0381] When multiple linkers for linking the respective domains are
used, they may all be of the same type, or may be of different
types. 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.
[0382] In order to link respective domains via peptide linkage,
polynucleotides encoding the domains are linked together 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 construct 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
genetic recombination techniques. Fusing in frame means, when two
or more elements or components are polypeptides, linking two or
more units of reading frames to form a continuous longer reading
frame while maintaining the correct reading frames of the
polypeptides. When two molecules of Fab are used as an
antigen-binding domain, an antibody, which is an antigen-binding
molecule of the present invention where the antigen-binding domain
is linked in frame to a constant region including an Fc region via
peptide bond without linker, can be used as a preferred
antigen-binding molecule of the present invention.
Low-Molecular-Weight Antibody
[0383] The antibodies used in the present invention are not limited
to full-length antibody molecules, and can be low-molecular-weight
antibodies (minibodies) and modified products thereof. A
low-molecular-weight antibody includes an antibody fragment that
lacks a portion of a full-length antibody (for example, whole
antibody such as whole IgG); and is not particularly limited as
long as it has an antigen-binding activity. The
low-molecular-weight antibody of the present invention is not
particularly limited as long as it is a portion of a full-length
antibody, but preferably comprises a heavy-chain variable region
(VH) and/or a light-chain variable region (VL). The amino acid
sequence of VH or VL may have substitution(s), deletion(s),
addition(s), and/or insertion(s). Furthermore, as long as it has an
antigen-binding activity, VH and/or VL can be partially deleted.
The variable region may be chimerized or humanized. Specific
examples of antibody fragments include Fab, Fab', F(ab')2, and Fv.
Specific examples of low-molecular-weight antibodies include Fab,
Fab', F(ab')2, Fv, scFv (single chain Fv), diabody, and sc(Fv)2
(single chain (Fv)2). Multimers of these antibodies (for example,
dimers, trimers, tetramers, and polymers) are also included in the
low-molecular-weight antibodies of the present invention.
[0384] Antibody fragments can be produced by treating an antibody
with an enzyme such as papain and pepsin. Alternatively, genes
encoding these antibody fragments can be constructed, inserted into
expression vectors, and then expressed in appropriate host cells
(see, for example, Co et al., (J. Immunol. (1994) 152, 2968-2976);
Better and Horwitz (Methods in Enzymology (1989) 178, 476-496),
Plueckthun and Skerra (Methods in Enzymology (1989) 178, 476-496);
Lamoyi (Methods in Enzymology (1989) 121, 652-663); Rousseaux
(Methods in Enzymology (1989) 121, 663-669); and Bird, et al.,
TIBTECH (1991) 9, 132-137).
[0385] A diabody refers to a bivalent low-molecular-weight antibody
constructed by gene fusion (Hollinger et al., (Proc. Natl. Acad.
Sci. USA 90, 6444-6448 (1993)); EP 404,097; WO 1993/11161; and
such). A diabody is a dimer composed of two polypeptide chains.
Generally, in each polypeptide chain constituting the dimer, VL and
VH are linked by a linker within the same chain. The linker in a
diabody is generally short enough to prevent binding between VL and
VH. Specifically, the amino acid residues constituting the linker
are, for example, about five residues. A linker between VL and VH
that are encoded by the same polypeptide chain is too short to form
a single-chain variable region fragment, and a dimer is formed
between the polypeptide chains. As a result, diabodies have two
antigen binding sites.
[0386] scFv can be obtained by linking the H-chain V region and
L-chain V region of an antibody. In scFv, the H-chain V region and
L-chain V region are ligated via a linker, preferably a peptide
linker (Huston, et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,
5879-5883). The H-chain V region and L-chain V region of scFv may
be derived from any of the antibodies described herein. The peptide
linker for ligating the V regions is not particularly limited; and
for example, any single-chain peptide consisting of 3 to 25
residues or so, or peptide linkers described later or such can be
used as the linker. PCR methods such as those described above can
be used for ligating the V regions. DNA encoding scFv can be
amplified by a PCR method using as a template either whole DNA or a
partial DNA encoding a desired amino acid sequence, which is
selected from a DNA sequence encoding the H chain or the H chain V
region of the above-mentioned antibody, and a DNA encoding the L
chain or the L chain V region of the above-mentioned antibody; and
using a pair of primers having sequences corresponding to the
sequences of the two ends. Next, a DNA having the desired sequence
can be obtained by performing a PCR reaction using a combination of
a DNA encoding the peptide linker portion, and a pair of primers
having sequences designed so that both ends of the DNA will be
ligated to the H chain and the L chain, respectively. Once the
scFv-encoding DNA is constructed, expression vectors having the
DNA, and recombinant cells transformed with the expression vector
can be obtained according to conventional methods. Furthermore, the
scFvs can be obtained by culturing the resulting recombinant cells
to express the scFv-encoding DNA.
[0387] sc(Fv)2 is a low-molecular-weight antibody prepared by
linking two VHs and two VLs with linkers or such to form a single
chain (Hudson et al. (J. Immunol. Methods 1999; 231: 177-189)).
sc(Fv)2 can be produced, for example, by linking scFvs with a
linker.
[0388] Moreover, antibodies in which two VHs and two VLs are
arranged in the order of VH, VL, VH, and VL starting from the
N-terminal side of a single chain polypeptide
([VH]-linker-[VL]-linker-[VH]-linker-[VL]) are preferred. The order
of the two VHs and the two VLs is not particularly limited to the
above-mentioned arrangement, and they may be arranged in any order.
Examples include the following arrangements: [0389]
[VL]-linker-[VH]-linker-[VH]-linker-[VL] [0390]
[VH]-linker-[VL]-linker-[VL]-linker-[VH] [0391]
[VH]-linker-[VH]-linker-[VL]-linker-[VL] [0392]
[VL]-linker-[VL]-linker-[VH]-linker-[VH] [0393]
[VL]-linker-[VH]-linker-[VL]-linker-[VH]
[0394] A linker similar to the linker described in the section
"Antigen-binding molecules" above may be used as the linker for
linking the antibody variable regions. A particularly preferred
embodiment of sc(Fv)2 in the present invention includes, for
example, the following sc(Fv)2: [0395] [VH]-peptide linker (15
amino acids)-[VL]-peptide linker (15 amino acids)-[VH]-peptide
linker (15 amino acids)-[VL]
[0396] Typically, three linkers are required to link four antibody
variable regions. The linkers to be used may be of the same type or
different types. Examples of a non-limiting embodiment of a
low-molecular-weight antibody in the present invention include a
diabody or sc(Fv)2, wherein the paratopes are different from each
other; one of the paratopes binds to an epitope in a membrane-type
molecule which binds to a cancer cell membrane; and the other
paratope binds to an epitope in the membrane-type molecule
expressed on the cell membrane of effector cells. In the
above-mentioned diabody or sc(Fv)2, the binding activity of one
paratope that binds to an epitope in a membrane-type molecule which
binds to a cancer cell membrane may depend on a cancer
tissue-specific compound, the binding activity of one of the
paratopes toward an epitope in a membrane-type molecule which binds
to an effector cell membrane may depend on a cancer tissue-specific
compound, or the binding activities of both paratopes may depend on
a cancer tissue-specific compound.
[0397] A non-limiting embodiment of a low-molecular-weight antibody
in the present invention includes, for example, a diabody or
sc(Fv)2, wherein the paratopes are different from each other; one
of the paratopes binds to an epitope in a membrane-type molecule
which binds to a cancer cell membrane; and the other paratope binds
to an epitope in a cytotoxic substance. In the diabody or sc(Fv)2
mentioned above, the binding activity of one of the paratopes that
binds to an epitope in a membrane-type molecule which binds to a
cancer cell membrane may depend on a cancer tissue-specific
compound, the binding activity of the other paratope that binds to
an epitope in a cytotoxic substance may depend on a cancer
tissue-specific compound, or the binding activities of both
paratopes may depend on a cancer tissue-specific compound.
[0398] Such low-molecular-weight antibody can be obtained by
treating an antibody with an enzyme such as papain or pepsin to
generate antibody fragments, or by constructing DNAs that encode
these antibody fragments or low-molecular-weight antibodies,
inserting them into expression vectors, and then expressing them in
appropriate host cells (see, for example, Co, M. S. et al., J.
Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,
Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A.,
Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.
(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986)
121, 663-669; and Bird, R. E. and Walker, B. W., Trends Biotechnol.
(1991) 9, 132-137).
FcRn
[0399] 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 .beta. or light chain (.beta.2
microglobulin) complexed with transmembrane a or heavy chain. Like
MHC, FcRn a 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).
[0400] 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.
[0401] Human FcRn whose precursor is a polypeptide having the
signal sequence of SEQ ID NO: 28 (the polypeptide with the signal
sequence is shown in SEQ ID NO: 29) 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. Fc 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 Fc
region of the present invention. Examples include a complex between
human FcRn and human .beta.2-microglobulin.
Binding Activity of the Fc Region to FcRn, in Particular, Human
FcRn
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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).
Fc Region Having FcRn-Binding Activity Under an Acidic pH Range
Condition
[0406] An Fc region having FcRn-binding activity under an acidic pH
range condition may also be preferably used as the Fc region
contained in an antigen-binding molecule provided by the present
invention. Generally, IgG antibodies are known to have long plasma
retention through binding to FcRn. Binding between IgG and FcRn is
observed only under acidic conditions (pH 6.0), and the binding is
hardly observed under neutral conditions (pH 7.4). IgG antibodies
are non-specifically incorporated into cells, but they return to
the cell surface by binding to FcRn in the endosome under endosomal
acidic conditions, and then dissociate from FcRn under neutral
conditions in plasma. When mutations are introduced into the Fc
region of IgG to eliminate the FcRn-binding under an acidic pH
range condition, the antibodies are not recycled from inside the
endosome into plasma. Therefore, plasma retention of the antibody
is remarkably impaired. A method for improving FcRn-binding under
an acidic pH range condition has been reported as a method for
improving plasma retention of IgG antibodies. Improving
FcRn-binding under an acidic pH range condition by introducing
amino acid substitutions into the IgG antibody Fc region can
increase the efficiency of recycling from inside the endosome into
plasma, and as a result, plasma retention is improved.
[0407] The present invention is not restricted to a particular
theory, but for example, when an antigen-binding molecule provided
by the present invention binds to a membrane-type antigen expressed
on cancer cells contained in cancer tissues, it may be possible to
continuously suppress cancer cell proliferation as described below.
Even after cancer cells expressing a membrane-type molecule, to
which an antigen-binding molecule of the present invention is bound
in the presence of a high concentration of a cancer tissue-specific
compound, are damaged by cytotoxic activity mediated by the
antigen-binding molecule, the antigen may still be bound to the
antigen-binding domain in the antigen-binding molecule. From the
antigen-binding molecules non-specifically incorporated into cells,
those that release the antigen in the presence of a low
concentration of the cancer tissue-specific compound return to the
cell surface by binding to FcRn in the endosome under acidic
conditions inside the endosome, and then dissociate from FcRn under
neutral conditions in plasma. In the presence of a high
concentration of a cancer tissue-specific compound, the
antigen-binding molecules of the present invention recycled in this
manner can bind again to their antigens which are membrane-type
molecules expressed on cancer cells.
[0408] The present invention is not restricted to a particular
theory, but for example, when a soluble antigen bound by
antigen-binding molecules provided by the present invention is a
ligand that positively regulates activation of inflammatory cells
or proliferation of target cells contained in a target tissue, it
may be possible to suppress proliferation of target cells or
activation of inflammatory cells as described below.
Antigen-binding molecules of the present invention bound to the
soluble molecule, i.e., its antigen, are non-specifically
incorporated into cells in the presence of a high concentration of
target tissue-specific compounds. This is followed by release of
the antigen in the presence of a low concentration of target
tissue-specific compounds, the antigen-binding molecules return to
the cell surface by binding to FcRn in the endosome under acidic
conditions inside the endosome, and then the antigen-binding
molecules dissociate from FcRn under neutral conditions in plasma.
The antigen-binding molecules of the present invention recycled in
this manner can bind again to soluble molecules, i.e., their
antigen, in the presence of a high concentration of target
tissue-specific compounds. On the other hand, the antigens that
dissociated from the antigen-binding molecules in the presence of a
low concentration of the target tissue-specific compounds are
degraded in the lysosome. The concentration of the soluble antigen
decreases as it passes through the recycling stage. Therefore, it
is considered that cancer cell proliferation or inflammatory cell
activation can be suppressed.
[0409] 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 altering 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
altering the amino acids of an Fc region. Amino acid alterations 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 alteration. Those
skilled in the art can appropriately alter the amino acids using
known techniques similar to the aforementioned techniques used to
modify the Fc.gamma.-receptor-binding activity.
[0410] 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 altering the amino acids of an
IgG-type human immunoglobulin used as a starting Fc region.
Preferred Fc regions of an IgG-type immunoglobulin for alteration
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 altered 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 an alteration 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 alteration 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
alteration 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 alteration 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 alteration 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 alteration 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. Alteration of these amino acids
enhances FcRn binding of the Fc region of an IgG-type
immunoglobulin under an acidic pH range condition.
[0411] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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
alterations 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 altered is not particularly limited; and amino acid may be
altered at only one site or amino acids may be altered at two or
more sites.
[0412] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration 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 alteration 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 alteration 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.
[0413] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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.
[0414] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration may
include His or Met for the amino acid of position 428, and/or His
or Met for the amino acid of position 434.
[0415] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration 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.
[0416] Furthermore, when the Fc region of human IgG1 is comprised
as the Fc region, a non-limiting embodiment of the alteration 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
alterations 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 altered is not particularly limited;
and amino acid may be altered at only one site or amino acids may
be altered at two sites.
[0417] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration may include Glu for the amino acid of
position 250, and/or Leu or Phe for the amino acid of position
428.
[0418] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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
alterations 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 altered is not
particularly limited; and amino acid may be altered at only two
sites or amino acids may be altered at three or more sites.
[0419] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration 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 alteration 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 alteration 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 alteration 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 alteration 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 alteration 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 alteration may include Gln for the
amino acid of position 307, and Ile for the amino acid of position
436.
[0420] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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 alteration
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
alteration 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
alteration 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.
[0421] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alteration 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 altered is not particularly limited
and amino acid at only two sites may be altered and amino acids at
three or more sites may be altered.
[0422] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the alteration 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 alterations 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
alteration 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 alteration may include Val for the amino acid of
position 376, and His for the amino acid of position 434.
Fc Regions Having FcRn-Binding Activity Under Neutral pH Range
Conditions
[0423] 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.
[0424] 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.
[0425] 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 altering
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 altering 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
altering 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 alterations 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 alteration. Those skilled in the
art can appropriately alter amino acids using known methods.
[0426] In the present invention, "alteration of amino acids" or
"amino acid alteration" of an Fc region includes alteration 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 FcRn
in an acidic pH range (i.e., the starting Fc region does not
necessarily need to have an activity to bind to human FcRn in a
neutral pH range). Examples of starting Fc regions preferably
include Fc regions of IgG antibodies, i.e., native Fc regions.
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: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO:
7), or IgG4 (SEQ ID NO: 8) 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 altered 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.
[0427] 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 amino acids of
starting Fc regions, or by deletion of one or more amino acids from
amino acids of starting Fc regions. 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 must
have sequence identity or similarity of less than 100% to their
starting Fc region. In a preferred embodiment, the variants have
amino acid sequence identity or similarity of 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 altered Fc region of the present
invention and its starting Fc region. Amino acid difference between
an altered Fc region and its starting Fc region can also be
preferably specified based on amino acid differences at the
above-described particular amino acid residue positions as
indicated by EU numbering. Methods for producing such variants are
exemplified in the section "Amino acid alterations".
[0428] Fc regions comprised in the antigen-binding molecules of the
present invention that have a human FcRn-binding activity in the
neutral 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 neutral 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 neutral 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 neutral pH range is 0.5 .mu.M (KD) or stronger as a result of
altering amino acids of an IgG-type human immunoglobulin used as a
starting Fc region. Preferred Fc regions of IgG-type
immunoglobulins for alteration include, for example, those of human
IgGs such as IgG1, IgG2, IgG3, and IgG4 shown in SEQ ID NOs: 5, 6,
7, and 8, respectively, and variants thereof.
[0429] When an antigen-binding molecule comprises the Fc region of
human IgG1 as the Fc region, suitable examples of amino acids that
may be altered to achieve the above-mentioned desired effects on
FcRn binding under a neutral pH range condition by altering 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 alteration 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 alteration
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
alteration 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. Alteration of these amino acids
enhances FcRn binding of the Fc region of an IgG-type
immunoglobulin under a neutral pH range condition.
[0430] Fc regions having human FcRn-binding activity in the neutral
pH range can also be obtained by altering amino acids of human
immunoglobulin of IgG type used as the starting Fc region. The Fc
regions of IgG type immunoglobulins adequate for alteration
include, for example, those of human IgGs such as IgG1, IgG2, IgG3,
and IgG4 respectively represented by SEQ ID NOs: 5, 6, 7, and 8,
and altered forms thereof. Amino acids of any positions may be
altered 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 alteration 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 alteration include, for example, amino acids
of the following 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. Alteration
of these amino acids augments the human FcRn binding of the Fc
region of IgG-type immunoglobulin in the neutral pH range.
[0431] From those described above, alterations 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 altered 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.
[0432] Particularly preferred alterations include, for example:
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 of the Fc region
according to EU numbering. Meanwhile, the number of amino acids to
be altered is not particularly limited and an amino acid at only
one site may be altered, and amino acids at two or more sites may
be altered. These combinations of amino acid alterations include,
for example, those described in Tables 2-1 to 2-33.
TABLE-US-00005 TABLE 2-1 Variant KD (M) Amino acid alteration site
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/N434W F11 3.10E-07 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 I322V 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/T256E/N434Y
Table 2-2 is a continuation of Table 2-1.
TABLE-US-00006 TABLE 2-2 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/N434W F48 3.20E-07 M252Y/T256Q/N434Y F49 5.10E-07
M252F/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 2-3 is a continuation of Table 2-2.
TABLE-US-00007 TABLE 2-3 F79 5.90E-06 S254T/T256E/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/T256E/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 2-4 is a continuation of Table 2-3.
TABLE-US-00008 TABLE 2-4 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 M252Y/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 F131 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/N434Y F135 7.50E-07 P257S/M428L/N434Y
F136 3.80E-07 P257T/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 2-5 is a continuation of Table 2-4.
TABLE-US-00009 TABLE 2-5 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 M252W/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 M252Y/T307I/N434Y
Table 2-6 is a continuation of Table 2-5.
TABLE-US-00010 TABLE 2-6 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 M252Y/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/N315H/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 M252Y/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 2-7 is a continuation of Table 2-6.
TABLE-US-00011 TABLE 2-7 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/N434Y 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-07 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/M428F/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 2-8 is a continuation of Table 2-7.
TABLE-US-00012 TABLE 2-8 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 2-9 is a continuation of Table 2-8.
TABLE-US-00013 TABLE 2-9 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
S239K/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 2-10 is a continuation of Table 2-9.
TABLE-US-00014 TABLE 2-10 F365 5.10E-08
P257V/V305A/T307Q/M428L/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 M252W/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 2-11 is a continuation of Table 2-10.
TABLE-US-00015 TABLE 2-11 F406 1.50E-07
P257A/T307Q/L309R/M428L/N434Y F407 1.60E-07
P257V/T307Q/L309R/M428L/N434Y F408 2.50E-07 P257V/N286E/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/K360H/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/N434Y 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/M428L/N434Y
Table 2-12 is a continuation of Table 2-11.
TABLE-US-00016 TABLE 2-12 F441 1.80E-07 P237A/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/M428L/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/N325G/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 2-13 is a continuation of Table 2-12.
TABLE-US-00017 TABLE 2-13 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 2-14 is a continuation of Table 2-13.
TABLE-US-00018 TABLE 2-14 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/N434Y 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 1.60E-06 M252Y/T256E/T307Q/Q311H
F577 1.30E-06 M252Y/N286E/T307A/Q311A F578 5.70E-07
M252Y/N286E/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/V308P F585 2.90E-07 S239K/M252Y/N286E/V308P
Table 2-15 is a continuation of Table 2-14.
TABLE-US-00019 TABLE 2-15 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/T256Q/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 S239K/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-08 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 2-16 is a continuation of Table 2-15.
TABLE-US-00020 TABLE 2-16 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/N434Y 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 2-17 is a continuation of Table 2-16.
TABLE-US-00021 TABLE 2-17 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/V308P/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/K288Y/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 2-18 is a continuation of Table 2-17.
TABLE-US-00022 TABLE 2-18 F712 6.00E-08
P238D/M252Y/T307Q/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 F719
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/L328E/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 P238D/T250L/M252Y/N434Y F750
3.00E-07 P238D/T250M/M252Y/N434Y F751 5.30E-07
P238D/T250N/M252Y/N434Y
Table 2-19 is a continuation of Table 2-18.
TABLE-US-00023 TABLE 2-19 F753 1.80E-07 P238D/T250Q/M252Y/N434Y
F755 3.50E-07 P238D/T250S/M252Y/N434Y F756 3.70E-07
P238D/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/M252Y/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/N434Y
Table 2-20 is a continuation of Table 2-19.
TABLE-US-00024 TABLE 2-20 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 M252Y/N389A/N390A/N434Y
F809 2.20E-07 M252Y/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
P238D/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/M252Y/I253V/N434Y
Table 2-21 is a continuation of Table 2-20.
TABLE-US-00025 TABLE 2-21 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/R255K/N434Y F839 3.40E-07
P238D/T250V/M252Y/R255L/N434Y F840 4.20E-07
P238D/T250V/M252Y/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 2-22 is a continuation of Table 2-21.
TABLE-US-00026 TABLE 2-22 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 M252Y/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
L235K/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/N434Y
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/N315D/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/N315D/G316V/N434Y F915
2.30E-07 M252Y/N315D/G316W/N434Y
Table 2-23 is a continuation of Table 2-22.
TABLE-US-00027 TABLE 2-23 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/N286E/N384A/N389A/N434Y F922 5.90E-08
L235K/S239K/M252Y/N286E/N434Y/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/Y436I 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/M252Y/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/M252Y/T307Q/Q311A/N434Y/Y436V F944 1.70E-07
T250V/M252Y/N434Y/Y436V F945 1.70E-08 T250V/M252Y/V308P/N434Y/Y436V
F946 4.30E-08 T250V/M252Y/T307Q/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/Y436V F960
8.00E-07 M252Y/N434Y/H435R/Y436V
Table 2-24 is a continuation of Table 2-23.
TABLE-US-00028 TABLE 2-24 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/S239K/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/N434Y/Y436V F993 3.80E-08
M252Y/T307Q/Q311A/N434Y/Y436V F994 2.80E-07 M252Y/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/N434Y/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 2-25 is a continuation of Table 2-24.
TABLE-US-00029 TABLE 2-25 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/I332E/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/Q311A/N325G/N434Y/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/V308P/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
2.00E-07 L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.20E-08
L235K/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/S239K/T250V/M252Y/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/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V
Table 2-26 is a continuation of Table 2-25.
TABLE-US-00030 TABLE 2-26 F1053 4.23E-08
L235R/S239K/T250V/M252Y/T307Q/Q311A/ N434Y/Y436V F1058 1.31E-07
M252Y/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/P387R/N434Y/Y436V F1063
1.43E-07 M252Y/P387S/N434Y/Y436V 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 N252Y/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 M252Y/Q386T/N434Y F1097 2.82E-07 M252Y/G385N/P387S/N434Y
F1098 2.58E-07 S239D/M252Y/N434Y F1099 2.57E-07
M252Y/K326D/L328Y/N434Y F1100 2.41E-07
S239D/M252Y/K326D/L328Y/N434Y F1101 6.59E-08
S239D/M2S2Y/T307Q/Q311A/N434Y F1102 6.46E-08
M252Y/T307Q/Q311A/K326D/L328Y/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
M252Y/V422S/S424R/N434Y/Y436V F1106 1.42E-07
M252Y/N434Y/Y436V/Q438R/S440E F1107 1.23E-07
M252Y/V422D/N434Y/Y436V
Table 2-27 is a continuation of Table 2-26.
TABLE-US-00031 TABLE 2-27 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/V422E/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/Q438R F1123
9.55E-09 T250V/M252Y/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
L235K/S239K/M252Y/V422S/N434Y/Y436V F1133 1.58E-07
L235R/S239K/M252Y/S424R/N434Y/Y436V F1134 1.66E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438R F1135 1.26E-07
L235R/S239K/M252Y/N434Y/Y436V/S440E F1136 1.63E-07
L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V F1137 1.58E-07
L235R/S239K/M252Y/V422S/S424R/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/Q438R/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 2-28 is a continuation of Table 2-27.
TABLE-US-00032 TABLE 2-28 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
L233R/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/S434N/M34Y/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/N431S F1188 1.72E-06
T307A/E380A/N434H F1189 1.57E-07
M252Y/H433D/N434Y/Y436V/Q138R/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/Q438K/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 2-29 is a continuation of Table 2-28.
TABLE-US-00033 TABLE 2-29 F1213 6.33E-07 L251N/M252Y/N434Y/Y436V
F1216 1.16E-06 L251S/M252Y/N434Y/Y436V F1217 1.14E-06
L251T/M252Y/N434Y/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/Q438K/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 L235R/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
L235R/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
L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E F1261
1.35E-07 L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D F1262 1.26E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1263 1.24E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D 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/Q438R/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 2-30 is a continuation of Table 2-29.
TABLE-US-00034 TABLE 2-30 F1276 1.02E-07
L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D F1277 1.69E-07
L235K/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E F1278 1.4E-07
L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D F1279
1.23E-07 L235R/S239K/M252Y/T256G/N436Y/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/S440D F1282
7.69E-08 L235P/S239K/M252Y/T256N/N434Y/Y436V/Q438K/S440D F1283
1.34E-07 L235R/S239K/M252Y/T256N/H433D/N434Y/V436V/Q438R/S440E
F1284 1.12E-07
L235R/S339K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D F1285
9.36E-08 L235R/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/Q438R/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/N434Y/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
G337R/S239K/M252Y/N434Y/Y436V
Table 2-31 is a continuation of Table 2-30.
TABLE-US-00035 TABLE 2-31 F1317 1.38E-07
S239K/M252Y/P329K/N434Y/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 L235R/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/Y436V/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/M232Y/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/Q438K/S440E F1340
5.58E-08 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E
F1341 3.22E-07 L235R/S339K/M252Y/S254T/N434Y/Y436T/Q438K/S440E
F1342 2.51E-07 L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E
F1343 2.01E-07
L235R/S239K/M252K/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/S339K/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
L235R/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/H433D/N434Y/Y436T/Q438R/S440E F1354 2.29E-07
L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E F1355 2.47E-07
L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E F1356 1.58E-07
G236R/M252Y/L328K/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 2-32 is a continuation of Table 2-31.
TABLE-US-00036 TABLE 2-32 F1359 1.28E-07
L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E F1360 3.12E-07
L235R/S239K/M252Y/S254T/H443D/N434Y/Y436T/Q438K/S440E F1361
3.52E-07 L235R/S239K/M252Y/S254T/H443D/N434Y/Y436F/Q438R/S440E
F1362 1.41E-07
L235R/S239K/M252Y/S254T/H443D/N434Y/Y436F/Q438K/S440E F1363 1.9E-07
L235R/S239K/M252Y/S254T/H443D/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/T256E/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/N343Y/Y436T/Q438R/S440E F1373
8.67E-08
L235R/S239K/M252Y/S254T/T256E/H433D/N343Y/Y436F/Q438K/S440E F1374
1.2E-07 L235R/S239K/M252Y/S254T/T256E/H433D/N343Y/Y436F/Q438R/S440E
F1375 1.03E-07 L235R/S239K/M252Y/S254T/N434Y/Y436V F1376 9.09E-08
L235R/S239K/M252Y/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/1332V F1411
1.70E-07 V308P/1332V/M428L/N434S F1413 3.70E-08
L235R/S239K/M252Y/S252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/Q438K/
S440E F1414 5.60E-08
L235R/S239K/M252Y/S252Y/S254T/T256E/T307Q/H433D/N434Y/Y436V/Q438K/S440E
F1415 5.90E-08
L235R/S239K/M252Y/S252Y/S254T/T256E/Q311A/H433D/N434Y/Y436V/Q438K/S440E
F1416 1.30E-08
L235R/S239K/M252Y/S252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/S440E
F1417 5.90E-08
L235R/S239K/M252Y/S252Y/S254T/T256E/H433D/N434W/Y436V/Q438K/S440E
F1418 7.50E-08
L235R/S239K/M252Y/S252Y/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/T256E/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 2-33 is a continuation of Table 2-32.
TABLE-US-00037 TABLE 2-33 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/M252Y/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
L235R/S239K/M252Y/T256E/T307D/V308P/H433D/N434Y/Y436V/Q438R/S440E
F1434 3.60E-08
L235R/S239K/M252Y/T256E/T307E/V308P/H433D/N434Y/Y436V/Q438R/S440E
Heterocomplex Comprising the Four Molecules Including Two Molecules
of FcRn and One Molecule of Activating Fc.gamma. Receptor
[0433] Crystallographic studies on FcRn with IgG antibodies
demonstrated that an FcRn-IgG complex is composed of one molecule
of IgG for two molecules of FcRn, and the two molecules are thought
to bind around the interface of the CH2 and CH3 domains located on
both sides of the IgG Fc region (Burmeister et al. (Nature (1994)
372, 336-343)). Meanwhile, as demonstrated in Example 3 of
PCT/JP2012/058603, the antibody Fc region was demonstrated to be
able to form a complex comprising the four molecules including two
molecules of FcRn and one molecule of activating Fc.gamma. receptor
(PCT/JP2012/058603). This heterocomplex formation is a phenomenon
which was revealed as a result of analyzing the properties of
antigen-binding molecules containing an Fc region having an
FcRn-binding activity under a neutral pH range condition.
[0434] While the present invention is not bound to a particular
principle, it can be considered that antigen-binding molecules
administered in vivo produce the effects described below on the in
vivo pharmacokinetics (plasma retention) of the antigen-binding
molecules and an immune response (immunogenicity) to the
administered antigen-binding molecules, as a result of the
formation of heterocomplexes containing the four molecules
including the Fc region contained in the antigen-binding molecules,
two molecules of FcRn, and one molecule of activating Fc.gamma.
receptor. In addition to the various types of activating Fc.gamma.
receptors, FcRn is expressed on immune cells. It is suggested that
the formation of such tetrameric complexes on immune cells by
antigen-binding molecules promotes incorporation of antigen-binding
molecules into immune cells by increasing affinity toward immune
cells and by causing association of intracellular domains to
enhance the internalization signal. The same also applies to
antigen-presenting cells and the possibility that antigen
binding-molecules are likely to be incorporated into
antigen-presenting cells by formation of tetrameric complexes on
the cell membrane of antigen-presenting cells. In general,
antigen-binding molecules incorporated into antigen-presenting
cells are degraded in the lysosomes of the antigen-presenting cells
and are presented to T cells. As a result, plasma retention of
antigen-binding molecules may be worsened because incorporation of
antigen-binding molecules into antigen-presenting cells is promoted
by the formation of the above-described tetrameric complexes on the
cell membrane of the antigen-presenting cells. Similarly, an immune
response may be induced (aggravated).
[0435] For this reason, it is conceivable that when an
antigen-binding molecule having lowered ability to form such
tetrameric complexes is administered in vivo, plasma retention of
the antigen-binding molecules would improve, and induction of in
vivo immune response would be suppressed. Preferred embodiments of
such antigen-binding molecules which inhibit the formation of these
complexes on immune cells including antigen-presenting cells are,
for example, the three embodiments described below.
Antigen-Binding Molecules which Inhibit the Formation of
Heterocomplexes
Embodiment 1
An Antigen-Binding Molecule Containing an Fc Region Having
FcRn-Binding Activity Under a Neutral pH Range Condition and Whose
Binding Activity Toward Activating Fc.gamma.R is Lower than the
Binding Activity of a Native Fc Region Toward Activating
Fc.gamma.R
[0436] The antigen-binding molecule of Embodiment 1 forms a
trimeric complex by binding to two molecules of FcRn; however, it
does not form any complex containing activating Fc.gamma.R. An Fc
region whose binding activity toward activating Fc.gamma.R is lower
than the binding activity of a native Fc region toward activating
Fc.gamma.R can be prepared by altering the amino acids of the
native Fc region as described above. Whether the binding activity
toward activating Fc.gamma.R of the altered Fc region is lower than
the binding activity toward activating Fc.gamma.R of the native Fc
region can be appropriately tested using the methods described in
the section "Binding Activity" above.
[0437] Preferred activating Fc.gamma. receptors include Fc.gamma.RI
(CD64) which includes Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc;
Fc.gamma.RIIa (including allotypes R131 and H131); and
Fc.gamma.RIII (CD16) which includes isoforms Fc.gamma.RIIIa
(including allotypes V158 and F158) and Fc.gamma.RIIIb (including
allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb-NA2).
[0438] Herein, "a binding activity of the Fc region variant toward
an activating Fc.gamma. receptor is lower than the binding activity
of the native Fc region toward an activating Fc.gamma. receptor"
means that the binding activity of the Fc region variant toward any
of the human Fc.gamma. receptors (Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb) is lower 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, the binding activity of the antigen-binding
molecule containing an Fc region variant as compared to the binding
activity of an antigen-binding molecule containing a native Fc
region as a control is 95% or less, preferably 90% or less, 85% or
less, 80% or less, 75% or less, and particularly preferably 70% or
less, 65% or less, 60% or less, 55% 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, 9% or less, 8% or less, 7% or less,
6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1%
or less. As a native Fc region, a starting Fc region may be used,
and Fc regions of wild-type antibodies of different isotypes may
also be used.
[0439] Meanwhile, the binding activity of the native form toward an
activating Fc.gamma.R is preferably a binding activity toward the
Fc.gamma. receptor for human IgG1. Other than performing the
above-described alterations, binding activity toward the Fc.gamma.
receptor can be lowered by changing the isotype to human IgG2,
human IgG3, or human IgG4. Alternatively, besides by performing the
above-described alterations, the binding activity toward an
Fc.gamma. receptor can also be lowered by expressing the
antigen-binding molecule containing an Fc region having a binding
activity toward the Fc.gamma. receptor in hosts that do not add
sugar chains such as Escherichia coli.
[0440] For the antigen-binding molecule containing a control Fc
region, an antigen-binding molecule having an Fc region of a
monoclonal IgG antibody may be appropriately used. The structures
of such Fc regions are shown in SEQ ID NO: 5 (A is added to the N
terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 6 (A is
added to the N terminus of RefSeq Accession No. AAB59393.1), SEQ ID
NO: 7 (RefSeq Accession No. CAA27268.1), and SEQ ID NO: 8 (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,
effect on the binding activity of the antigen-binding molecule
containing the Fc region toward an Fc.gamma. receptor is tested by
using the antigen-binding molecule having an Fc region of a
monoclonal IgG antibody of a particular isotype as a control. In
this way, antigen-binding molecules containing an Fc region whose
binding activity toward the Fc.gamma. receptor was demonstrated to
be high are suitably selected.
[0441] In a non-limiting embodiment of the present invention,
preferred examples of Fc regions whose binding activity toward an
activating Fc.gamma.R is lower than the binding activity of the
native Fc region toward an activating Fc.gamma.R include Fc regions
with alteration of one or more amino acids at any of positions 234,
235, 236, 237, 238, 239, 270, 297, 298, 325, 328, and 329 as
indicated by EU numbering in the amino acids of an above-described
Fc region to be different from those of the native Fc region. The
alterations in the Fc region are not limited to the above example,
and they may be, for example, modifications such as deglycosylation
(N297A and N297Q), IgG1-L234A/L235A, IgG1-A325A/A330S/P331S,
IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A,
IgG1-L234F/L235E/P331S, IgG1-S267E/L328F, IgG2-V234A/G237A,
IgG2-H268QN309L/A330S/A331S, IgG4-L235A/G237A/E318A, and IgG4-L236E
described in Cur. Opin. in Biotech. (2009) 20 (6), 685-691;
alterations such as G236R/L328R, L235G/G236R, N325A/L328R, and
N325L/L328R described in WO 2008/092117; amino acid insertions at
positions 233, 234, 235, and 237 according to EU numbering; and
alterations at the positions described in WO 2000/042072.
[0442] In a non-limiting embodiment of the present invention,
examples of a preferred Fc region include Fc regions having one or
more of the following alterations as indicated by EU numbering in
an aforementioned Fc region:
Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser,
Thr, or Trp for the amino acid at position 234; Ala, Asn, Asp, Gln,
Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg for the
amino acid at position 235; Arg, Asn, Gln, His, Leu, Lys, Met, Phe,
Pro, or Tyr for the amino acid at position 236; Ala, Asn, Asp, Gln,
Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg for
the amino acid at position 237; Ala, Asn, Gln, Glu, Gly, His, Ile,
Lys, Thr, Trp, or Arg for the amino acid at position 238; Gln, His,
Lys, Phe, Pro, Trp, Tyr, or Arg for the amino acid at position 239;
Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr,
Trp, Tyr, or Val for the amino acid at position 265; Ala, Arg, Asn,
Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr for
the amino acid at position 266; Arg, His, Lys, Phe, Pro, Trp, or
Tyr for the amino acid at position 267; Ala, Arg, Asn, Gln, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val for
the amino acid at position 269; Ala, Arg, Asn, Gln, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val for the amino
acid at position 270; Arg, His, Phe, Ser, Thr, Trp, or Tyr for the
amino acid at position 271; Arg, Asn, Asp, Gly, His, Phe, Ser, Trp,
or Tyr for the amino acid at position 295; Arg, Gly, Lys, or Pro
for the amino acid at position 296; Ala for the amino acid at
position 297; Arg, Gly, Lys, Pro, Trp, or Tyr for the amino acid at
position 298; Arg, Lys, or Pro for the amino acid at position 300;
Lys or Pro for the amino acid at position 324; Ala, Arg, Gly, His,
Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val for the amino acid at
position 325; Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val for the amino acid at position 327; Arg, Asn,
Gly, His, Lys, or Pro for the amino acid at position 328; Asn, Asp,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr,
Val, or Arg for the amino acid at position 329; Pro or Ser for the
amino acid at position 330; Arg, Gly, or Lys for the amino acid at
position 331; or Arg, Lys, or Pro for the amino acid at position
332.
Embodiment 2
An Antigen-Binding Molecule Containing an Fc Region Having
FcRn-Binding Activity Under a Neutral pH Range Condition and Whose
Binding Activity Toward an Inhibitory Fc.gamma.R is Higher than the
Binding Activity Toward an Activating Fc.gamma. Receptor
[0443] By binding to two molecules of FcRn and one molecule of
inhibitory Fc.gamma.R, the antigen-binding molecule of Embodiment 2
can form a complex comprising these four molecules. However, since
a single antigen-binding molecule can bind with only one molecule
of Fc.gamma.R, the single antigen-binding molecule in a state bound
to an inhibitory Fc.gamma.R cannot bind to other activating
Fc.gamma.Rs. Furthermore, it has been reported that an
antigen-binding molecule that is incorporated into cells in a state
bound to an inhibitory Fc.gamma.R is recycled onto the cell
membrane, and thus escapes from degradation inside the cells
(Immunity (2005) 23, 503-514). More specifically, it is considered
that antigen-binding molecules having selective binding activity
toward an inhibitory Fc.gamma.R cannot form heterocomplexes
containing an activating Fc.gamma.R and two molecules of FcRn,
which cause an immune response.
[0444] Preferred activating Fc.gamma. receptors include Fc.gamma.RI
(CD64) which includes Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc;
Fc.gamma.RIIa (including allotypes R131 and H131); and
Fc.gamma.RIII (CD16) which includes isoforms Fc.gamma.RIIIa
(including allotypes V158 and F158) and Fc.gamma.RIIIb (including
allotypes Fc.gamma.RIIIb-NA1 and Fc.gamma.RIIIb-NA2). Meanwhile,
examples of preferred inhibitory Fc.gamma. receptors include
Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2).
[0445] Herein, "a binding activity toward an inhibitory Fc.gamma.R
is higher than the binding activity toward an activating Fc.gamma.
receptor" means that the binding activity of the Fc region variant
toward Fc.gamma.RIIb is higher than the binding activity toward any
of the human Fc.gamma. receptors, Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb. For example, it means that
based on an above-described analytical method, the binding activity
toward Fc.gamma.RIIb of the antigen-binding molecule containing an
Fc region variant as compared with the binding activity toward any
of the human Fc.gamma. receptors, Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb is 105% or more, preferably
110% or more, 120% or more, 130% or more, 140% or more, and
particularly preferably 150% or more, 160% or more, 170% or more,
180% or more, 190% or more, 200% or more, 250% or more, 300% or
more, 350% or more, 400% or more, 450% or more, 500% or more, 750%
or more, 10 times or more, 20 times or more, 30 times or more, 40
times or more, 50 times or more.
[0446] Most preferably, the binding activity toward Fc.gamma.RIIb
is higher than each of the binding activities toward Fc.gamma.RIa,
Fc.gamma.RIIa (including allotypes R131 and H131), and
Fc.gamma.RIIIa (including allotypes V158 and F158). Fc.gamma.RIa
shows a markedly high affinity toward native IgG1; thus, the
binding is thought to be saturated in vivo due to the presence of a
large amount of endogenous IgG1. For this reason, inhibition of
complex formation may be possible even if the binding activity
toward Fc.gamma.RIIb is greater than the binding activities toward
Fc.gamma.RIIa and Fc.gamma.RIIIa, and lower than the binding
activity toward Fc.gamma.RIa.
[0447] As a control antigen-binding molecule containing an Fc
region, antigen-binding molecules having an Fc region of a
monoclonal IgG antibody may be appropriately used. The structures
of such Fc regions are shown in SEQ ID NO: 5 (A is added to the N
terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 6 (A is
added to the N terminus of RefSeq Accession No. AAB59393.1), SEQ ID
NO: 7 (RefSeq Accession No. CAA27268.1), and SEQ ID NO: 8 (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,
effect on the binding activity of the Fc region-containing
antigen-binding molecule toward an Fc.gamma. receptor is tested by
using an antigen-binding molecule having the Fc region of a
monoclonal IgG antibody of a particular isotype as a control. In
this way, antigen-binding molecules containing an Fc region whose
binding activity toward the Fc.gamma. receptor was demonstrated to
be high are appropriately selected.
[0448] In a non-limiting embodiment of the present invention,
preferred examples of Fc regions having a selective binding
activity toward an inhibitory Fc.gamma.R include Fc regions in
which among the amino acids of an above-described Fc region, the
amino acid at 238 or 328 as indicated by EU numbering is altered to
an amino acid different from that of the native Fc region.
Furthermore, as an Fc region having a selective binding activity
toward an inhibitory Fc.gamma. receptor, the Fc regions or
alterations described in US 2009/0136485 can be appropriately
selected.
[0449] In a non-limiting embodiment of the present invention, a
preferred example is an Fc region having one or more of the
following alterations as indicated by EU numbering in an
aforementioned Fc region: the amino acid at position 238 is Asp; or
the amino acid at position 328 is Glu.
[0450] In still another non-limiting embodiment of the present
invention, examples of a preferred Fc region include Fc regions
having a substitution of Pro at position 238 according to EU
numbering with Asp and having one or more of the alterations:
alteration of the amino acid at position 237 according to EU
numbering to Trp, the amino acid at position 237 according to EU
numbering is Phe, the amino acid at position 267 according to EU
numbering is Val, the amino acid at position 267 according to EU
numbering is Gln, the amino acid at position 268 according to EU
numbering is Asn, the amino acid at position 271 according to EU
numbering is Gly, the amino acid at position 326 according to EU
numbering is Leu, the amino acid at position 326 according to EU
numbering is Gln, the amino acid at position 326 according to EU
numbering is Glu, the amino acid at position 326 according to EU
numbering is Met, the amino acid at position 239 according to EU
numbering is Asp, the amino acid at position 267 according to EU
numbering is Ala, the amino acid at position 234 according to EU
numbering is Trp, the amino acid at position 234 according to EU
numbering is Tyr, the amino acid at position 237 according to EU
numbering is Ala, the amino acid at position 237 according to EU
numbering is Asp, the amino acid at position 237 according to EU
numbering is Glu, the amino acid at position 237 according to EU
numbering is Leu, the amino acid at position 237 according to EU
numbering is Met, the amino acid at position 237 according to EU
numbering is Tyr, the amino acid at position 330 according to EU
numbering is Lys, the amino acid at position 330 according to EU
numbering is Arg, the amino acid at position 233 according to EU
numbering is Asp, the amino acid at position 268 according to EU
numbering is Asp, the amino acid at position 268 according to EU
numbering is Glu, the amino acid at position 326 according to EU
numbering is Asp, the amino acid at position 326 according to EU
numbering is Ser, the amino acid at position 326 according to EU
numbering is Thr, the amino acid at position 323 according to EU
numbering is Ile, the amino acid at position 323 according to EU
numbering is Leu, the amino acid at position 323 according to EU
numbering is Met, the amino acid at position 296 according to EU
numbering is Asp, the amino acid at position 326 according to EU
numbering is Ala, the amino acid at position 326 according to EU
numbering is Asn, and the amino acid at position 330 according to
EU numbering is Met.
Embodiment 3
An Antigen-Binding Molecule Containing an Fc Region, in which One
of the Two Polypeptides Constituting the Fc Region has an
FcRn-Binding Activity Under a Neutral pH Range Condition and the
Other Polypeptide does not have FcRn-Binding Activity Under a
Neutral pH Range Condition
[0451] By binding to one molecule of FcRn and one molecule of
Fc.gamma.R, the antigen-binding molecule of Embodiment 3 can form a
trimeric complex; however, it does not form any heterocomplex
comprising four molecules including two molecules of FcRn and one
molecule of Fc.gamma.R. As an Fc region in which one of the two
polypeptides constituting the Fc region has an FcRn-binding
activity under a neutral pH range condition and the other does not
have any FcRn-binding activity under a neutral pH range condition
contained in the antigen-binding molecule of Embodiment 3, Fc
regions derived from bispecific antibodies may be suitably used.
Bispecific antibodies are two types of antibodies having
specificities toward different antigens. Bispecific antibodies of
an IgG type can be secreted from hybrid hybridomas (quadromas)
resulting from fusion of two types of hybridomas producing IgG
antibodies (Milstein et al. (Nature (1983) 305, 537-540).
[0452] When an antigen-binding molecule of Embodiment 3 described
above is produced by using recombination techniques such as those
described in the section "Antibodies" above, one can use a method
in which genes encoding the polypeptides that constitute the two
types of Fc regions of interest are transfected into cells to
co-express them. However, the produced Fc regions will be a mixture
in which the following will exist at a molecular ratio of 2:1:1: an
Fc region in which one of the two polypeptides constituting the Fc
region has an FcRn-binding activity under a neutral pH range
condition and the other polypeptide does not have any FcRn-binding
activity under a neutral pH range condition; an Fc region in which
the two polypeptides constituting the Fc region both have an
FcRn-binding activity under a neutral pH range condition; and an Fc
region in which both of the two polypeptides constituting the Fc
region do not have FcRn-binding activity under a neutral pH range
condition. It is difficult to purify antigen-binding molecules
containing the desired combination of Fc regions from the three
types of IgGs.
[0453] When producing the antigen-binding molecules of Embodiment 3
using such recombination techniques, antigen-binding molecules
comprising a heteromeric combination of Fc regions can be
preferentially secreted by adding appropriate amino acid
substitutions to the CH3 domains constituting the Fc regions.
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 arranged in the hole. This promotes
heteromeric H chain formation and simultaneously inhibits homomeric
H chain formation (WO 1996027011; Ridgway et al., (Protein
Engineering (1996) 9, 617-621); Merchant et al., (Nature
Biotechnology (1998) 16, 677-681)).
[0454] 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 two polypeptides that
constitute an Fc region. Specifically, methods for controlling
polypeptide association may be employed to produce a bispecific
antibody (WO 2006/106905), in which amino acid residues forming the
interface between two polypeptides that constitute the Fc region
are altered to inhibit the association between Fc regions having
the same sequence, and to allow the formation of polypeptide
complexes formed by two Fc regions of different sequences.
Specifically, the methods in the above-described section on
bispecific antibodies and methods for producing them can be used as
a non-limiting embodiment for preparing the antigen-binding
molecule of Embodiment 3 of the present invention.
[0455] These antigen-binding molecules of Embodiments 1 to 3 are
all expected to be able to reduce immunogenicity and improve plasma
retention as compared to antigen-binding molecules capable of
forming tetrameric complexes.
Methods for Producing Antigen-Binding Domains
[0456] The present invention provides methods for producing
antigen-binding domains whose antigen-binding activity in the
presence of a target tissue-specific compound is higher than the
antigen-binding activity in the absence of the compound.
[0457] More specifically, the present invention provides a method
for producing an antigen-binding domain, which comprises steps (a)
to (e) below: [0458] (a) determining the antigen-binding activitiy
of an antigen-binding domain in the absence of a target
tissue-specific compound; [0459] (b) determining the
antigen-binding activity of an antigen-binding domain in the
presence of the target tissue-specific compound; [0460] (c)
selecting an antigen-binding domain whose antigen-binding activity
in the absence of a target tissue-specific compound is lower than
in the presence of the compound; [0461] (d) culturing cells
transfected with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (c) is operably linked; and
[0462] (e) collecting an antigen-binding domain from a culture
medium of the cells cultured in (d).
[0463] The present invention also provides a method for producing
an antigen-binding domain, which comprises steps (a) to (e) below:
[0464] (a) determining the antigen-binding activity of an
antigen-binding domain in the presence of a low concentration of a
target tissue-specific compound; [0465] (b) determining the
antigen-binding activity of an antigen-binding domain in the
presence of a high concentration of the target tissue-specific
compound; [0466] (c) selecting an antigen-binding domain whose
antigen-binding activity in the presence of a low concentration of
the target tissue-specific compound is lower than in the presence
of a high concentration of the compound; [0467] (d) culturing cells
transfected with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (c) is operably linked; and
[0468] (e) collecting an antigen-binding domain from a culture
medium of the cells cultured in (d).
[0469] Furthermore, the present invention provides a method for
producing an antigen-binding domain, which comprises steps (a) to
(e) below: [0470] (a) contacting antigen-binding domains or a
library thereof with an antigen in the presence of a target
tissue-specific compound; [0471] (b) placing the antigen-binding
domains that bound to the antigen in said step (a) in the absence
of the compound; [0472] (c) isolating an antigen-binding domain
that was dissociated in said step (b); [0473] (d) culturing cells
transfected with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (c) is operably linked; and
[0474] (e) collecting an antigen-binding domain from a culture
medium of the cells cultured in (d).
[0475] In addition, the present invention provides a method for
producing an antigen-binding domain, which comprises steps (a) to
(e) below: [0476] (a) contacting antigen-binding domains or a
library thereof to an antigen in the presence of a high
concentration of a target tissue-specific compound; [0477] (b)
placing the antigen-binding domains that bind to the antigen in
said step (a) in the presence of a low concentration of the
compound; [0478] (c) isolating an antigen-binding domain that
dissociates in said step (b); [0479] (d) culturing cells
transfected with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (c) is operably linked; and
[0480] (e) collecting an antigen-binding domain from a culture
medium of the cells cultured in (d).
[0481] The present invention provides a method for producing an
antigen-binding domain, which comprises steps of (a) to (f) below:
[0482] (a) contacting a library of antigen-binding domains with an
antigen in the absence of a target tissue-specific compound; [0483]
(b) selecting antigen-binding domains that do not bind to the
antigen in said step (a); [0484] (c) allowing the antigen-binding
domains selected in said step (b) to bind to the antigen in the
presence of the compound; [0485] (d) isolating an antigen-binding
domain that bind to the antigen in said step (c); [0486] (e)
culturing cells transfected with a vector to which a polynucleotide
encoding the antigen-binding domain selected in (d) is operably
linked; and [0487] (f) collecting an antigen-binding domain from a
culture medium of the cells cultured in (e).
[0488] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (f) below:
[0489] (a) contacting a library of antigen-binding domains with an
antigen in the presence of a low concentration of a target
tissue-specific compound; [0490] (b) selecting antigen-binding
domains that do not bind to the antigen in said step (a); [0491]
(c) allowing the antigen-binding domains selected in said step (b)
to bind to the antigen in the presence of a high concentration of
the compound; [0492] (d) isolating an antigen-binding domain that
bind to the antigen in said step (c); [0493] (e) culturing cells
transfected with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (d) is operably linked; and
[0494] (f) collecting an antigen-binding domain from a culture
medium of the cells cultured in (e).
[0495] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (e) below:
[0496] (a) contacting a library of antigen-binding domains with an
antigen-immobilized column in the presence of a target
tissue-specific compound; [0497] (b) eluting antigen-binding
domains that bind to the column in said step (a) from the column in
the absence of the compound; [0498] (c) isolating the
antigen-binding domain eluted in said step (b); [0499] (d)
culturing cells transfected with a vector to which a polynucleotide
encoding the antigen-binding domain selected in (c) is operably
linked; and [0500] (e) collecting an antigen-binding domain from a
culture medium of the cells cultured in (d).
[0501] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (e) below:
[0502] (a) contacting a library of antigen-binding domains with an
antigen-immobilized column in the presence of a high concentration
of a target tissue-specific compound; [0503] (b) eluting
antigen-binding domains that bind to the column in said step (a)
from the column in the presence of a low concentration of the
compound; [0504] (c) isolating an antigen-binding domain eluted in
said step (b); [0505] (d) culturing cells transfected with a vector
to which a polynucleotide encoding the antigen-binding domain
selected in (c) is operably linked; and [0506] (e) collecting an
antigen-binding domain from a culture medium of the cells cultured
in (d).
[0507] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (f) below:
[0508] (a) allowing a library of antigen-binding domains to pass
through an antigen-immobilized column in the absence of a target
tissue-specific compound; [0509] (b) collecting antigen-binding
domains that are eluted without binding to the column in step (a);
[0510] (c) allowing the antigen-binding domains collected in step
(b) to bind to the antigen in the presence of the compound; [0511]
(d) isolating an antigen-binding domain that bind to the antigen in
step (c); [0512] (e) culturing cells transfected with a vector to
which a polynucleotide encoding the antigen-binding domain selected
in (d) is operably linked; and [0513] (f) collecting an
antigen-binding domain from a culture medium of the cells cultured
in (e).
[0514] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (f) below:
[0515] (a) allowing a library of antigen-binding domains to pass
through an antigen-immobilized column in the presence of a low
concentration of a target tissue-specific compound; [0516] (b)
collecting antigen-binding domains that are eluted without binding
to the column in said step (a); [0517] (c) allowing the
antigen-binding domains collected in said step (b) to bind to the
antigen in the presence of a high concentration of the compound;
[0518] (d) isolating an antigen-binding domain that binds to the
antigen in said step (c); [0519] (e) culturing cells transfected
with a vector to which a polynucleotide encoding the
antigen-binding domain selected in (d) is operably linked; and
[0520] (f) collecting an antigen-binding domain from a culture
medium of the cells cultured in (e).
[0521] Furthermore, the present invention provides a method for
producing an antigen-binding domain, which comprises steps (a) to
(f) below: [0522] (a) contacting an antigen with a library of
antigen-binding domains in the presence of a target tissue-specific
compound; [0523] (b) obtaining antigen-binding domains that bind to
the antigen in step (a); [0524] (c) placing the antigen-binding
domain obtained in step (b) in the absence of the compound; [0525]
(d) isolating an antigen-binding domain whose antigen-binding
activity in step (c) is weaker than the reference selected in step
(b); [0526] (e) culturing cells transfected with a vector to which
a polynucleotide encoding the antigen-binding domain selected in
(d) is operably linked; and [0527] (f) collecting an
antigen-binding domain from a culture medium of the cells cultured
in (e).
[0528] The present invention provides a method for producing an
antigen-binding domain, which comprises steps (a) to (f) below:
[0529] (a) contacting an antigen with a library of antigen-binding
domains in the presence of a high concentration of a target
tissue-specific compound; [0530] (b) obtaining antigen-binding
domains that bind to the antigen in step (a); [0531] (c) placing
the antigen-binding domains obtained in step (b) in the presence of
a low concentration of the compound; [0532] (d) isolating an
antigen-binding domain whose antigen-binding activity in step (c)
is weaker than the reference selected in step (b); [0533] (e)
culturing cells transfected with a vector to which a polynucleotide
encoding the antigen-binding domain selected in (d) is operably
linked; and [0534] (f) collecting an antigen-binding domain from a
culture medium of the cells cultured in (e).
[0535] The terms "cells", "cell line", and "cell culture" are used
synonymously herein, and such naming may include all progenies of
the cells or cell line. This way, for example, the terms
"transformant" and "transformed cells" include cultures and primary
target cells derived from them regardless of the number of
passages. Furthermore, it is understood that due to intentional or
accidental mutations, the DNA content is not always exactly the
same in all progenies. Progenies of mutants having substantially
the same function or biological activity such as those screened for
in the initially transformed cells may also be included. When the
description is intended to refer to a different naming, that
intention may become obvious from the context of the description.
Cells that are appropriate for use are suitably selected from cells
described in the section "Antibodies" above.
[0536] When referring to the expression of a coding sequence, the
term "control sequences" refers to DNA nucleotide sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes include, for example, a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancers for the expression of a coding sequence.
[0537] For a nucleic acid, the term "operably linked" means that
the nucleic acid is placed into a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence
or secretory leader is operably linked to DNA for a polypeptide if
it is expressed as a precursor protein that participates in the
secretion of the polypeptide. A promoter or enhancer is operably
linked to a coding sequence if it affects the transcription of the
sequence. A ribosome binding site is operably linked to a coding
sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being
linked are contiguous and, in the case of a secretory leader,
contiguous and in 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, the 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.
[0538] "Ligation" refers to the process of forming phosphodiester
bonds between two nucleic acid fragments. For ligation of the two
fragments, the ends of the fragments must be compatible with each
other. In some cases, the ends will be directly compatible after
endonuclease digestion. However, it may be necessary first to
convert the staggered ends commonly produced after endonuclease
digestion to blunt ends to make them compatible for ligation. For
blunting the ends, the DNA is treated in a suitable buffer for at
least 15 minutes at 15.degree. C. with about 10 units of the Klenow
fragment of DNA polymerase I or T4 DNA polymerase in the presence
of the four deoxyribonucleotide triphosphates. The DNA is then
purified by phenol-chloroform extraction and ethanol precipitation,
or by silica purification. The DNA fragments that are to be ligated
together are put in solution in equimolar amounts. The solution
will contain ATP, ligase buffer, and a ligase such as T4 DNA ligase
at about 10 units per 0.5 .mu.g of DNA. If the DNA is to be ligated
into a vector, the vector is first linearized by digestion with the
appropriate restriction endonuclease(s). The linearized fragment is
then treated with bacterial alkaline phosphatase or calf intestinal
phosphatase to prevent self-ligation of the fragment during the
ligation step.
[0539] In the production methods of the present invention, an
antigen-binding domain which has a higher antigen-binding activity
in the presence of a target tissue-specific compound than in its
absence, which has been selected by the method described in the
above section "Antigen-binding domain dependent on a compound
specific to a target tissue" is isolated. For example, when an
antigen-binding domain isolated in this manner has been selected
from a library, the polynucleotide encoding the antigen-binding
domain is isolated by general gene amplification from a virus such
as a phage, as described in the Examples below. Furthermore, when
an antigen-binding domain or an antibody isolated in this manner
has been selected from culture media of cells such as hybridomas,
the antibody gene or such can be isolated by general gene
amplification from the cells as shown in the section "Antibodies"
above.
Methods for Producing Antigen-Binding Molecules
[0540] The present invention provides methods for producing
antigen-binding molecules whose antigen-binding activity in the
presence of a target tissue-specific compound is higher than the
antigen-binding activity in the absence of the compound.
[0541] More specifically, the present invention provides a method
for producing antigen-binding molecules, which comprises the steps
of:
[0542] (a) determining the antigen-binding activity of an
antigen-binding domain in the absence of a target tissue-specific
compound;
[0543] (b) determining the antigen-binding activity of the
antigen-binding domain in the presence of the target
tissue-specific compound;
[0544] (c) selecting an antigen-binding domain with lower
antigen-binding activity in the absence of the target
tissue-specific compound than in the presence of the compound;
[0545] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0546] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0547] (f) collecting antigen-binding molecules from a culture
medium of the cells cultured in (e).
[0548] The present invention also provides a method for producing
an antigen-binding molecule, which comprises the steps of:
[0549] (a) determining the antigen-binding activity of an
antigen-binding domain in the presence of a low concentration of a
target tissue-specific compound;
[0550] (b) determining the antigen-binding activity of the
antigen-binding domain in the presence of a high concentration of
the target tissue-specific compound;
[0551] (c) selecting an antigen-binding domain with lower
antigen-binding activity in the presence of a low concentration of
the target tissue-specific compound than in the presence of a high
concentration of the compound;
[0552] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0553] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0554] (f) collecting antigen-binding molecules from a culture
medium of the cells cultured in (e).
[0555] Furthermore, the present invention provides a method for
producing an antigen-binding molecule, which comprises the steps
of:
[0556] (a) contacting antigen-binding domains or a library thereof
with an antigen in the presence of a target tissue-specific
compound;
[0557] (b) placing the antigen-binding domains that bind to the
antigen in said step (a) in the absence of the compound;
[0558] (c) isolating an antigen-binding domain that dissociates in
said step (b);
[0559] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0560] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0561] (f) collecting an antigen-binding molecule from a culture
medium of the cells cultured in (e).
[0562] In addition, the present invention provides a method for
producing an antigen-binding molecule, which comprises the steps
of:
[0563] (a) contacting antigen-binding domains or a library thereof
with an antigen in the presence of a high concentration of a target
tissue-specific compound;
[0564] (b) placing the antigen-binding domains that bind to the
antigen in said step (a) in the presence of a low concentration of
the compound;
[0565] (c) isolating an antigen-binding domain that dissociates in
said step (b);
[0566] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0567] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0568] (f) collecting antigen-binding molecules from a culture
medium of the cells cultured in (e).
[0569] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0570] (a) contacting a library of antigen-binding domains with an
antigen in the absence of a target tissue-specific compound;
[0571] (b) selecting antigen-binding domains that do not bind to
the antigen in said step (a);
[0572] (c) allowing the antigen-binding domains selected in said
step (b) to bind to the antigen in the presence of the
compound;
[0573] (d) isolating an antigen-binding domain that binds to the
antigen in said step (c);
[0574] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0575] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0576] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0577] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0578] (a) contacting a library of antigen-binding domains with an
antigen in the presence of a low concentration of a target
tissue-specific compound;
[0579] (b) selecting antigen-binding domains that do not bind to
the antigen in said step (a);
[0580] (c) allowing the antigen-binding domains selected in said
step (b) to bind to the antigen in the presence of a high
concentration of the compound;
[0581] (d) isolating an antigen-binding domain that binds to the
antigen in said step (c);
[0582] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0583] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0584] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0585] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0586] (a) contacting a library of antigen-binding domains with an
antigen-immobilized column in the presence of a target
tissue-specific compound;
[0587] (b) eluting antigen-binding domains that bind to the column
in said step (a) from the column in the absence of the
compound;
[0588] (c) isolating an antigen-binding domain eluted in said step
(b);
[0589] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0590] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0591] (f) collecting antigen-binding molecules from a culture
medium of the cells cultured in (e).
[0592] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0593] (a) contacting a library of antigen-binding domains with an
antigen-immobilized column in the presence of a high concentration
of a target tissue-specific compound;
[0594] (b) eluting antigen-binding domains that bind to the column
in said step (a) from the column in the presence of a low
concentration of the compound;
[0595] (c) isolating an antigen-binding domain eluted in said step
(b);
[0596] (d) linking a polynucleotide encoding the antigen-binding
domain selected in (c) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0597] (e) culturing cells introduced with a vector to which the
polynucleotide obtained in (d) is operably linked; and
[0598] (f) collecting antigen-binding molecules from a culture
medium of the cells cultured in (e).
[0599] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0600] (a) allowing a library of antigen-binding domains to pass
through an antigen-immobilized column in the absence of a target
tissue-specific compound;
[0601] (b) collecting antigen-binding domains that are eluted
without binding to the column in said step (a);
[0602] (c) allowing the antigen-binding domains collected in step
(b) to bind to the antigen in the presence of the compound;
[0603] (d) isolating an antigen-binding domain that binds to the
antigen in step (c);
[0604] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0605] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0606] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0607] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0608] (a) allowing a library of antigen-binding domains to pass
through an antigen-immobilized column in the presence of a low
concentration of a target tissue-specific compound;
[0609] (b) collecting antigen-binding domains that are eluted
without binding to the column in said step (a);
[0610] (c) allowing the antigen-binding domains collected in said
step (b) to bind to the antigen in the presence of a high
concentration of the compound;
[0611] (d) isolating an antigen-binding domain that binds to the
antigen in said step (c);
[0612] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0613] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0614] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0615] Furthermore, the present invention provides a method for
producing an antigen-binding molecule, which comprises the steps
of:
[0616] (a) contacting a library of antigen-binding domains with an
antigen in the presence of a target tissue-specific compound;
[0617] (b) obtaining antigen-binding domains that bind to the
antigen in said step (a);
[0618] (c) placing the antigen-binding domains obtained in said
step (b) in the absence of the compound;
[0619] (d) isolating an antigen-binding domain whose
antigen-binding activity in said step (c) is weaker than the
reference selected in step (b);
[0620] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0621] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0622] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0623] The present invention provides a method for producing an
antigen-binding molecule, which comprises the steps of:
[0624] (a) contacting a library of antigen-binding domains with an
antigen in the presence of a high concentration of a target
tissue-specific compound;
[0625] (b) obtaining antigen-binding domains that bind to the
antigen in said step (a);
[0626] (c) placing the antigen-binding domains obtained in step (b)
in the presence of a low concentration of the compound;
[0627] (d) isolating an antigen-binding domain whose
antigen-binding activity in step (c) is weaker than the reference
selected in step (b);
[0628] (e) linking a polynucleotide encoding the antigen-binding
domain selected in (d) to a polynucleotide encoding a polypeptide
containing an Fc region;
[0629] (f) culturing cells introduced with a vector to which the
polynucleotide obtained in (e) is operably linked; and
[0630] (g) collecting antigen-binding molecules from a culture
medium of the cells cultured in (f).
[0631] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain is, for example, the Fc region contained in
the constant region of a human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID
NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8) 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 EU numbering, and contains the
hinge, CH2, and CH3 domains. The Fc region may be obtained from
human IgG1, but it is not limited to any particular subclass of
IgG.
[0632] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain includes, for example, Fc regions whose
Fc.gamma. receptor-binding activity is higher than the Fc.gamma.
receptor-binding activity of the Fc region of a native human IgG.
Examples of such Fc regions include Fc regions in which at least
one or more amino acids selected from the group consisting of amino
acids at 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,
according to EU numbering, are different from the corresponding
amino acid residues according to EU numbering in the Fc region
contained in the antibody constant region of SEQ ID NO: 5, 6, 7, or
8.
[0633] Furthermore, a non-limiting embodiment of the
above-mentioned Fc region includes, for example, Fc regions
comprising at least one or more amino acid alterations selected
from the group consisting of:
alteration of the amino acid at position 221 to Lys or Tyr;
alteration of the amino acid at position 222 to Phe, Trp, Glu, or
Tyr; alteration of the amino acid at position 223 to Phe, Trp, Glu,
or Lys; alteration of the amino acid at position 224 to Phe, Trp,
Glu, or Tyr; alteration of the amino acid at position 225 to Glu,
Lys, or Trp; alteration of the amino acid at position 227 to Glu,
Gly, Lys, or Tyr; alteration of the amino acid at position 228 to
Glu, Gly, Lys, or Tyr; alteration of the amino acid at position 230
to Ala, Glu, Gly, or Tyr; alteration of the amino acid at position
231 to Glu, Gly, Lys, Pro, or Tyr; alteration of the amino acid at
position 232 to Glu, Gly, Lys, or Tyr; alteration of the amino acid
at position 233 to Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; alteration of the amino
acid at position 234 to Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; alteration of
the amino acid at position 235 to Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 236 to Ala, Asp, Glu, Phe,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or
Tyr; alteration of the amino acid at position 237 to Asp, Glu, Phe,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or
Tyr; alteration of the amino acid at position 238 to Asp, Glu, Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or
Tyr; alteration of the amino acid at position 239 to Asp, Glu, Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or
Tyr; alteration of the amino acid at position 240 to Ala, Ile, Met,
or Thr; alteration of the amino acid at position 241 to Asp, Glu,
Leu, Arg, Trp, or Tyr; alteration of the amino acid at position 243
to Leu, Glu, Leu, Gln, Arg, Trp, or Tyr; alteration of the amino
acid at position 244 to His; alteration of the amino acid at
position 245 to Ala; alteration of the amino acid at position 246
to Asp, Glu, His, or Tyr; alteration of the amino acid at position
247 to Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr;
alteration of the amino acid at position 249 to Glu, His, Gln, or
Tyr; alteration of the amino acid at position 250 to Glu, or Gln;
alteration of the amino acid at position 251 to Phe; alteration of
the amino acid at position 254 to Phe, Met, or Tyr; alteration of
the amino acid at position 255 to Glu, Leu, or Tyr; alteration of
the amino acid at position 256 to Ala, Met, or Pro; alteration of
the amino acid at position 258 to Asp, Glu, His, Ser, or Tyr;
alteration of the amino acid at position 260 to Asp, Glu, His, or
Tyr; alteration of the amino acid at position 262 to Ala, Glu, Phe,
Ile, or Thr; alteration of the amino acid at position 263 to Ala,
Ile, Met, or Thr; alteration of the amino acid at position 264 to
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr; alteration of the amino acid at position 265
to Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 266 to Ala, Ile, Met, or Thr; alteration of the amino acid
at position 267 to Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Thr, Val, Trp, or Tyr; alteration of the amino acid
at position 268 to Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro,
Gln, Arg, Thr, Val, or Trp; alteration of the amino acid at
position 269 to Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 270 to Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr; alteration of the amino acid at position 271
to Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 272 to Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 273 to Phe or Ile; alteration of the amino acid at
position 274 to Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 275 to Leu or Trp; alteration of the amino acid at
position 276 to Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr; alteration of the amino acid at
position 278 to Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, or Trp; alteration of the amino acid at
position 279 to Ala; alteration of the amino acid at position 280
to Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr; alteration of
the amino acid at position 281 to Asp, Lys, Pro, or Tyr; alteration
of the amino acid at position 282 to Glu, Gly, Lys, Pro, or Tyr;
alteration of the amino acid at position 283 to Ala, Gly, His, Ile,
Lys, Leu, Met, Pro, Arg, or Tyr; alteration of the amino acid at
position 284 to Asp, Glu, Leu, Asn, Thr, or Tyr; alteration of the
amino acid at position 285 to Asp, Glu, Lys, Gln, Trp, or Tyr;
alteration of the amino acid at position 286 to Glu, Gly, Pro, or
Tyr; alteration of the amino acid at position 288 to Asn, Asp, Glu,
or Tyr; alteration of the amino acid at position 290 to Asp, Gly,
His, Leu, Asn, Ser, Thr, Trp, or Tyr; alteration of the amino acid
at position 291 to Asp, Glu, Gly, His, Ile, Gln, or Thr; alteration
of the amino acid at position 292 to Ala, Asp, Glu, Pro, Thr, or
Tyr; alteration of the amino acid at position 293 to Phe, Gly, His,
Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 294 to Phe, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 295 to Asp, Glu, Phe, Gly,
His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 296 to Ala, Asp, Glu, Gly,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val;
alteration of the amino acid at position 297 to Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 298 to Ala, Asp, Glu, Phe,
His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 299 to Ala, Asp, Glu, Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or
Tyr; alteration of the amino acid at position 300 to Ala, Asp, Glu,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or
Trp; alteration of the amino acid at position 301 to Asp, Glu, His,
or Tyr; alteration of the amino acid at position 302 to Ile;
alteration of the amino acid at position 303 to Asp, Gly, or Tyr;
alteration of the amino acid at position 304 to Asp, His, Leu, Asn,
or Thr; alteration of the amino acid at position 305 to Glu, Ile,
Thr, or Tyr; alteration of the amino acid at position 311 to Ala,
Asp, Asn, Thr, Val, or Tyr; alteration of the amino acid at
position 313 to Phe; alteration of the amino acid at position 315
to Leu; alteration of the amino acid at position 317 to Glu or Gln;
alteration of the amino acid at position 318 to His, Leu, Asn, Pro,
Gln, Arg, Thr, Val, or Tyr; alteration of the amino acid at
position 320 to Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr,
Val, Trp, or Tyr; alteration of the amino acid at position 322 to
Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 323 to Ile; alteration of
the amino acid at position 324 to Asp, Phe, Gly, His, Ile, Leu,
Met, Pro, Arg, Thr, Val, Trp, or Tyr; alteration of the amino acid
at position 325 to Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,
Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; alteration of the
amino acid at position 326 to Ala, Asp, Glu, Gly, Ile, Leu, Met,
Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; alteration of the amino
acid at position 327 to Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr; alteration of the
amino acid at position 328 to Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 329 to Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 330 to Cys, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;
alteration of the amino acid at position 331 to Asp, Phe, His, Ile,
Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr; alteration of the amino
acid at position 332 to Ala, Asp, Glu, Phe, Gly, His, Lys, Leu,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; alteration of
the amino acid at position 333 to Ala, Asp, Glu, Phe, Gly, His,
Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr; alteration of the amino
acid at position 334 to Ala, Glu, Phe, Ile, Leu, Pro, or Thr;
alteration of the amino acid at position 335 to Asp, Phe, Gly, His,
Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr; alteration of
the amino acid at position 336 to Glu, Lys, or Tyr; alteration of
the amino acid at position 337 to Glu, His, or Asn; alteration of
the amino acid at position 339 to Asp, Phe, Gly, Ile, Lys, Met,
Asn, Gln, Arg, Ser, or Thr; alteration of the amino acid at
position 376 to Ala or Val; alteration of the amino acid at
position 377 to Gly or Lys; alteration of the amino acid at
position 378 to Asp; alteration of the amino acid at position 379
to Asn; alteration of the amino acid at position 380 to Ala, Asn,
or Ser; alteration of the amino acid at position 382 to Ala or Ile;
alteration of the amino acid at position 385 to Glu; alteration of
the amino acid at position 392 to Thr; alteration of the amino acid
at position 396 to Leu; alteration of the amino acid at position
421 to Lys; alteration of the amino acid at position 427 to Asn;
alteration of the amino acid at position 428 to Phe or Leu;
alteration of the amino acid at position 429 to Met; alteration of
the amino acid at position 434 to Trp; alteration of the amino acid
at position 436 to Ile; and alteration of the amino acid at
position 440 to Gly, His, Ile, Leu, or Tyr; according to EU
numbering, in the amino acid residues of the Fc region contained in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. The
number of altered amino acids is not particularly limited; an amino
acid at only one site may be altered, or amino acids at two or more
sites may be altered. Combinations of amino acid alterations at two
or more sites include, for example, those described in Table 1
(Table 1-1 to Table 1-3).
[0634] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain is, for example, an Fc region having binding
activity toward an inhibitory Fc.gamma. receptor that is higher
than the binding activity toward an activating Fc.gamma. receptor.
Specifically, a non-limiting embodiment of such Fc regions is an Fc
region whose binding activity to Fc.gamma.RIIb is higher than the
binding activity toward any of the human Fc.gamma. receptors
Fc.gamma.RIa, Fc.gamma.RIIa, Fc.gamma.RIIIa, and/or
Fc.gamma.RIIIb.
[0635] A non-limiting embodiment of the above-mentioned Fc region
preferably includes, for example, an Fc region in which the amino
acid at 238 or 328 according to EU numbering in the Fc region
contained in the antibody constant region of SEQ ID NO: 5, 6, 7, or
8, is altered to an amino acid different from that of the native Fc
region. A preferred example of such Fc regions is an Fc region
having one or more of the following alterations: alteration of the
amino acid at position 238 to Asp, and alteration of the amino acid
at position 328 to Glu, according to EU numbering, in the
aforementioned Fc region.
[0636] In still another non-limiting embodiment of the
above-mentioned Fc region, preferred examples include Fc regions
having one or more of the alterations exemplified in
PCT/JP2012/054624: substitution of Pro at position 238 according to
EU numbering with Asp, alteration of the amino acid at position 237
according to EU numbering to Trp, alteration of the amino acid at
position 237 according to EU numbering to Phe, alteration of the
amino acid at position 267 according to EU numbering to Val,
alteration of the amino acid at position 267 according to EU
numbering to Gln, alteration of the amino acid at position 268
according to EU numbering to Asn, alteration of the amino acid at
position 271 according to EU numbering to Gly, alteration of the
amino acid at position 326 according to EU numbering to Leu,
alteration of the amino acid at position 326 according to EU
numbering to Gln, alteration of the amino acid at position 326
according to EU numbering to Glu, alteration of the amino acid at
position 326 according to EU numbering to Met, alteration of the
amino acid at position 239 according to EU numbering to Asp,
alteration of the amino acid at position 267 according to EU
numbering to Ala, alteration of the amino acid at position 234
according to EU numbering to Trp, alteration of the amino acid at
position 234 according to EU numbering to Tyr, alteration of the
amino acid at position 237 according to EU numbering to Ala,
alteration of the amino acid at position 237 according to EU
numbering to Asp, alteration of the amino acid at position 237
according to EU numbering to Glu, alteration of the amino acid at
position 237 according to EU numbering to Leu, alteration of the
amino acid at position 237 according to EU numbering to Met,
alteration of the amino acid at position 237 according to EU
numbering to Tyr, alteration of the amino acid at position 330
according to EU numbering to Lys, alteration of the amino acid at
position 330 according to EU numbering to Arg, alteration of the
amino acid at position 233 according to EU numbering to Asp,
alteration of the amino acid at position 268 according to EU
numbering to Asp, alteration of the amino acid at position 268
according to EU numbering to Glu, alteration of the amino acid at
position 326 according to EU numbering to Asp, alteration of the
amino acid at position 326 according to EU numbering to Ser,
alteration of the amino acid at position 326 according to EU
numbering to Thr, alteration of the amino acid at position 323
according to EU numbering to Ile, alteration of the amino acid at
position 323 according to EU numbering to Leu, alteration of the
amino acid at position 323 according to EU numbering to Met,
alteration of the amino acid at position 296 according to EU
numbering to Asp, alteration of the amino acid at position 326
according to EU numbering to Ala, alteration of the amino acid at
position 326 according to EU numbering to Asn, and alteration of
the amino acid at position 330 according to EU numbering to
Met.
[0637] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain includes Fc regions having binding activity
to FcRn in the acidic pH range. Amino acids that can undergo such
alteration include, for example, amino acids at positions 252, 254,
256, 309, 311, 315, 433, and/or 434 according to EU numbering, and
amino acids at positions 253, 310, 435, and/or 426 which are
combined with the above amino acids, as described in WO
1997/034631. Preferred examples include amino acids at 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 (EU
numbering) as described in WO 2000/042072. Similarly, preferred
examples of amino acids that can undergo such alteration include
amino acids at 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 can undergo such alteration are, for example, amino acids at
positions 250, 314, and 428 according to EU numbering as described
in WO2004/092219. In addition, preferred examples of amino acids
that can undergo such alteration include amino acids at 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, preferred examples of amino acids that can undergo
such alteration include amino acids at positions 251, 252, 307,
308, 378, 428, 430, 434, and/or 436 according to EU numbering as
described in WO 2010/045193.
[0638] A non-limiting embodiment of the above-mentioned Fc region
includes, for example, Fc regions having at least one or more amino
acid alterations selected from the group consisting of:
alteration of the amino acid of position 251 to Arg or Leu;
alteration of the amino acid of position 252 to Phe, Ser, Thr, or
Tyr; alteration of the amino acid of position 254 to Ser or Thr;
alteration of the amino acid of position 255 to Arg, Gly, Ile, or
Leu; alteration of the amino acid of position 256 to Ala, Arg, Asn,
Asp, Gln, Glu, or Thr; alteration of the amino acid of position 308
to Ile or Thr; alteration of the amino acid of position 309 to Pro;
alteration of the amino acid of position 311 to Glu, Leu, or Ser;
alteration of the amino acid of position 312 to Ala or Asp;
alteration of the amino acid of position 314 to Ala or Leu;
alteration of the amino acid of position 385 to Ala, Arg, Asp, Gly,
His, Lys, Ser, or Thr; alteration of the amino acid of position 386
to Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr; alteration of the
amino acid of position 387 to Ala, Arg, His, Pro, Ser, or Thr;
alteration of the amino acid of position 389 to Asn, Pro, or Ser;
alteration of the amino acid of position 428 to Leu, Met, Phe, Ser,
or Thr; alteration of the amino acid of position 433 to Arg, Gln,
His, Ile, Lys, Pro, or Ser; alteration of the amino acid of
position 434 to His, Phe, or Tyr; and alteration of the amino acid
of position 436 to Arg, Asn, His, Lys, Met, or Thr, according to EU
numbering, in the amino acid residues of the Fc region contained in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. The
number of amino acids to be altered is not particularly limited; an
amino acid at only one site may be altered or amino acids at two or
more sites may be altered.
[0639] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes Fc regions in which the amino acid at position 308 is Ile,
the amino acid at position 309 is Pro, and/or the amino acid at
position 311 is Glu, according to EU numbering, in the amino acid
residues of the Fc region contained in the antibody constant region
of SEQ ID NO: 5, 6, 7, or 8. Another non-limiting embodiment of
this Fc region may include Fc regions containing 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, yet another non-limiting embodiment of this alteration
may include Fc regions containing 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.
A different non-limiting embodiment of this alteration may include
Fc regions containing 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.
[0640] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes, for example, Fc regions containing 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, in the amino acid residues of the Fc
region included in the antibody constant region of SEQ ID NO: 5, 6,
7, or 8.
[0641] A different non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes, for example, Fc regions containing 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, in the amino acid residues of the Fc region included in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. Moreover,
another non-limiting embodiment of this alteration includes Fc
regions containing His or Met for the amino acid of position 428
and/or His or Met for the amino acid of position 434.
[0642] Another different non-limiting embodiment of the Fc region
whose binding activity to FcRn in the acidic pH range is stronger
than the binding activity of the starting Fc region of human IgG1
may be, for example, alterations 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, in the amino acid residues
of the Fc region included in the antibody constant region of SEQ ID
NO: 5, 6, 7, or 8. Another non-limiting embodiment of this
alteration include Fc regions containing 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.
[0643] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes Fc regions containing at least one or more amino acids
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, in the
amino acid residues of the Fc region contained in the antibody
constant region of SEQ ID NO: 5, 6, 7, or 8.
[0644] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes, for example, Fc regions containing Gln for the amino acid
of position 250, and/or Leu or Phe for the amino acid of position
428, according to EU numbering, in the amino acid residues of the
Fc region contained in the antibody constant region of SEQ ID NO:
5, 6, 7, or 8. Another non-limiting embodiment of this alteration
may include Glu for the amino acid of position 250, and/or Leu or
Phe for the amino acid of position 428.
[0645] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes Fc regions containing at least two or more amino acids
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; according to EU
numbering, in the amino acid residues of the Fc region contained in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.
[0646] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes, for example, Fc regions containing Gln for the amino acid
of position 307, and Ala or Ser for the amino acid of position 434,
according to EU numbering, in the amino acid residues of the Fc
region contained in the antibody constant region of SEQ ID NO: 5,
6, 7, or 8. Another non-limiting embodiment of this Fc region
includes Fc regions containing Pro for the amino acid of position
308, and Ala for the amino acid of position 434. Furthermore,
another non-limiting embodiment of this Fc region includes Fc
regions containing Tyr for the amino acid of position 252, and Ala
for the amino acid of position 434. A different non-limiting
embodiment of this Fc region includes Fc regions containing Val for
the amino acid of position 378, and Ala for the amino acid of
position 434. Another different non-limiting embodiment of this Fc
region includes alterations including Leu for the amino acid of
position 428, and Ala for the amino acid of position 434. Another
different non-limiting embodiment of this Fc region includes Fc
regions containing Ala for the amino acid of position 434, and Ile
for the amino acid of position 436. Furthermore, another
non-limiting embodiment of this alteration includes Fc regions
containing 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 alteration includes Fc regions containing Gln
for the amino acid of position 307, and Ile for the amino acid of
position 436.
[0647] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes Fc regions containing 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, in the
amino acid residues of the Fc region contained in the antibody
constant region of SEQ ID NO: 5, 6, 7, or 8. Another non-limiting
embodiment of this Fc region includes Fc regions containing 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 Fc region includes Fc
regions containing 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 Fc region
includes Fc regions containing 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.
[0648] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
includes at least one or more amino acid alterations selected from
the group consisting of:
alteration of the amino acid of position 238 to Leu; alteration of
the amino acid of position 244 to Leu; alteration of the amino acid
of position 245 to Arg; alteration of the amino acid of position
249 to Pro; alteration of the amino acid of position 252 to Tyr;
alteration of the amino acid of position 256 to Pro; alteration of
the amino acid of position 257 to Ala, Ile, Met, Asn, Ser, or Val;
alteration of the amino acid of position 258 to Asp; alteration of
the amino acid of position 260 to Ser; alteration of the amino acid
of position 262 to Leu; alteration of the amino acid of position
270 to Lys; alteration of the amino acid of position 272 to Leu or
Arg; alteration of the amino acid of position 279 to Ala, Asp, Gly,
His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr; alteration of the
amino acid of position 283 to Ala, Asp, Phe, Gly, His, Ile, Lys,
Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr; alteration of the
amino acid of position 285 to Asn; alteration of the amino acid of
position 286 to Phe; alteration of the amino acid of position 288
to Asn or Pro; alteration of the amino acid of position 293 to Val;
alteration of the amino acid of position 307 to Ala, Glu, or Met;
alteration of the amino acid of position 311 to Ala, Ile, Lys, Leu,
Met, Val, or Trp; alteration of the amino acid of position 312 to
Pro; alteration of the amino acid of position 316 to Lys;
alteration of the amino acid of position 317 to Pro; alteration of
the amino acid of position 318 to Asn or Thr; alteration of the
amino acid of position 332 to Phe, His, Lys, Leu, Met, Arg, Ser, or
Trp; alteration of the amino acid of position 339 to Asn, Thr, or
Trp; alteration of the amino acid of position 341 to Pro;
alteration of the amino acid of position 343 to Glu, His, Lys, Gln,
Arg, Thr, or Tyr; alteration of the amino acid of position 375 to
Arg; alteration of the amino acid of position 376 to Gly, Ile, Met,
Pro, Thr, or Val; alteration of the amino acid of position 377 to
Lys; alteration of the amino acid of position 378 to Asp or Asn;
alteration of the amino acid of position 380 to Asn, Ser, or Thr;
alteration of the amino acid of position 382 to Phe, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr; alteration of
the amino acid of position 423 to Asn; alteration of the amino acid
of position 427 to Asn; alteration of the amino acid of position
430 to Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,
Thr, Val, or Tyr; alteration of the amino acid of position 431 to
His or Asn; alteration of the amino acid of position 434 to Phe,
Gly, His, Trp, or Tyr; alteration of the amino acid of position 436
to Ile, Leu, or Thr; alteration of the amino acid of position 438
to Lys, Leu, Thr, or Trp; alteration of the amino acid of position
440 to Lys; and alteration of the amino acid of position 442 to
Lys; according to EU numbering, in the amino acid residues of the
Fc region contained in the antibody constant region of SEQ ID NO:
5, 6, 7, or 8. The number of amino acids to be altered is not
particularly limited and amino acids at only two sites may be
altered and amino acids at three or more sites may be altered.
[0649] Another non-limiting embodiment of the Fc region whose
binding activity to FcRn in the acidic pH range is stronger than
the binding activity of the starting Fc region of human IgG1
include Fc regions containing Ile for the amino acid of position
257, and Ile for the amino acid of position 311, according to EU
numbering, in the amino acid residues of the Fc region contained in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. Another
non-limiting embodiment of this Fc region includes Fc regions
containing Ile for the amino acid of position 257 and His for the
amino acid of position 434. Another non-limiting embodiment of this
Fc region includes Fc regions containing Val for the amino acid of
position 376 and His for the amino acid of position 434.
[0650] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain includes, for example, Fc regions having
binding activity to human FcRn in the neutral pH range. Examples of
Fc regions having binding activity to human FcRn in the neutral pH
range include Fc regions in which at least one or more amino acids
at positions selected from the group consisting of positions
221-225, 227, 228, 230, 232, 233-241, 243-252, 254-260, 262-272,
274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343,
345, 360, 362, 370, 375-378, 380, 382, 385-387, 389, 396, 414, 416,
423, 424, 426-438, 440, and 442, according to EU numbering, are
substituted in the amino acid residues of the Fc region included in
the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.
[0651] Another non-limiting embodiment of the aforementioned Fc
region having binding activity to FcRn in the neutral pH range
includes Fc regions in which 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,
according to EU numbering, are substituted in the amino acid
residues of the Fc region contained in the antibody constant region
of SEQ ID NO: 5, 6, 7, or 8. By substituting at least one amino
acid selected from these amino acids with a different amino acid,
the Fc region included in the antigen-binding molecule can bind to
human FcRn in the neutral pH range.
[0652] Another non-limiting embodiment of the aforementioned Fc
region having binding activity to FcRn in the neutral pH range
includes Fc regions containing 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; according to EU
numbering. The number of amino acids to be altered is not
particularly limited and an amino acid at only one site may be
altered or amino acids at two or more sites may be altered.
Combinations of these amino acid alterations include, for example,
those described in Table 2-1 to 2-33.
[0653] A non-limiting embodiment of the Fc region whose
polynucleotide sequence is linked to a polynucleotide encoding an
antigen-binding domain includes, for example, Fc regions whose
binding activity toward an activating Fc.gamma.R is lower than that
of the native Fc region toward an activating Fc.gamma.R. Another
non-limiting embodiment of the Fc region preferably includes, for
example, Fc regions in which one or more amino acids at positions
234, 235, 236, 237, 238, 239, 270, 297, 298, 325, 328, and 329
according to EU numbering are altered to amino acids that are
different from those of the native Fc region of SEQ ID NO: 5, 6, 7,
or 8. The alterations in the Fc region are not limited to the above
example, and they may be, for example, alterations such as
deglycosylation (N297A and N297Q), IgG1-L234A/L235A,
IgG1-A325A/A330S/P331S, IgG1-C226S/C229S,
IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,
IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268QN309L/A330S/A331S,
IgG4-L235A/G237A/E318A, and IgG4-L236E described in Cur. Opin. in
Biotech. (2009) 20 (6), 685-691; alterations such as G236R/L328R,
L235G/G236R, N325A/L328R, and N325L/L328R described in WO
2008/092117; amino acid insertions at positions 233, 234, 235, and
237 according to EU numbering; and alterations at the positions
described in WO 2000/042072.
[0654] Another non-limiting embodiment of the aforementioned Fc
region whose binding activity toward activating Fc.gamma.R is lower
than the binding activity of the native Fc region toward activating
Fc.gamma.R includes Fc regions comprising at least one or more
amino acids selected from the group consisting of:
Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser,
Thr, or Trp for the amino acid at position 234; Ala, Asn, Asp, Gln,
Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg for the
amino acid at position 235; Arg, Asn, Gln, His, Leu, Lys, Met, Phe,
Pro, or Tyr for the amino acid at position 236; Ala, Asn, Asp, Gln,
Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg for
the amino acid at position 237; Ala, Asn, Gln, Glu, Gly, His, Ile,
Lys, Thr, Trp, or Arg for the amino acid at position 238; Gln, His,
Lys, Phe, Pro, Trp, Tyr, or Arg for the amino acid at position 239;
Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr,
Trp, Tyr, or Val for the amino acid at position 265; Ala, Arg, Asn,
Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr for
the amino acid at position 266; Arg, His, Lys, Phe, Pro, Trp, or
Tyr for the amino acid at position 267; Ala, Arg, Asn, Gln, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val for
the amino acid at position 269; Ala, Arg, Asn, Gln, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val for the amino
acid at position 270; Arg, His, Phe, Ser, Thr, Trp, or Tyr for the
amino acid at position 271; Arg, Asn, Asp, Gly, His, Phe, Ser, Trp,
or Tyr for the amino acid at position 295; Arg, Gly, Lys, or Pro
for the amino acid at position 296; Ala for the amino acid at
position 297; Arg, Gly, Lys, Pro, Trp, or Tyr for the amino acid at
position 298; Arg, Lys, or Pro for the amino acid at position 300;
Lys or Pro for the amino acid at position 324; Ala, Arg, Gly, His,
Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val for the amino acid at
position 325; Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val for the amino acid at position 327; Arg, Asn,
Gly, His, Lys, or Pro for the amino acid at position 328; Asn, Asp,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr,
Val, or Arg for the amino acid at position 329; Pro or Ser for the
amino acid at position 330; Arg, Gly, or Lys for the amino acid at
position 331; or Arg, Lys, or Pro for the amino acid at position
332; according to EU numbering. The number of amino acids to be
altered is not particularly limited, and an amino acid at only one
site may be altered or amino acids at two or more sites may be
altered.
[0655] In a non-limiting embodiment of the present invention, two
polypeptides forming an Fc region that are derived from a
bispecific antibody as described above can be suitably used as the
Fc region to be included in an antigen-binding molecule. More
specifically, it is preferable to use two polypeptides that
constitute an Fc region, and which comprise Cys for the amino acid
at position 349 and Trp for the amino acid at position 366
according to EU numbering in the amino acid sequence of one of the
polypeptides; and Cys for the amino acid at position 356, Ser for
the amino acid at position 366, Ala for the amino acid at position
368, and Val for the amino acid at position 407 as indicated by EU
numbering in the amino acid sequence of the other polypeptide.
[0656] In another non-limiting embodiment of the present invention,
two polypeptides that constitute an Fc region and which comprises
Asp for the amino acid at position 409 according to EU numbering in
the amino acid sequence of one of the polypeptides, and Lys for the
amino acid at position 399 according to EU numbering in the amino
acid sequence of the other polypeptide, 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 the amino acid at
position 360 or Asp may suitably be added as the amino acid at
position 392.
[0657] In still another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region and which
comprise Glu for the amino acid at position 370 according to EU
numbering in the amino acid sequence of one of the polypeptides,
and Lys for the amino acid at position 357 according to EU
numbering in the amino acid sequence of the other polypeptide, may
be suitably used as the Fc region.
[0658] In yet another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region and which
comprise Glu for the amino acid at position 439 according to EU
numbering in the amino acid sequence of one of the polypeptides,
and Lys for the amino acid at position 356 according to EU
numbering in the amino acid sequence of the other polypeptide, may
be suitably used as the Fc region.
[0659] In still yet another non-limiting embodiment of the present
invention, any of combinations of the above-mentioned embodiments,
as shown below, may be suitably used as the Fc region:
[0660] (i) two polypeptides that constitute an Fc region and which
comprise Asp for the amino acid at position 409 and Glu for the
amino acid at position 370 according to EU numbering in the amino
acid sequence of one of the polypeptides, and Lys for the amino
acid at position 399 and Lys for the amino acid at position 357
according to EU numbering in the amino acid sequence of the other
polypeptide (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);
[0661] (ii) two polypeptides that constitute an Fc region, and
which comprise Asp for the amino acid at position 409 and Glu for
the amino acid at position 439 according to EU numbering of the
amino acid sequence of one of the polypeptides; and Lys for the
amino acid at position 399 and Lys for the amino acid at position
356 according to EU numbering in the amino acid sequence of the
other polypeptide (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);
[0662] (iii) two polypeptides that constitute an Fc region, and
which comprise Glu for the amino acid at position 370 and Glu for
the amino acid at position 439 according to EU numbering in the
amino acid sequence of one of the polypeptides, and Ly for the
amino acid at position 357 and Lys for the amino acid at position
356 according to EU numbering in the amino acid sequence of the
other polypeptide; or
two polypeptides that constitute an Fc region, and which comprise
Asp the amino acid at position 409, Glu for the amino acid at
position 370, and Glu for the amino acid at position 439 according
to EU numbering in the amino acid sequence of one of the
polypeptides; and Lys for the amino acid at position 399, Lys for
the amino acid at position 357, and Lys for the amino acid at
position 356 according to EU numbering in the amino acid sequence
of the other polypeptide (in this embodiment, the amino acid at
position 370 according to EU numbering may not be substituted with
Glu, and furthermore, when the amino acid at position 370 is not
substituted with 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).
[0663] Further, in another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region and which
comprise Lys for the amino acid at position 356 according to EU
numbering in the amino acid sequence of one of the polypeptides,
and Arg for the amino acid at position 435 and Glu for the amino
acid at position 439 according to EU numbering in the amino acid
sequence of the other polypeptide may also be suitably used.
[0664] In still another non-limiting embodiment of the present
invention, two polypeptides that constitute an Fc region and which
comprise Lys for the amino acid at position 356 and Lys for the
amino acid at position 357 according to EU numbering in the amino
acid sequence of one of the polypeptides, and Glu for the amino
acid at position 370, Arg for the amino acid at position 435, and
Glu for the amino acid at position 439 according to EU numbering in
the amino acid sequence of the other polypeptide may also be
suitably used.
[0665] Antigen-binding molecules of the present invention are
isolated from culture media of cells transformed with a desired
expression vector in which a polynucleotide encoding an
antigen-binding domain and a polynucleotide encoding a polypeptide
containing an Fc region, which have been linked in the
above-described manner, are operably linked.
[0666] When the Fc region contained in the antigen-binding molecule
of the present invention is an Fc region that has been modified so
that the percentage of the Fc region to which a fucose-deficient
sugar chain has been attached, or bisecting N-acetylglucosamine has
been attached, will become higher, the above-mentioned transformed
host cells that are suitably used are host cells that have low
ability to add fucose to a sugar chain as a result of modification
of the activity to form the sugar chain structure of a polypeptide
to be modified with a sugar chain (for example, WO 2000/061739, WO
2002/031140, and WO 2006/067913). In a non-limiting embodiment of
such host cells, host cells deficient in the activity of an enzyme
or transporter selected from the group consisting of
fucosyltransferase (EC 2.4.1.152), fucose transporter (SLC35C1),
GMD (GDP-mannose-4,6-dehydratase) (EC 4.2.1.47), Fx
(GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase) (EC
1.1.1.271), and GFPP (GDP-.beta.-L-fucose pyrophosphorylase (EC
2.7.7.30), may be suitably used (for example, WO 2000/061739, WO
2002/031140, and WO 2006/067913). Host cells deficient in such
activity can be produced, for example, by a method that destroys
the genes of these functional proteins endogenous to CHO cells, BHK
cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse
myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridoma
cells, or such so that they are unable to function.
[0667] When the Fc region contained in the antigen-binding molecule
of the present invention is an Fc region having a sugar chain
containing a bisecting GlcNAc, the above-described transformed
cells that are suitably used are host cells expressing a gene
encoding a functional protein having GnTIII
(.beta.-1,4-mannosyl-glycoprotein
4-.beta.-N-acetylglucosaminyltransferase) (EC2.4.1.144) activity or
GalT (.beta.-1,4-galactosyltransferase) (EC 2.4.1.38) activity to
produce antibodies which have bisecting GlcNAc-containing sugar
chains (WO2002/079255 and such). In another suitable non-limiting
embodiment, host cells that co-express, in addition to the
aforementioned functional proteins, a gene encoding a functional
protein having human ManII (manosidase II) (3.2.1.114) activity, a
gene encoding a functional protein having GnTI
(.beta.-1,2-acetylglucosaminyltransferase I) (EC 2.4.1.94)
activity, a gene encoding a functional protein having GnTII
(.beta.-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143)
activity, a gene encoding a functional protein having ManI
(mannosidase) (EC 3.2.1.113) activity, and .alpha.-1,6-fucosyl
transferase (EC 2.4.1.68), are suitably used (WO2004/065540).
[0668] Antigen-binding molecules of the present invention are
produced using methods that follow the methods for producing
antibodies, such as isolation from culture media of the
above-mentioned cells, which are described in the section
"Antibodies" above. A non-limiting embodiment of the aforementioned
polypeptides containing an Fc region includes, for example, the
antibody constant region of SEQ ID NO: 5, 6, 7, or 8. A
non-limiting embodiment of the antigen-binding molecules of the
present invention is for example, a full-length antibody
molecule.
Pharmaceutical Compositions
[0669] The present invention provides pharmaceutical compositions
comprising an antigen-binding molecule that does not act
systemically in the blood or normal tissues, but acts on lesions
such as cancer and inflamed sites, to exhibit drug efficacy while
avoiding side effects. The antigen-binding molecule contained in
the pharmaceutical composition of the present invention binds to an
antigen expressed in cancer cells, immune cells, stromal cells, or
such in cancer tissues; an antigen secreted in cancer tissues; or
an antigen expressed by immune cells or such in inflammatory
tissues; and an antigen secreted in inflammatory tissues; and
cannot bind to antigens expressed in normal tissues; therefore,
side effects due to cytotoxic activity, neutralizing activity, or
such against normal tissues are avoided; and at the same time,
potent cytotoxic effects, growth suppressing effects, and
immunity-enhancing action on cancers, or immunosuppressive effects
against inflammatory cells in inflammatory tissues, are exhibited.
For example, a bispecific or biparatopic antigen-binding molecule
containing an antigen-binding domain that binds to EGFR expressed
on cancer cells and an antigen-binding domain that binds to CD3
expressed on T cells in a manner dependent on a cancer
tissue-specific compound, does not bind to EGFR expressed on normal
tissues but bind to EGFR expressed on cancer cells; thereby
exhibiting potent antitumor effects while avoiding side effects.
Specifically, while the antigen-binding molecule binds to CD3
expressed on T cells in the vicinity of cancer cells in a manner
dependent on a cancer tissue-specific compound, the molecule does
not bind to CD3 expressed on T cells that are not in the vicinity
of cancer cells. Therefore, the molecule activates T cells in the
vicinity of cancer cells, exhibiting potent antitumor effects while
avoiding side effects.
[0670] Such antigen-binding molecules that bind to an antigen in
target tissues but not in other normal tissues and blood exhibit
drug efficacy while avoiding side effects. Antigen-binding
molecules provided by the present invention, which bind to an
antigen by using a small molecule present at high concentrations in
target tissues in vivo as a switch, namely, small molecule switch
antigen-binding molecules, do not bind to the antigen in a normal
environment where the small molecule is not present, but can bind
to the antigen in target tissues where the small molecule is
present at high concentrations.
[0671] A non-limiting embodiment of such small molecule switch
antigen-binding molecules includes cancer tissue-specific, or
inflammatory tissue-specific, compound-dependent antigen-binding
molecules; and a cancer tissue-specific or inflammatory
tissue-specific compound such as adenosine, adenosine
5'-triphosphate (ATP), inosine, kynurenine, prostaglandin E2
(PGE2), succinic acid, and lactic acid, which are present at a high
concentration in cancer tissues or inflammatory tissues and capable
of functioning as a switch, provides a switch function by being
sandwiched between the antigen-binding molecule of the present
invention (the paratope contained therein) and the antigen (the
epitope contained therein). In the absence of the compound, the
interaction between the paratope in the antigen-binding molecule of
the present invention and the epitope in the antigen is not
sufficient for the antigen-binding molecule of the present
invention to be able to bind to the antigen. In the presence of the
compound, the compound interposes between the paratope in the
antigen-binding molecule of the present invention and the epitope
in the antigen; and the antigen-binding molecule that has bound to
the antigen in a target tissue such as cancer tissue or
inflammatory tissue, where the compound is present at a high
concentration, can exhibit drug efficacy on cells expressing the
antigen. Moreover, since this binding of the switch compound is
reversible, the binding of an antigen-binding molecule of the
present invention to an antigen by means of these switch compounds
may be controlled in a reversible manner. Thus, antigen-binding
molecules of the present invention which can exhibit drug efficacy
in a lesion site such as cancer tissue or inflammatory tissue by
binding to pathogenic cells such as cancer cells or immune cells in
a cancer tissue or inflammatory tissue or by binding to an antigen
secreted in a cancer tissue or inflammatory tissue are useful as
pharmaceutical compositions. The pharmaceutical compositions of the
present invention may comprise a pharmaceutically acceptable
carrier.
[0672] In the present invention, pharmaceutical compositions
generally refer to pharmaceutical agents for treating or
preventing, or testing and diagnosing diseases. Furthermore, in the
present invention, the phrase "pharmaceutical composition
containing an antigen-binding molecule whose antigen-binding
activity varies depending on the concentration of a target
tissue-specific compound" can be rephrased as "method for treating
a disease which comprises administering to a subject to be treated
an antigen-binding molecule whose antigen-binding activity varies
depending on the concentration of a target tissue-specific
compound", or rephrased as "use of an antigen-binding molecule
whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound in the
production of a pharmaceutical for treating a disease".
Furthermore, the phrase "pharmaceutical composition containing an
antigen-binding molecule whose antigen-binding activity varies
depending on the concentration of a target tissue-specific
compound" can be rephrased as "use of an antigen-binding molecule
whose antigen-binding activity varies depending on the
concentration of a target tissue-specific compound, for treating a
disease".
[0673] 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.
[0674] 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).
[0675] 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.
[0676] 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.
[0677] 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.
[0678] 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.
[0679] All prior art documents cited in this specification are
incorporated herein by reference.
[0680] Herein below, the present invention will be specifically
described with the Examples; however, the present invention should
not be limited thereto.
EXAMPLES
Example 1
Concept of Antibodies that Bind to Antigens Via Small Molecules
Serving as a Switch, which are Present at High Concentrations in
Target Tissues
[0681] In order to exert drug efficacy while avoiding adverse
effects, there is a need for drug discovery technology that works
in lesions such as cancer or inflammatory sites without acting
systemically in normal tissues or blood. Antibody molecules that
can bind to antigens expressed on cancer cells but are incapable of
binding to the antigens expressed on normal tissues after
administration can exert strong cytotoxic effects against cancer
while avoiding adverse effects on normal tissues as a result of
cytotoxic action. For example, antigen-binding molecules that have
been altered from the above-described EGFR-BiTE (Non-patent
Document 9), which cannot bind to EGFR expressed on normal tissues
but are capable of binding to EGFR expressed on cancer cells, can
exert strong an antitumor effect while avoiding adverse effects.
Meanwhile, BiTE exerts an antitumor effect by recruiting and
activating T cells via CD3 (Non-patent Document 8); and if it is
possible to confer EGFR-BiTE with the property of binding to CD3
expressed on T cells in the vicinity of cancer cells but not to CD3
expressed on T cells outside the vicinity of cancer cells,
EGFR-BiTE altered to have the property can activate T cells in
cancer and thus can exert strong antitumor effects while avoiding
adverse effects.
[0682] However, this is not limited to only antibody
pharmaceuticals against cancer. When an antibody molecule binds and
inhibits cytokines in the synovial fluid of inflamed joints in
rheumatoid arthritis but does not systemically inhibit the
cytokines, the molecule can exert potent therapeutic effects
against inflammatory/autoimmune diseases such as rheumatoid
arthritis while avoiding increased risks of infection due to
systemic neutralization of cytokines.
[0683] As described above, antibodies that bind to antigens in
cancer tissues but not to antigens in other tissues such as normal
tissues and blood can exert drug efficacy while avoiding adverse
effects. However, ideal antibodies having such properties have not
been reported so far. Meanwhile, as shown in FIG. 1, antibody
molecules that bind to antigens via small molecules, as a switch,
that are present at high concentrations in cancer tissues in vivo
(i.e., small molecule switch antibodies), do not bind to antigens
in environments in the absence of such small molecules; and they
can bind to antigens in target tissues where the small molecules
are present at high concentrations.
[0684] In developing such small-molecule switch antibodies, first
it was to search for small molecules that are present at high
concentration in cancer tissues and are considered to be usable as
a switch. The result suggested that adenosine, adenosine
triphosphate (adenosine 5'-triphosphate (ATP)), inosine,
kynurenine, prostaglandin E2 (PGE2), succinic acid, and lactic acid
were promising as a switch. Each of these small molecules is either
produced by cancer cells, or released from cancer cells after cell
death, or produced by immune cells etc infiltrating cancer tissues,
and thus they are present at high concentrations in cancer tissues;
however, they are present at lower concentrations in normal tissues
and blood in comparison to cancer tissues. If these small molecules
can be sandwiched in a complex between an antibody and an antigen
in such a way as shown in FIG. 2, the small molecules can achieve
the switch function. Specifically, in the absence of small
molecules, the antigen-antibody interaction is insufficient and the
antibody cannot bind to its antigen. Meanwhile, in the presence of
a small molecule, the antibody can bind to its antigen via the
small molecule sandwiched between the antibody and antigen. In
other words, in the presence of a low concentration of small
molecules, the antigen-antibody interaction is insufficient and the
antibody cannot bind to its antigen, while in the presence of a
high concentration of small molecules, the antibody can bind to its
antigen via a small molecule sandwiched between the antibody and
antigen. Furthermore, the binding of small molecules as a switch is
reversible, and the regulation of antigen binding by the small
molecule switch is also reversible.
[0685] In this context, first, the present inventors attempted to
isolate small molecule switch antibodies against IL-6 (Br. J.
Haematol. (2011) 152 (5), 579-92) which is reported to be involved
in cancer cell growth.
Example 2
Acquisition of Antibodies that Bind to Human IL-6 in the Presence
of Small Molecules from a Human Antibody Library Using
Phage-Display Techniques
[0686] (2-1) Construction of a Phage-Display Library of Naive Human
Antibodies
[0687] A phage-display library of human antibodies consisting of
multiple phages that present the Fab domains of human antibodies
whose sequences were different from one another was constructed
using as a template, polyA RNA prepared from human PBMC,
commercially available human polyA RNA, or such according to a
method known to those skilled in the art.
(2-2) Acquirement of Antibodies that Bind to Human IL-6 in the
Presence of Small Molecules from the Library by Bead Panning
[0688] The phage-display library of naive human antibodies
constructed as described in (2-1) was screened for antibodies that
exhibit antigen-binding activity in the presence of small
molecules, specifically, by collecting phages displaying antibodies
that in the presence of small molecules exhibit antigen-binding
activity to antigens captured by beads. Phages were collected from
a phage suspension eluted from the beads in the absence of small
molecules. In this preparation method, the antigen used was
biotin-labeled human IL-6.
[0689] Phages produced in E. coli containing the phagemid vector
constructed for phage display were purified by a conventional
method. Then, a phage library suspension was prepared by dialyzing
the phages against TBS. Next, BSA was added at a final
concentration of 4% to the phage library suspension. Panning was
performed using antigen-immobilized magnetic beads. The magnetic
beads used were NeutrAvidin coated beads (Sera-Mag SpeedBeads
NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280
Streptavidin).
[0690] To efficiently isolate small molecule switch antibodies
which depend on small molecules which can serve as a switch in
cancer tissues, panning was carried out to enrich antibodies that
bind to antigens in the presence of a mixed solution of small
molecules (adenosine, adenosine triphosphate (adenosine
5'-triphosphate (ATP)), inosine, kynurenine, prostaglandin E2
(PGE2), succinic acid, and lactic acid (hereinafter referred to as
small molecule cocktail (SC)) but not in the absence of SC.
[0691] Specifically, together with 250 pmol of biotin-labeled
antigen, SC containing adenosine triphosphate sodium salt (ATP-Na),
adenosine, inosine, succinic acid, and lactic acid at a final
concentration of 1 mM, prostaglandin E2 (PGE2) at a final
concentration of 1 .mu.M, and kynurenine at a final concentration
of 100 .mu.M, which had been adjusted to be pH 7.4 with NaOH, was
contacted with the prepared phage library suspension for 60 minutes
at room temperature. Then, BSA-blocked magnetic beads were added to
the phage library suspension, and the antigen-phage complex was
allowed to bind to the magnetic beads at room temperature for 15
minutes. After washing once with SC/TBS (TBS containing SC), the
beads were combined with 0.5 ml of 1 mg/ml trypsin solution.
Immediately after suspending the beads at room temperature for 15
minutes, the phage suspension was collected from the isolated beads
using a magnetic stand. The collected phage suspension was added to
10 ml of E. coli cells of strain ER2738 at the logarithmic growth
phase (OD600=0.4 to 0.7). The E. coli was infected with the phage
by incubating the above E. coli with gently stirring at 37.degree.
C. for one hour. The infected E. coli was seeded in a 225
mm.times.225 mm plate. Then, phages were collected from the culture
medium of the seeded E. coli to prepare a liquid stock of phage
library.
[0692] The first round of panning was carried out to collect phages
that are capable of binding in the presence of small molecules,
while the second and subsequent rounds of panning were performed to
enrich phages that are capable of binding to antigens in the
presence of SC. Specifically, the prepared phage library suspension
was mixed with 40 pmol biotin-labeled antigen, SC, and NaOH, and
contacted with the small molecules and antigens for 60 minutes at
room temperature. BSA-blocked magnetic beads were added and allowed
to bind to the antigen-phage complex for 15 minutes at room
temperature. The beads were washed with 1 ml of SC/TBST and SC/TBS.
Then, immediately after 0.5 ml of TBS was added to suspend the
beads at room temperature, a phage suspension was collected from
the isolated beads using a magnetic stand. After this treatment was
repeated, the two separately eluted phage suspensions were mixed
together. Then, the resultant beads were combined with 0.5 ml of
TBS and stirred at room temperature for five minutes. A phage
suspension was collected from the isolated beads using a magnetic
stand. By addition of 5 .mu.l of 100 mg/ml trypsin to the collected
phage suspension, the pIII protein (helper phage-derived protein
pIII) that does not display Fab was cleaved off from phages, and
the ability of phages that do not display Fab to infect E. coli was
eliminated. The phages collected from the trypsinized phage
suspension were added to 10 ml of E. coli strain ER2738 at the
logarithmic growth phase (OD600=0.4 to 0.7). The E. coli was
incubated at 37.degree. C. for one hour under gentle stirring to
infect phage. The infected E. coli was seeded in a 225 mm.times.225
mm plate. The two types of infected E. coli obtained through the
second round of panning were mixed in equal amounts at this time
point. Then, phages were collected from the culture medium of the
seeded E. coli to prepare a phage library suspension. Panning was
performed three times to isolate antibodies that have
antigen-binding activity in the presence of SC.
(2-3) Acquisition of Antibodies that Bind to Human IL-6 in the
Presence of Small Molecules from the Library Using a Negative
Selection Method
[0693] The constructed phage-display library of naive human
antibodies was screened for antibodies that exhibit antigen-binding
activity in the presence of small molecules. As a first step of
screening, the phage-display library of naive human antibodies was
contacted with biotin-labeled antigen-streptavidin in the absence
of small molecules to eliminate phages displaying antibodies that
have antigen-binding activity even in the absence of small
molecules. Then, panning was performed in the presence of small
molecules in the same manner. Thus, screening was carried out for
antibodies that have antigen-binding activity in the presence of
small molecules. Biotin-labeled IL-6 was used as the antigen.
[0694] Phages were produced in E. coli retaining the constructed
phage-display phagemid. The produced phages were purified by a
conventional method, and then a phage library suspension was
prepared by dialyzing the phages against TBS. Then, BSA was added
to the phage library suspension at a final concentration of 4%. The
magnetic beads used were NeutrAvidin coated beads (Sera-Mag
SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads
(Dynabeads M-280 Streptavidin). Panning was performed using
antigen-immobilized magnetic beads.
[0695] Together with 250 pmol of biotin-labeled antigen, SC
containing ATP-Na, adenosine, inosine, succinic acid, and lactic
acid at a final concentration of 1 mM, PGE2 at a final
concentration of 1 .mu.M, and kynurenine at a final concentration
of 100 .mu.M, whose pH was adjusted to 7.4 with NaOH, was added and
contacted with the prepared phage library suspension for 60 minutes
at room temperature. Then, BSA-blocked magnetic beads were added to
the phage library suspension, and the antigen-phage complex was
allowed to bind to the magnetic beads at room temperature for 15
minutes. After washing once with SC/TBS, the beads were combined
with 0.5 ml of 1 mg/ml trypsin solution. Immediately after
suspending the beads at room temperature for 15 minutes, the phage
suspension was collected from the isolated beads using a magnetic
stand. The collected phage suspension was added to 10 ml of E. coli
cells of strain ER2738 at the logarithmic growth phase (OD600=0.4
to 0.7). The E. coli was incubated at 37.degree. C. for one hour
with gentle stirring to be infected by phage. The infected E. coli
was seeded in a 225 mm.times.225 mm plate. Then, phages were
collected from the culture medium of the seeded E. coli to prepare
a liquid stock of phage library.
[0696] The first round of panning was carried out to collect phages
that are capable of binding in the presence of SC, while the second
and subsequent rounds of panning were performed to enrich phages
that are capable of binding to antigens in the presence of SC.
Specifically, 250 pmol of biotinylated antigen was added to
BSA-blocked Sera-Mag NeutrAvidin beads for binding at room
temperature for 15 minutes. The beads were washed three times with
TBS. The phage library suspension subjected to BSA blocking was
added to the beads, and allowed to bind thereto at room temperature
for one hour. Phages that did not bind to the antigens or beads
were collected by isolating the beads using a magnetic stand. 40
pmol of biotin-labeled antigen, SC, and NaOH were added to the
collected phages. Thus, the phage library was contacted with the
small molecules in SC at room temperature for 60 minutes. Then,
BSA-blocked magnetic beads were added to the mixture of labeled
antigen, SC, and phage library, and allowed to bind to the
antigen-phage complex for 15 minutes at room temperature. The beads
were washed with 1 ml of SC/TBST and SC/TBS. Then, 0.5 ml of 1
mg/ml trypsin solution was added to the mixture. After the mixed
suspension was stirred at room temperature for 20 minutes, phages
were collected from the beads that had been separated using a
magnetic stand. The collected phages were added to 10 ml of E. coli
strain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7).
The E. coli was incubated at 37.degree. C. for one hour under
gentle stirring to be infected by phage. The infected E. coli was
seeded in a 225 mm.times.225 mm plate. Panning was performed three
times to isolate antibodies that have antigen-binding activity in
the presence of SC.
(2-4) Assessment of Binding Activity in the Presence of Small
Molecules by Phage ELISA
[0697] Culture supernatants containing phages were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained by the method
described above. The collected culture supernatants were treated by
ultrafiltration using NucleoFast 96 (MACHEREY-NAGEL). 100 .mu.l of
the collected culture supernatants were added to each well of
NucleoFas96 and centrifuged (4500 g for 45 minutes) to remove the
flow-through portion. 100 .mu.l of H.sub.2O was added to each well,
and again the NucleoFast 96 was centrifuged (4500 g for 30 minutes)
for washing. After 100 .mu.l of TBS was added, the NucleoFast 96
was allowed to stand for five minutes at room temperature. Finally,
a phage suspension was collected from the supernatant in each
well.
[0698] After addition of TBS or SC/TBS, the purified phages were
subjected to ELISA by the following procedure. A StreptaWell 96
microtiter plate (Roche) was coated overnight with 100 .mu.l of TBS
containing the biotin-labeled antigen. After the antigen was
removed by washing each well of the plate with TBST, the wells were
blocked with 250 .mu.l of 2% skim milk-TBS for one hour or more. 2%
skim milk-TBS was removed, and then the prepared, purified phages
were added to each well. The plate was allowed to stand at
37.degree. C. for one hour to allow binding of antibody-displaying
phages to the antigen in the presence or absence of SC in each
well. After each well was washed with TBST or SC/TBST, the
HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech)
diluted with TBS or SC/TBS was added thereto, and the plate was
incubated for one hour. Following washing with TBST or SC/TBST, the
TMB single solution (ZYMED) was added to each well, and the
chromogenic reaction in the solution was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm.
[0699] Phage ELISA of isolated 96 clones revealed a clone
"I6NMSC1-3_A11", which has binding activity to human IL-6 as an
antigen in the presence of a small molecule cocktail.
Example 3
Assessment of Antibodies that Bind to Antigens in the Presence of
Small Molecules
[0700] (3-1) Expression and Purification of Antibodies that Bind to
Human IL-6
[0701] Genes were amplified from clone I6NMSC1-3_A11 which had been
assessed to have antigen-binding activity in the presence of SC
using specific primers (SEQ ID NOs: 110 and 112) by phage ELISA as
described in Example 2. The nucleotide sequences of the genes were
analyzed (the heavy chain and light chain sequences are shown in
SEQ ID NOs: 30 and 31, respectively). The gene encoding the
variable region of I6NMSC1-3_A11 was inserted into an animal
expression plasmid for human IgG1/Lambda, while each of the genes
encoding the variable regions of known anti-human IL-6 antibody
CLB8-F1 (the heavy chain and light chain are SEQ ID NOs: 32 and 33,
respectively) and the variable regions of anti-human glypican 3
antibody GC413 (the heavy chain and light chain are SEQ ID NOs: 34
and 35, respectively) as a negative control were inserted into an
animal expression plasmid for human IgG1/kappa. Antibodies were
expressed using the method described below. FreeStyle 293-F
(Invitrogen) which is derived from human fetal kidney cells were
suspended at a cell density of 1.33.times.10.sup.6 cells/ml in
FreeStyle 293 Expression Medium (Invitrogen) and aliquoted at 3 ml
into each well of a 6-well plate. The plasmid DNA was transfected
into the cells by lipofection. From the culture supernatants after
four days of culture in a CO.sub.2 incubator (37.degree. C., 8%
CO.sub.2, 90 rpm), antibodies were purified by a method known to
those skilled in the art using rProtein A Sepharose.TM. Fast Flow
(Amersham Biosciences). Absorbance of solutions of purified
antibodies was measured at 280 nm using a spectrophotometer. From
the values obtained by measurement, the concentrations of purified
antibodies were calculated using an extinction coefficient
determined by the PACE method (Protein Science (1995) 4,
2411-2423).
(3-2) Identification of Small Molecules Necessary for Human IL-6
Binding of the Obtained Antibodies
[0702] Three types of antibodies: isolated I6NMSC1-3_A11
(hereinafter abbreviated as A11), and CLB8-F1 and GC413 as controls
were subjected to ELISA under the nine conditions described in
Table 3. Meanwhile, each small molecule was appropriately prepared
at the concentrations shown in Table 3 using the buffers indicated
in Table 4. Biotin-labeled human IL-6 was used as the antigen.
TABLE-US-00038 TABLE 3 Condition Small molecule Concentration 1
ATP-Na 1 mM 2 Adenosine 1 mM 3 Inosine 1 mM 4 PGE2 1 .mu.M 5
Succinic acid 1 mM 6 Lactic acid 1 mM 7 Kynurenine 100 .mu.M 8 ATP
1 mM, Adenosine 1 mM, Inosine 1 mM, PGE2 1 .mu.M, Succinic acid 1
mM, Lactic acid 1 mM, Kynurenine 100 .mu.M 9 -- --
TABLE-US-00039 TABLE 4 Wash buffer 10 mM ACES, 150 mM NaCl, 0.05%
Tween20, pH7.4 Blocking Buffer 10 mM ACES, 150 mM NaCl, 2% BSA,
pH7.4 Sample Buffer 10 mM ACES, 150 mM NaCl, Each small molecule,
pH7.4
[0703] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing the biotin-labeled antigen. After washing with the Wash
buffer to remove unbound antigen from the plate, each well was
blocked for one hour or more with 250 .mu.l of the Blocking Buffer.
The Blocking Buffer was removed from each well. The purified IgGs
were prepared to 2.5 .mu.g/ml in a Sample Buffer containing small
molecules at the final concentrations shown in Table 3, and each
was aliquoted at 100 .mu.l to each well of the plate. The plate was
allowed to stand at room temperature for one hour to allow binding
of each IgG to the antigen in each well. After washing with a Wash
Buffer containing the small molecules at the final concentrations
shown in Table 3, an HRP-conjugated anti-human IgG antibody
(BIOSOURCE) diluted with a Sample Buffer containing the same small
molecules was added to each well. The plate was incubated for one
hour. Following wash with a Wash Buffer containing each small
molecule, the TMB single solution (ZYMED) was added to each well.
The chromogenic reaction in the solution of each well was
terminated by adding sulfuric acid. Then, the developed color was
assessed by measuring absorbance at 450 nm.
[0704] The measurement result is shown in FIG. 3. The result showed
that the absorbance of CLB8-F1 was constant regardless of the type
or presence of small molecule, whereas the absorbance of
I6NMSC1-3_A11 was markedly lower under condition 9 (without small
molecules) as compared to under condition 8 (the complete small
molecule cocktail solution). Similar to phage ELISA, this result
showed that I6NMSC1-3_A11 had the property that its antigen binding
is altered depending on the presence of small molecules. Meanwhile,
I6NMSC1-3_A11 showed equivalent absorbance under condition 7 (in
the presence of 100 .mu.M kynurenine) to that under condition 8;
however, the absorbance was markedly lower under other conditions.
This result demonstrates that I6NMSC1-3_A11 is an antibody that
binds to human IL-6 as an antigen in the presence of kynurenine but
not in the absence of kynurenine.
Example 4
Assessment of the Effect of Kynurenine on Human IL6 Binding by
Surface Plasmon Resonance
(4-1) Assessment of Kynurenine for its Switch Function in Human
IL-6 Binding
[0705] Using Biacore T200 (GE Healthcare), A11 was analyzed for its
interaction with human IL-6 (Kamakura Techno-Science, Inc.) in
antigen-antibody reaction. Sensor chip CM5 (GE Healthcare) was
immobilized with an appropriate amount of protein A/G (Invitrogen)
by amine coupling. Antibodies of interest were captured by the chip
to allow interaction to IL-6 as an antigen. The two types of
running buffers used were 10 mmol/l ACES, 150 mmol/l NaCl, 0.05%
(w/v) Tween20, 100 .mu.mol/l kynurenine, pH 7.4, and 10 mmol/l
ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. The interaction
with IL-6 as an antigen was assessed at 37.degree. C. The buffer
used to dilute IL-6 was the same running buffer as described
above.
[0706] A diluted solution of human IL-6 and a running buffer as a
blank were injected at a flow rate of 5 .mu.l/min for three minutes
to allow interaction of human IL-6 with A11 captured on the sensor
chip. Then, the running buffer was injected at a flow rate of 5
.mu.l/min for three minutes. After observation of human IL-6
dissociation from the antibody, 10 mmol/l glycine-HCl (pH 1.5) was
injected at a flow rate of 30 .mu.l/min for 30 seconds to
regenerate the sensor chip. The dissociation constant K.sub.D (M)
of A11 was calculated for human IL-6 based on the association rate
constant ka (1/Ms) and dissociation rate constant kd (1/s), both of
which are kinetic parameters calculated from the sensorgram
obtained by the measurement. Each parameter was calculated using
the Biacore T200 Evaluation Software (GE Healthcare).
[0707] The sensorgrams for the interaction between A11 and 4
.mu.mol/l human IL-6 obtained by the measurement in the presence or
absence of 100 .mu.mol/l kynurenine are shown in FIG. 4. As shown
in FIG. 4, A11 bound to IL-6 in the presence of 100 .mu.mol/l
kynurenine; however, in the absence of kynurenine, the IL-6 binding
was undetectable. This demonstrates that A11 has the property that
it binds to IL-6 via kynurenine as a switch. Meanwhile, the
dissociation constant K.sub.D of A11 was 1.0E.sup.-6 mol/l in the
presence of 100 .mu.mol/l kynurenine.
(4-2) Assessment for the Effect of Kynurenine Concentration on
Human IL-6 Binding
[0708] Then, the effect of kynurenine concentration on
antigen-antibody reaction between A11 and human IL-6 was assessed
using Biacore T200 (GE Healthcare). The running buffer used was 10
mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4.
Antigen-antibody reaction between A11 and human IL-6 was assessed
at 25.degree. C. A11 was immobilized onto sensor chip CM5 by amine
coupling, and as an analyte IL-6 was diluted to 1 .mu.mol/l with 10
mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4
containing kynurenine at various concentrations, and allowed to
interact for 60 seconds to observe changes in the amount of
binding. The result is shown in FIG. 5. This result demonstrated
that the higher the concentration of kynurenine as a switch, the
more IL-6 binds to A11.
[0709] Next, the same experiment as described above was carried out
to assess the effect of kynurenine concentration on
antigen-antibody reaction between human IL-6 and the human
IL-6-binding antibody H01 (the heavy chain and light chain are SEQ
ID NOs: 36 and 37, respectively) immobilized on sensor chip CM5,
which was derived from a library and served as a control for the
switch function of kynurenine in A11. The result is shown in FIG.
6. This result confirmed that for the control anti-IL-6 antibody
H01 derived from a library, its binding to IL-6 is not altered even
if the kynurenine concentration changes.
[0710] Then, the effect of the concentration of kynurenine as a
switch in the divalent binding of A11 to IL-6 was assessed using
Biacore T200 (GE Healthcare). The running buffer used was 10 mmol/l
ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4.
Antigen-antibody reaction between A11 and human IL-6 was assessed
at 25.degree. C. IL-6 was immobilized onto sensor chip CM5 by amine
coupling, and as an analyte A11 was diluted to 0.1 .mu.mol/l with
10 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4
containing various concentrations of kynurenine, and allowed to
interact for 60 seconds to observe changes in the amount of
divalent binding of A11 to IL-6. The result is shown in FIG. 7. In
this assay system, A11 is expected to bind in a divalent manner
since IL-6 is immobilized on a sensor chip. With such an assay
system where A11 recognizes IL-6 in a divalent manner, the amount
of A11 bound to IL-6 was also observed to increase with a higher
kynurenine concentration. This result demonstrated that A11 has the
property that in its divalent binding, it also binds to IL-6 via
kynurenine as a switch.
(4-3) Effect of Kynurenine as a Switch on the Dissociation of
Antibodies from Human IL-6
[0711] Using Biacore T200 (GE Healthcare), A11 bound to IL-6 in the
presence of kynurenine was tested to assess whether in the absence
of kynurenine, it dissociates in a kynurenine
concentration-dependent manner. The running buffer used was 10
mmol/l ACES, 150 mmol/1 NaCl, 0.05% (w/v) Tween20, pH 7.4, and 10
mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4, 100
.mu.mol/l kynurenine. Assay was carried out at 25.degree. C. IL-6
was immobilized onto sensor chip CM5 by amine coupling, and as an
analyte A11 was diluted to 0.1 .mu.mol/l with 10 mmol/l ACES, 150
mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4, containing 100 .mu.mol/l
kynurenine, and allowed to interact for 60 seconds. Then, the
dissociation of IL-6 was monitored with each type of running
buffer. In order to compare the degree of dissociation between
respective running buffer conditions, the amounts of IL-6 bound to
A11 were normalized and compared by taking the value in the
presence of 100 .mu.mol/l kynurenine as 100. A sensorgram
representing the interaction between A11 and IL-6 after
normalization is shown in FIG. 8. The result shown in FIG. 8
demonstrates that A11 has the property that it binds to IL-6 in the
presence of kynurenine and then rapidly dissociates from IL-6 in
the absence of kynurenine. Specifically, the kynurenine-mediated
regulation of the antibody binding to human IL-6 by kynurenine was
demonstrated to be completely reversible.
[0712] These results demonstrated that A11 is an antibody that
binds to IL-6 in the presence of kynurenine via kynurenine as a
switch, but is dissociated from IL-6 in the absence of kynurenine.
It was also confirmed that it is possible to have full ON/OFF
regulation of A11 so that it has no human IL-6-binding activity in
the absence of kynurenine. The switch function was expected to be
achieved in the manner such as shown in FIG. 2.
(4-4) Assessment of Kynurenine for its Binding to Human IL-6
[0713] The interaction between IL-6 (Kamakura Techno-Science, Inc.)
and kynurenine was analyzed using Biacore T200 (GE Healthcare).
Sensor chip CM5 (GE Healthcare) was immobilized with about 5000 RU
of IL-6 by amine coupling, and 800, 400, 200, 100, 50, or 25 nmol/l
kynurenine was allowed to interact with IL-6. The running buffer
used was 10 mmol/1 ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH
7.4. A11 measurements for the interaction described above were
carried out at 25.degree. C. Kynurenine was diluted using the
running buffer. The obtained sensorgram showing the interaction
between IL-6 and kynurenine is shown in FIG. 9.
[0714] About 5000 RU of IL-6 was immobilized in the above-described
experiment. The molecular weights of IL-6 and kynurenine were about
20000 g/mol and about 200 g/mol, respectively. Thus, at maximum
about 50 RU of kynurenine was expected to interact. Under the
measurement condition described above, however, obvious interaction
with IL-6 was not detectable even when kynurenine was allowed to
interact at a maximal concentration of 800 nmol/l.
[0715] Based on the result of the Example described above, the KD
of kynurenine for formation of the complex consisting of A11, IL-6,
and kynurenine is estimated to be several tens nM to several nM.
This also suggests that if hypothetically kynurenine interacts
directly with IL-6, the interaction would be observed unambiguously
when kynurenine was allowed to interact at 800 nmol/l. The result
described above implies the possibility that kynurenine does not
interact directly with IL-6 but interacts with A11 or the A11-IL-6
complex at several tens nM.
Example 5
Acquisition of Anti-Adenosine Antibodies by Rabbit B Cell
Cloning
(5-1) Design of Immunogen to Construct Adenosine-Binding
Library
[0716] The immunogens used in immunizing rabbits were
2'-Adenosine-PEG-Tetanus toxin p30 helper peptide
(2'-Adenosine-PEG-peptide) shown in FIG. 10 and
5'-Adenosine-PEG-Tetanus toxin p30 helper peptide
(5'-Adenosine-PEG-peptide) shown in FIG. 11. The Tetanus toxin p30
helper peptide consists of the amino acid sequence
FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 4), and is a peptide identified
as an epitope of T cell receptor expressed on helper T cells (Eur.
J. Immunol. (1989) 19, 2237-2242). The peptide is known to activate
antibody production (J. Immunol. (1992) 149, 717-721). When linked
to adenosine, the peptide serves as an adjuvant and thus is
expected to enhance the production of antibodies against adenosine.
The linkage between adenosine and the Tetanus toxin p30 helper
peptide was designed to be through PEG so that epitopes of
antibodies against adenosine can hardly contain the Tetanus toxin
p30 helper peptide. Adenosine is an ATP metabolite, and since the
phosphate groups of ATP are attached to the 5' hydroxyl group of
adenosine, antibodies that do not recognize the 5' hydroxyl group
of adenosine as an epitope may also bind to ATP in addition to
adenosine. That is, it would be easier to obtain antibodies that
can bind to both adenosine and ATP by using as an immunogen the
5'-Adenosine-PEG-Tetanus toxin p30 helper peptide, while it would
be easier to obtain antibodies that bind to adenosine but not to
ATP by using as an immunogen the 2'-Adenosine-PEG-Tetanus toxin p30
helper peptide. For this reason, the two types of immunogens which
contain the Tetanus toxin p30 helper peptide linked to the 2' or 5'
position of adenosine were prepared in the manner described in
(5-2). In addition, 2'-Adenosine-PEG-biotin (FIG. 12) and
5'-Adenosine-PEG-biotin (FIG. 13), in which biotin is conjugated
instead of the Tetanus toxin p30 helper peptide, were produced as
described below. By assessing the binding to these two types of
Adenosine-PEG-biotin, antibodies can be tested to demonstrate that
their epitopes do not contain the Tetanus toxin p30 helper
peptide.
(5-2) Synthesis of Immunogens to Prepare Adenosine-Binding
Library
[0717] 2'-Adenosine-PEG-peptide (adenosine 2'-PEG-peptide conjugate
or 2'-(PEG-peptide)adenosine) and 2'-Adenosine-PEG-biotin
(adenosine 2'-PEG-biotin conjugate or 2'-(PEG-biotin)adenosine)
were synthesized in the manner described below. The synthesized
2'-Adenosine-PEG-peptide and 2'-Adenosine-PEG-biotin were analyzed
or fractionated under the conditions below.
[0718] The conditions of LCMS analysis are noted as below.
TABLE-US-00040 TABLE 5 Column Analysis Column Flow rate temperature
condition Apparatus (length, mm) Mobile phase Gradient (A/B)
(ml/min) (.degree. C.) Wavelength SQDAA05 Acquity UPLC/SQD Aldrich
Ascentis Express A) 10 mM AcONH4, 95/5 => 0/100 1.0 35 210-400
nm C18 (2.1 .times. 50) H2O (1.0 min) => 0/100 PDA total B) MeOH
(0.4 min) SQDAA50 Acquity UPLC/SQD Aldrich Ascentis Express A) 10
mM AcONH4, 50/50 => 0/100 1.0 35 210-400 nm C18 (2.1 .times. 50)
H2O (0.7 min) => 0/100 PDA total B) MeOH (0.7 min) SQDFA05
Acquity UPLC/SQD Aldrich Ascentis Express A) 0.1% FA, H2O 95/5
=> 0/100 1.0 35 210-400 nm C18 (2.1 .times. 50) B) 0.1% FA CH3CN
(1.0 min => 0/100 PDA total (0.4 min) SQDFA50 Acquity UPLC/SQD
Aldrich Ascentis Express A) 0.1% FA, H2O 50/50 => 0/100 1.0 35
210-400 nm C18 (2.1 .times. 50) B) 0.1% FA CH3CN (0.7 min) =>
0/100 PDA total (0.7 min)
[0719] The conditions of preparative HPLC are described as
below.
TABLE-US-00041 TABLE 6 Column Preparative Column Flow rate
temperature condition Apparatus (length, mm) Mobile phase Gradient
(A/B) (ml/min) (.degree. C.) Wavelength A Preparative HPLC Aldrich
Ascentis RP- A) 0.1% FA H2O isocratic (A/B): 15/85 20.0 40 254, 258
nm system with Amide B) 0.1% FA MeCN injection/fractionation (21.2
.times. 150 mm 5 .mu.m) (Gilson, Inc.) B Preparative HPLC YMC Actus
ODS-A A) 20 mM AcONH4, isocratic (A/B): 47/53 20.0 40 254, 258 nm
system with (20 .times. 100 mm 5 .mu.m) H2O injection/fractionation
B) 20 mM AcONH4 (Gilson, Inc.) MeOH/MeCN (1/1)
(5-2-1) Synthesis of Compound 006 (Boc-Phe-Asn-Asn-Phe-Thr
(tBu)-Val-Ser (tBu)-Phe-Trp (Boc)-Lue-Arg (Pbf)-Val-Pro-Lys
(Boc)-Val-Ser (tBu)-Ala-Ser (tBu)-His (Trt)-Leu-Glu (tBu)-OH)
##STR00005##
[0720] Peptide synthesis was performed by the Fmoc method using a
peptide synthesizer (Multipep RS; Intavis). All Fmoc amino acids
were purchased from WATANABE CHEMICAL INDUSTRIES, LTD. The detailed
procedure of the treatment was in the manual attached to the
synthesizer.
[0721] Fmoc-Glu(tBu)-OH linked at its C terminus to 2-chlorotrityl
resin (250 mg/column, 30 columns, 11.7 mmol), an
N,N-dimethylformamide solution containing various Fmoc amino acids
(0.6 mol/l) and 1-hydroxy-7-azabenzotriazole (0.375 mol/l), and an
N,N-dimethylformamide solution (10% v/v) of diisopropylcarbodiimide
were loaded in the synthesizer. The synthesis reaction was
performed using as an Fmoc-deprotection solution, an
N,N-dimethylformamide solution (20% v/v) containing piperidine and
5% (wt/v) urea. After the resin was washed with
N,N-dimethylformamide, Fmoc deprotection was carried out, followed
by one cycle of Fmoc amino acid condensation reaction. This cycle
was repeated to elongate peptides on the resin surface. After
elongation, the resin was washed with trifluoroethanol. Peptides
were cleaved off from the resin by adding
trifluoroethanol/dichloromethane (=1/1). Thus, compound 006 (7.2 g)
was obtained as a crude product.
[0722] LCMS(ESI)m/z=1185(M+3H)3+
[0723] Retention time: 1.24 minute (Analysis condition,
SQDAA05)
(5-2-2) Synthesis of Compound 007
##STR00006##
[0725] A suspension of adenosine (2.00 g, 7.48 mmol) in
N,N-dimethylformamide (40 ml) was cooled down to 0.degree. C., and
60% sodium hydride (0.42 g, 10.48 mol) was added thereto. The
reaction mixture was stirred for one hour at 0.degree. C. After
adding methyl bromoacetate (0.76 ml, 8.01 mmol), the resulting
reaction mixture was stirred for five hours at room temperature,
and acetic acid (1 ml) and methanol (3 ml) were added thereto. The
reaction mixture was concentrated under reduced pressure. The
resulting residue was purified by normal phase silica gel column
chromatography (dichloromethane/methanol). Thus, compound 007 (0.93
g, 37%) was obtained.
[0726] LCMS(ESI) m/z=340(M+H)+
[0727] Retention time: 0.27 minute (Analysis condition,
SQDFA05)
(5-2-3) Synthesis of Compound 008
##STR00007##
[0729] t-Butyldimethylsilyl chloride (999 mg, 6.63 mol) and
imidazole (722 mg, 10.61 mol) were added to a pyridine solution (8
ml) of compound 007 (900 mg, 2.65 mmol). The reaction mixture was
stirred for four hours at room temperature, and extracted with
ethyl acetate/water. The extracted organic layer was washed with a
saturated sodium chloride solution, and dried over anhydrous sodium
sulfate. After filtration, the organic layer was concentrated under
reduced pressure. The resulting residue was purified by normal
phase silica gel column chromatography (dichloromethane/methanol).
Thus, compound 008 (1.17 g, 78%) was obtained.
[0730] LCMS(ESI)m/z=568(M+H)+
[0731] Retention time: 1.10 minute (Analysis condition,
SQDFA05)
(5-2-4) Synthesis of Compound 009
##STR00008##
[0733] Lithium hydroxide (61 mg, 2.55 mol) dissolved in water (0.17
ml) was added to a solution of compound 008 (290 mg, 0.511 mmol) in
methanol (0.34 ml)/tetrahydrofuran (0.34 ml). The reaction mixture
was stirred for 30 minutes at room temperature. The mixture was
neutralized with 1 M hydrochloric acid, and concentrated under
reduced pressure. The concentrated residue was extracted with ethyl
acetate/water. The resulting organic layer was washed with a
saturated sodium chloride solution, and dried over anhydrous sodium
sulfate. After filtration, the organic layer was concentrated under
a reduced pressure. Thus, compound 009 (319 mg, 90%) was
obtained.
[0734] LCMS(ESI)m/z=552(M-H)-
[0735] Retention time: 0.97 minute (Analysis condition,
SQDFA05)
(5-2-5) Synthesis of Compounds 010 and 011
##STR00009##
[0737] 1-Hydroxybenzotriazole (75 mg, 0.553 mol) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (106
mg, 0.553 mol) were added to an N,N-dimethylformamide (1.5 ml)
solution of compound 009 (255 mg, 0.460 mmol), and it was stirred
for three minutes at room temperature.
O-(2-aminoethyl)-O'-2-azidoethyl) nonaethylene glycol (291 mg,
0.553 mmol) was added to the reaction mixture, and it was stirred
for three hours at room temperature. The reaction mixture was
concentrated under a reduced pressure, and the resulting residue
was purified by reverse phase silica gel column chromatography
(aqueous 10 mM ammonium acetate solution/methanol. Compounds 010
(177 mg, 4%) and 011 (72 mg, 19%) were obtained.
Compound 010
[0738] LCMS(ESI)m/z=1063(M+H)+
[0739] Retention time: 0.98 minute (Analysis condition,
SQDFA05)
Compound 011
[0740] LCMS(ESI)m/z=949(M+H)+
[0741] Retention time: 0.67 minute (Analysis condition,
SQDFA05)
(5-2-6) Synthesis of Compound 012
##STR00010##
[0743] 10% palladium carbon (34 mg) was added to a solution of
compound 010 (170 mg, 0.160 mmol) in ethanol (1 ml). The reaction
mixture was stirred for two hours under hydrogen atmosphere, and
again 10% palladium carbon (34 mg) was added thereto. The reaction
mixture was stirred for two hours under a hydrogen atmosphere to
complete the reaction. The filtrate of the reaction solution was
concentrated under a reduced pressure. Compound 012 (34 mg, 95%)
was obtained.
[0744] LCMS(ESI)m/z=1037(M+H)+
[0745] Retention time: 0.70 minute (Analysis condition,
SQDFA05)
(5-2-7) Synthesis of Compounds 013 and 014
##STR00011## ##STR00012##
[0747] Compound 006 (354 mg, 0.110 mmol), 1-hydroxybenzotriazole
(13 mg, 0.100 mol), and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (19 mg, 0.100 mol) were added to a
solution of compound 012 (86 mg, 0.083 mmol) in
N,N-dimethylformamide (1.5 ml), and it was stirred for two hours at
room temperature. The filtrate of the reaction mixture was purified
by preparative condition A described in Table 6. A mixture of
compounds 013 and 014 (72 mg) was obtained.
Compound 013
[0748] LCMS(ESI)m/z=1525(M+3H)3+, 1144(M+4H)4+
[0749] Retention time: 1.13 minute (Analysis condition,
SQDAA50)
Compound 014
[0750] LCMS(ESI)m/z=1444(M+3H)3+, 1083(M+4H)4+
[0751] Retention time: 1.02 minute (Analysis condition,
SQDAA50)
(5-2-8) Synthesis of 2'-Adenosine-PEG-Peptide (Adenosine
2'-PEG-Peptide Conjugate or 2'-(PEG-Peptide)Adenosine) (Compound
015)
##STR00013##
[0753] Trifluoroacetic acid (16 ml), dichloromethane (8 ml), water
(1.3 ml), and tetraisopropylsilane (1.3 ml) were added to the
mixture of compounds 013 and 014 (42 mg), and it was stirred for
six hours at room temperature. The residue obtained by
concentrating the reaction mixture under reduced pressure was
purified by preparative condition B described in Table 6. Thus,
compound 015 (10 mg) was obtained.
[0754] LCMS(ESI)m/z=1090(M+3H)3+, 818(M+4H)4+
[0755] Retention time: 0.52 minute (Analysis condition,
SQDAA50)
(5-2-9) Synthesis of Compound 016
##STR00014##
[0757] 10% palladium carbon (34 mg) was added to a solution of
compound 011 (70 mg, 0.074 mmol) in ethanol (1 ml), and the
reaction mixture was stirred for five hours under hydrogen
atmosphere. The filtrate of the reaction mixture was concentrated
under reduced pressure. Thus, compound 016 (58 mg, 85%) was
obtained.
[0758] LCMS(ESI)m/z=923(M+H)+
[0759] Retention time: 0.50 minute (Analysis condition,
SQDFA05)
(5-2-10) Synthesis of Compound 017
##STR00015##
[0761] D-biotin N-succinimidyl (24 mg, 0.069 mmol) and
triethylamine (13 .mu.l, 0.094 mol) were added to a solution of
compound 016 (58 mg, 0.063 mmol) in N,N-dimethylformamide (1 ml),
and it was stirred for two hours at room temperature. Then, after
D-biotin N-succinimidyl (5 mg, 0.015 mmol) was added, the reaction
was completed upon 1.5 hours of stirring at room temperature. The
reaction mixture was purified by reverse phase silica gel column
chromatography (aqueous 10 mM ammonium acetate solution/methanol.
Compound 017 (50 mg, 69%) was obtained.
[0762] LCMS(ESI)m/z=1149(M+H)+
[0763] Retention time: 1.04 minute (Analysis condition,
SQDFA05)
(5-2-11) Synthesis of 2'-Adenosine-PEG-Biotin (Adenosine
2'-PEG-Biotin Conjugate or 2'-(PEG-Biotin)Adenosine) (Compound
018)
##STR00016##
[0765] A solution of 1 M tetra-n-butylammonium fluoride in
tetrahydrofuran (65 .mu.l, 0.065 mmol) was added to a solution of
compound 017 (62 mg, 0.054 mmol) in tetrahydrofuran (2 ml), and it
was stirred at room temperature for one hour. Then, 1 M
tetra-n-butylammonium fluoride in tetrahydrofuran solution (20
.mu.l, 0.020 mmol) was added, and the reaction was completed by
stirring at room temperature for one hour. The reaction mixture was
concentrated under a reduced pressure, and the residue was purified
by reverse phase silica gel column chromatography (aqueous 0.1%
formic acid solution/0.1% formic acid in acetonitrile). Compound
018 (12 mg, 21%) was obtained.
[0766] LCMS(ESI)m/z=1035(M+H)+
[0767] Retention time: 0.71 minute (Analysis condition,
SQDAA05)
[0768] Furthermore, 5'-Adenosine-PEG-peptide and
5'-Adenosine-PEG-biotin were also synthesized by the same
reaction.
(5-3) Production of Adenosine-Binding Antibodies in Animals and
Antibody Screening
[0769] Rabbits were immunized with 2'-Adenosine-PEG-peptide and/or
5'-Adenosine-PEG-peptide by a conventional method. Candidates for
cells with adenosine-binding activity were selected from
suspensions of cells collected from blood of the immunized rabbits,
by using autoMACS Pro Separator and FACSAria (BD) which uses
Adenosine-PEG-biotin-binding activity and rabbit IgG expression as
indicators. Then, screening was carried out with antibodies
secreted in the culture supernatants of the selected cells. In the
screening, ELISA was performed to assess the presence of binding
activity to Adenosine-PEG-biotin. ELISA was also performed to
assess whether adenosine, when added in combination with
Adenosine-PEG-biotin at a level 1000 times or more of that of
Adenosine-PEG-biotin, suppresses the binding to
Adenosine-PEG-biotin. The H-chain and L-chain variable regions were
isolated by PCR from cells selected using as an indicator the
presence of the Adenosine-PEG-biotin-binding activity as well as
suppression of the binding to Adenosine-PEG-biotin by adenosine
added in combination with Adenosine-PEG-biotin. The obtained
variable regions were expressed in combination with a human IgG1
heavy chain constant region and a human light chain constant
region.
(5-4) Acquisition of B Cells to Prepare Adenosine-Binding Immune
Library
[0770] Cells were collected from the spleens of rabbits immunized
with the 2'-Adenosine-PEG-Tetanus toxin peptide and
5'-Adenosine-PEG-Tetanus toxin peptide. From the cell suspensions,
candidates for cells with adenosine-binding activity were selected
using autoMACS Pro Separator and FACSAria (BD) with the presence of
binding to Adenosine-PEG-biotin as well as the expression of rabbit
IgG or IgM as indicators. The selected cells were washed with
PBS(-), and the prepared cell pellets were used to construct immune
libraries.
Example 6
Assessment of Clones Obtained by Rabbit B Cell Cloning
[0771] (6-1) Assessment of Clones Obtained by Rabbit B Cell Cloning
for their Binding Activity to 2'-Adenosine-PEG-Biotin
[0772] Clones obtained by rabbit B cell cloning were assessed for
their binding activity to adenosine by the SPR method.
Antigen-antibody reaction between the clones and
2'-Adenosine-PEG-Biotin was kinetically analyzed using Biacore 4000
(GE Healthcare). Sensor chip CM5 (GE Healthcare) was immobilized
with an appropriate amount of protein A/G (Invitrogen) by amine
coupling. Antibodies of interest were captured by the chip. Then,
after 100 nmol/12'-adenosine-PEG-Biotin was interacted as an
analyte for 60 seconds, the dissociation of the analyte was
monitored and measured for 60 seconds. The running buffer used was
HBS-P+(GE Healthcare). All measurements were carried out at
25.degree. C. The analyte was diluted using the running buffer.
[0773] The respective antibodies were compared for their binding
activity to 2'-Adenosine-PEG-Biotin using as an indicator the value
(N_binding.sub.--100) of dividing the amount of binding upon
interaction with 2'-Adenosine-PEG-Biotin by the amount of capture
(RU) for each antibody, and the value (N_stability.sub.--100) of
dividing the amount of dissociation of 2'-Adenosine-PEG-Biotin from
each antibody for 60 seconds after interaction with
2'-Adenosine-PEG-Biotin by the amount of capture (RU) for each
antibody. Regarding antibodies for which the amount of capture was
1500 RU or less, their binding was not sufficiently detectable and
thus they were excluded from the subjects to be tested. The result
is shown in FIG. 14. The result shown in FIG. 14 demonstrates that
the B cell cloning method yielded adenosine-binding clones with
various affinity.
(6-2) Assessment of 2'-Adenosine-PEG-Biotin-Binding Clones for
their Binding Activity to Adenosine and ATP, and Sequence Analysis
of the Clones
[0774] Clones which were demonstrated to bind to
2'-Adenosine-PEG-Biotin were assessed for their binding to
adenosine and ATP by SPR and competitive ELISA.
(6-2-1) Assessment by SPR of 2'-Adenosine-PEG-Biotin-Binding Clones
for their Binding to Adenosine and ATP
[0775] Using Biacore T200 (GE Healthcare), antibodies SMB0002,
SMB0089, and SMB0104 obtained by the B cell cloning method were
analyzed for their interaction with adenosine and ATP in
antigen-antibody reaction. Sensor chip CM5 (GE Healthcare) was
immobilized with an appropriate amount of protein A/G (Invitrogen)
by amine coupling. Antibodies of interest were captured by the chip
to allow interaction to adenosine or ATP as an antigen. The running
buffer used was 10 mmol/l ACES, 150 mmol/lNaCl, 0.05% (w/v)
Tween20, pH 7.4. All measurements were carried out at 25.degree. C.
The antigens were diluted using the running buffer.
[0776] Regarding SMB0002, SMB0089, and SMB0104, the diluted antigen
solutions and the running buffer as a blank were injected at a flow
rate of 20 .mu.l/min for two minutes to allow interaction of each
antigen with the antibodies captured on the sensor chip. Then, the
running buffer was injected at a flow rate of 20 .mu.l/min for
three minutes, and dissociation of the antigens from the antibodies
was observed. Next, 10 mmol/l glycine-HCl (pH 1.5) was injected at
a flow rate of 30 .mu.l/min for 30 seconds to regenerate the sensor
chip. The association rate constant ka (1/Ms) and dissociation rate
constant kd (1/s), both of which are kinetic parameters, were
calculated from the sensorgrams obtained by the measurement. The
dissociation constant KD (M) was calculated based on these
constants. Each parameter was calculated using the Biacore T200
Evaluation Software (GE Healthcare).
[0777] The result showed that multiple clones including SMB0002,
SMB0089, and SMB0104 bound to both adenosine and ATP. The
sensorgrams observed to assess each clone for its binding at
adenosine concentrations of 500, 125, 31.3, and 7.81 nM or at ATP
concentrations of 5000, 1250, 313, and 78.1 nM are shown in FIG.
15. As shown in FIG. 15, SMB0002, SMB0089, and SMB0104 were
demonstrated to bind to both adenosine and ATP. KDs of SMB0002,
SMB0089, and SMB0104 were 9.3E.sup.-9, 6.9E.sup.-9, 4.1E.sup.-8
(mol/l), and 1.0E.sup.-5 (mol/l) for adenosine, and 1.0E.sup.-5,
8.8E.sup.-7, and 1.4E.sup.-7 (mol/l) for ATP, respectively.
[0778] In the same manner using Biacore 4000 (GE Healthcare),
antibody SMB0171 obtained by B cell cloning was analyzed for its
interaction with adenosine and ATP in antigen-antibody reaction.
Sensor chip CM5 (GE Healthcare) was immobilized with an appropriate
amount of protein A/G (Invitrogen) by amine coupling. Antibodies of
interest were captured by the chip to allow interaction with
adenosine or ATP as an antigen. The running buffer used was
HBS-P+(GE Healthcare). All measurements were carried out at
25.degree. C. The antigens were diluted using the running
buffer.
[0779] Regarding SMB0171, diluted antigen solutions and the running
buffer as a blank were injected at a flow rate of 10 .mu.l/min for
one minute to allow interaction of each antigen with antibodies
captured on the sensor chip. Then, the running buffer was injected
at a flow rate of 10 .mu.l/min for three minutes, and dissociation
of the antibody from the antigens was observed. Then, 10 mmol/l
glycine-HCl (pH 1.5) was injected at a flow rate of 30 .mu.l/min
for 30 seconds to regenerate the sensor chip. The association rate
constant ka (1/Ms) and dissociation rate constant kd (1/s), both of
which are kinetic parameters, were calculated from the sensorgrams
obtained by measurement. The dissociation constant KD (M) was
calculated based on these constants. Each parameter was calculated
using the Biacore 4000 Evaluation Software (GE Healthcare).
[0780] The result showed that SMB0171 binds to ATP. The sensorgrams
observed in assessing each clone for its binding at ATP
concentrations of 50 and 5 .mu.M are shown in FIG. 16. As shown in
FIG. 16, SMB0171 was demonstrated to bind to ATP. KD of SMB0171 for
ATP was 5.9E.sup.-6 (mol/l).
(6-2-2) Assessment of 2'-Adenosine-PEG-Biotin-Binding Clones for
their Binding to Adenosine and ATP by Competitive ELISA
[0781] Antibodies demonstrated to bind to 2'-Adenosine-PEG-Biotin
were diluted to 1 .mu.g/ml with PBS, and added to each well of a
384-well MAXISorp (Nunc). To immobilize the antibodies, the plate
was allowed to stand for one hour or more at room temperature.
After the antibodies diluted with PBS were removed from each well,
TBS containing 1% BSA was added thereto and the plate was allowed
to stand for one hour or more. Then, the TBS (pH 7.4) containing 1%
BSA was removed from the plate. 2'-Adenosine-PEG-Biotin diluted to
50 nM with PBS, a mixture of 2'-Adenosine-PEG-Biotin and adenosine
diluted to 50 nM and 500 .mu.M respectively with PBS, a mixture of
2'-Adenosine-PEG-Biotin and ATP diluted to 50 nM and 500 .mu.M
respectively with PBS, or PBS alone was added to the plate. The
plate was allowed to stand at room temperature for one hour, and
then washed three times with 80 .mu.l of PBS containing 0.05%
Tween-20. Then, Streptavidin-HRP (Thermo fisher scientific) diluted
20000 times with PBS was added to each well, and the plate was
allowed to stand for one hour or more at room temperature. After
the plate was washed three times with 80 .mu.l of PBS containing
0.05% Tween-20, a chromogenic substrate (ABTS peroxidase substrate)
was added to each well. After the plate was incubated for one hour,
color development in the solution of each well was assessed by
measuring absorbance at 405 nm using SpectraMax from Molecular
Device.
[0782] As shown in FIG. 17, the result showed that the binding of
SMB0002 to 2'-Adenosine-PEG-Biotin was inhibited by adding excess
amounts of adenosine and ATP. Thus, the antibody clones were
demonstrated to bind not only to 2'-Adenosine-PEG-Biotin but also
to both adenosine and ATP.
(6-2-3) Sequence Analysis of Adenosine- and ATP-Binding Clones by
SPR
[0783] The amino acid sequences of clones demonstrated to bind to
both adenosine and ATP are shown in Table 7.
TABLE-US-00042 TABLE 7 Clone name Heavy chain SEQ ID NO Light chain
SEQ ID NO SMB0002 SEQ ID NO: 38 SEQ ID NO: 39 SMB0089 SEQ ID NO: 40
SEQ ID NO: 41 SMB0104 SEQ ID NO: 42 SEQ ID NO: 43 SMB0171 SEQ ID
NO: 44 SEQ ID NO: 45
Example 7
Acquisition of Antibodies that Bind to Adenosine and/or ATP from
Human Antibody Library Using Phage Display Techniques
[0784] (7-1) Construction of Phage-Display Library of Naive Human
Antibodies
[0785] A phage-display library of human antibodies consisting of
multiple phages that present the Fab domains of human antibodies
whose sequences were different from one another was constructed
using, as a template, polyA RNA prepared from human PBMC,
commercially available human polyA RNA, or such according to a
method known to those skilled in the art.
(7-2) Acquisition of Antibodies that Bind to Adenosine and/or ATP
from Library by Bead Panning
[0786] The phage-display library of naive human antibodies
constructed as described in (7-1) was screened for antibodies that
exhibit antigen-binding activity, specifically, by collecting
phages that display antibodies with binding activity to antigens
captured by beads. Biotinylated ATP, 2'-adenosine-PEG-Biotin, and
5'-adenosine-PEG-Biotin were used as antigens.
[0787] Phages produced in E. coli containing the phagemid vector
constructed for phage display were purified by a conventional
method, and then dialyzed against TBS to prepare a phage library
suspension. Then, BSA was added at a final concentration of 4% to
the suspension. Panning was performed using antigen-immobilized
magnetic beads. The magnetic beads used were NeutrAvidin coated
beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin
coated beads (Dynabeads M-280 Streptavidin).
[0788] Next, 250 pmol of biotinylated ATP, 2'-adenosine-PEG-Biotin,
and 5'-adenosine-PEG-Biotin were added to the prepared phage
library suspension. Thus, the phage library suspension was
contacted with adenosine and ATP for 60 minutes at room
temperature. Then, BSA-blocked magnetic beads were added to the
phage library suspension, and the complex of phage with adenosine
and/or ATP was allowed to bind to the magnetic beads at room
temperature for 15 minutes. The beads were washed once with TBS.
Then, the beads were combined with 0.5 ml of 1 mg/ml trypsin
solution. Immediately after the beads were suspended at room
temperature for 15 minutes, a phage suspension was collected from
the isolated beads using a magnetic stand. The collected phage
suspension was added to 10 ml of E. coli cells of strain ER2738 at
the logarithmic growth phase (OD600=0.4 to 0.7). The E. coli was
incubated at 37.degree. C. for one hour under gentle stirring to be
infected by phage. The infected E. coli was seeded in a 225
mm.times.225 mm plate. Then, phages were collected from the culture
medium of the seeded E. coli to prepare a liquid stock of phage
library.
[0789] A second round of panning was performed to also enrich
phages that are capable of binding to adenosine and/or ATP. The
prepared phage library suspension was contacted with adenosine and
ATP for 60 minutes at room temperature by adding 50 pmol each of
biotinylated ATP, 2'-Adenosine-PEG-Biotin, and
5'-Adenosine-PEG-Biotin. Then, the BSA-blocked magnetic beads were
added to the phage library suspension, and the complex of phage
with adenosine and/or ATP was allowed to bind to the magnetic beads
at room temperature for 15 minutes. The beads were washed three
times with TBST and twice with TBS. Then, the beads were combined
with 0.5 ml of 1 mg/ml trypsin solution. Immediately after the
beads were suspended at room temperature for 15 minutes, a phage
suspension was collected from the isolated beads using a magnetic
stand. The collected phage suspension was added to 10 ml of E. coli
cells of strain ER2738 at the logarithmic growth phase (OD600=0.4
to 0.7). The E. coli was incubated at 37.degree. C. for one hour
with gentle stirring to be infected by phage. The infected E. coli
was seeded in a 225 mm.times.225 mm plate. Then, phages were
collected from the culture medium of the seeded E. coli to prepare
a liquid stock of phage library.
[0790] By the same procedure, panning was performed three times to
obtain antibodies that are capable of binding to adenosine and/or
ATP. In the fourth round of panning, TBST wash and TBS wash were
each performed five times.
(7-3) Assessment of Adenosine- and ATP-Binding Activity by Phage
ELISA
[0791] From single colonies of E. coli obtained by panning as
described in the Example above, culture supernatants containing
phages were collected according to a conventional method (Method
Mol. Biol. (2002) 178, 133-145). The collected culture supernatants
were treated by ultrafiltration using NucleoFast 96
(MACHERY-NAGEL). 100 .mu.l of the culture supernatants were added
to each well of NucleoFast 96 and centrifuged at 4500 g for 45
minutes to remove flow through. After addition of 100 .mu.l of
H.sub.2O, the NucleoFast 96 was washed by centrifugation at 4500 g
for 30 minutes. After addition of 100 .mu.l of TBS, the NucleoFast
96 was allowed to stand for five minutes at room temperature. Then,
phage suspensions were collected from the supernatants.
[0792] Purified phages, to which TBS was added, were subjected to
ELISA by the following procedure. A StreptaWell 96 microtiter plate
(Roche) was coated at room temperature for one hour with 100 .mu.l
of TBS containing biotin-labeled antigens (a mixture of equal
amounts of 2'-adenosine-PEG-biotin, 5'-adenosine-PEG-biotin, and
ATP-PEG-biotin). After antigens were removed from each well of the
plate by washing with TBST (TBS containing 0.1% Tween20), the wells
were blocked with 250 .mu.l of 2% skim milk/TBS for one hour or
more. 2% skim milk/TBS was removed, and then the prepared, purified
phages were added to each well. The plate was allowed to stand at
room temperature for one hour to allow the phage-displayed antibody
to bind antigens in each well. After washing with TBST, an
HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech)
diluted with TBS was added to each well. The plate was incubated
for one hour. Following TBST wash, the TMB single solution (ZYMED)
was added to each well. The chromogenic reaction in the solution of
each well was terminated by adding sulfuric acid. Then, the
developed color was assessed by measuring absorbance at 450 nm.
[0793] From the 192 clones subjected to phage ELISA, 106 clones
that have the ability to bind to any one or two, or all three of
2'-Adenosine-PEG-biotin, 5'-Adenosine-PEG-biotin, and
ATP-PEG-biotin were obtained.
[0794] Next, for the purpose of confirming to which antigen of
2'-adenosine-PEG-biotin, 5'-adenosine-PEG-biotin, and
ATP-PEG-biotin these clones have binding ability, the purified
phages diluted with TBS were subjected to ELISA by the following
procedure. A StreptaWell 96 microtiter plate (Roche) was coated at
room temperature for one hour with 100 .mu.l of TBS containing a
biotin-labeled antigen (2'-adenosine-PEG-biotin,
5'-adenosine-PEG-biotin, or ATP-PEG-biotin). After the antigens
were removed by washing each well of the plate with TBST, the wells
were blocked with 250 .mu.l of 2% skim milk/TBS for one hour or
more. 2% skim milk/TBS was removed, and then the prepared, purified
phages were added to each well. The plate was allowed to stand at
room temperature for one hour to allow binding of
antibody-displaying phages to antigens in each well. After TBST
wash, an HRP-conjugated anti-M13 antibody (Amersham Pharmacia
Biotech) diluted with TBS was added to each well. The plate was
incubated for one hour. Following TBST wash, the TMB single
solution (ZYMED) was added to each well. The chromogenic reaction
in the solution of each well was terminated by adding sulfuric
acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The result of phage ELISA is shown in Table 8
below.
TABLE-US-00043 TABLE 8 Antigen binding ability Enrichment indicator
(S/N ratio > 1.5) Number of panning 4 Number of clones subjected
to ELISA 192 Number of Combination of 2'-Adenosine-PEG- 106 ELISA-
biotin, 5'-Adenosine-PEG-biotin, and positive ATP-PEG-biotin clones
2'-Adenosine-PEG-biotin 0 5'-Adenosine-PEG-biotin 6 ATP-PEG-biotin
76 Bind to two or more of 2'-Adenosine- 1 PEG-biotin,
5'-Adenosine-PEG-biotin, and ATP-PEG-biotin
[0795] Among the clones subjected to phage ELISA, a clone was
demonstrated to bind to two or more types of antigens. Its gene was
amplified with specific primers using the antibody fragment as a
template. The nucleotide sequence of the gene was analyzed. This
clone had the ability to bind to both 5'-Adenosine-PEG-biotin and
ATP-PEG-biotin, and was named ATNLSA1-4_D12. The heavy-chain
variable region sequence of antibody ATNLSA1-4_D12 is shown in SEQ
ID NO: 46, and its light-chain variable region sequence is shown in
SEQ ID NO: 47.
(7-4) Assessment of Adenosine- and ATP-Binding Activity by
Competitive Phage ELISA
[0796] Based on the structures of 5'-Adenosine-PEG-biotin and
ATP-PEG-biotin, there remained the possibility that clone
ATNLSA1-4_D12 (heavy chain variable region, SEQ ID NO: 46; light
chain, SEQ ID NO: 47), which was demonstrated by the result of
phage ELISA to have the ability to bind to both
5'-Adenosine-PEG-biotin and ATP-biotin, recognizes the biotin tag
or PEG moiety. Thus, to demonstrate that ATNLSA1-4_D12 is not an
antibody that recognizes the biotin tag or PEG, whether the antigen
binding is inhibited by adenosine or ATP was tested by phage ELISA
using ATNLSA1-4_D12, and IL-6R-binding clone PF1 (heavy chain, SEQ
ID NO: 48; light chain, SEQ ID NO: 49) prepared as a negative
control. ATNLSA1-4_D12 and PF1 were each diluted with TBS and
subjected to ELISA by the following procedure.
[0797] A StreptaWell 96 microtiter plate (Roche) was coated at room
temperature for one hour with 100 .mu.l of TBS containing
biotin-labeled antigens (a mixture of 5'-adenosine-PEG-biotin and
ATP-PEG-biotin). After the antigens were removed by washing each
well of the plate with TBST, the wells were blocked with 250 .mu.l
of 2% skim milk/TBS for one hour or more. 2% skim milk/TBS was
removed, and then the prepared, purified phages were added to each
well. The plate was allowed to stand at room temperature for one
hour to allow binding of the antibody-displaying phages to the
antigens in each well. Then, TBS that does not contain antigen or
that contains serial dilutions of ATP from an equal amount up to
10000 times that of the antigen was added to the wells. For the
competition of the immobilized antigen with ATP, the plate was
allowed to stand at room temperature for one hour. Then, after TBST
wash, an HRP-conjugated anti-M13 antibody (Amersham Pharmacia
Biotech) diluted with TBS was added to each well. The plate was
incubated for one hour. Following TBST wash, the TMB single
solution (ZYMED) was added to each well. The chromogenic reaction
in the solution of each well was terminated by adding sulfuric
acid. Then, the developed color was assessed by measuring
absorbance at 450 nm.
[0798] The measurement result is shown in FIG. 18. It was
demonstrated that the higher the ATP concentration, the smaller the
degree of color development for ATNLSA1-4_D12 in the presence of an
excess amount of ATP. Thus, the binding between ATNLSA1-4_D12 and
its antigen was demonstrated to be inhibited in an ATP
concentration-dependent manner. Meanwhile, in a control experiment
with PF1 as a negative control, its antigen binding was not
detected regardless of the ATP concentration. The above finding
demonstrates that ATNLSA1-4_D12 is an antibody that has the ability
to bind to ATP but does not recognize the biotin tag or PEG.
(7-5) Expression and Purification of Antibodies that Bind to ATP
and Adenosine
[0799] Using specific primers, genes were amplified from clone
ATNLSA1-4_D12 that had been assessed to have binding activity to
ATP and adenosine by the phage ELISA described in Example 7. The
nucleotide sequences of the genes were analyzed (the heavy-chain
and light-chain sequences are shown in SEQ ID NOs: 46 and 47,
respectively). The gene encoding the variable region of
ATNLSA1-4_D12 was inserted into an animal expression plasmid for
human IgG1/Lambda. The antibody was expressed using the method
described below. Cells of human fetal kidney cell-derived FreeStyle
293-F (Invitrogen) were suspended at a cell density of
1.33.times.10.sup.6 cells/ml in FreeStyle 293 Expression Medium
(Invitrogen) and aliquoted at 3 ml into each well of a 6-well
plate. The constructed plasmid was introduced into the cells by
lipofection. After four days of culture in a CO.sub.2 incubator
(37.degree. C., 8% CO.sub.2, 90 rpm), the antibody was purified
from the culture supernatants by a method known to those skilled in
the art using rProtein A Sepharose.TM. Fast Flow (Amersham
Biosciences). Absorbance of the purified antibody solutions was
measured at 280 nm using a spectrophotometer. From the values
obtained by the measurement, the concentration of the purified
antibody was calculated using an extinction coefficient determined
by the PACE method (Protein Science (1995) 4, 2411-2423).
(7-6) Assessment of the ATP- and Adenosine-Binding Antibody for its
ATP and Adenosine Binding by Surface Plasmon Resonance
[0800] Biacore T200 (GE Healthcare) was used to analyze the
interaction of D12, in which the constant region of IgG is linked
to the variable region of clone ATNLSA1-4_D12 with ATP- and
adenosine-binding activity, in antigen-antibody reaction. Sensor
chip CM5 or CM4 (GE Healthcare) was immobilized with an appropriate
amount of protein A (Life technologies) by amine coupling. The
antibody of interest was captured by the chip to allow interaction
with ATP (Wako), adenosine (Wako), or ADP (adenosine diphosphate)
(Wako) as an antigen. The running buffer used was 50 mM Tris-HCl
(Takara, T903), 500 mM NaCl, 0.01% (w/v) Tween20. The antigen was
allowed to interact for 30 seconds at a flow rate of 30 .mu.l/min,
and was dissociated for 30 seconds. The interaction with the
antigen was assessed at 15.degree. C. The antigen was diluted using
the same running buffer.
[0801] The dissociation constant K.sub.D (M) was calculated based
on the association rate constant ka (1/Ms) and dissociation rate
constant kd (1/s), both of which are kinetic parameters calculated
from the sensorgram obtained by the measurement. Alternatively, the
dissociation constant K.sub.D (M) was calculated using steady state
analysis. Each parameter was calculated using the Biacore T200
Evaluation Software (GE Healthcare).
[0802] To calculate the K.sub.D for adenosine, the binding response
was assessed at various concentrations of adenosine in the presence
or absence of 20 .mu.mol/l ADP. In addition, the binding response
was separately assessed in the presence of 20 .mu.mol/l ADP. The
response (R) for specific adenosine binding was obtained by
subtracting the value of binding response in the presence of ADP
alone from the binding response to various concentrations of
adenosine in the presence of ADP, and then subtracting the
resultant value, which is assumed to correspond to the non-specific
binding components, from the value of binding response to adenosine
in the absence of ADP. From a curve in which adenosine
concentration is plotted on the X axis and R calculated according
to Formula 2 is plotted on the Y axis, the value of K.sub.D for
adenosine was determined by the least squares method using the
Solver function of Office Excel 2007 (Microsoft).
R=Rmax.times.conc/(K.sub.D+conc) (Formula 2)
[0803] In Formula 2, conc represents adenosine concentration
(mol/l) while Rmax represents the value of response expected for
the maximal binding of adenosine to antibody. Measured response
values were extracted by using Scrubber2 (BioLogics. Inc).
[0804] The KD of D12 determined by the measurement described above
was 8.5 .mu.mol/l for ATP, 0.25 .mu.mol/l for ADP, or 1100
.mu.mol/l for adenosine. This result demonstrates that D12 has
binding activity to ATP, ADP, and adenosine; and it also suggests
that D12 has binding activity to AMP (adenosine monophosphate) and
cAMP (cyclic adenosine monophosphate).
Example 8
Design of Library Using Anti-ATP/Adenosine Antibodies to Prepare
ATP/Adenosine Switch Antibodies
[0805] In cancer tissues and inflamed tissues, not only the
adenosine but also the ATP concentration is known to be high. Thus,
it is beneficial to use antibodies for which both adenosine and ATP
(referred to as ATP/adenosine in this Example) can serve as a
switch (specifically, antibodies that can bind to antigens when
adenosine or ATP is present at a high concentration) as well as
antibodies for which either adenosine or ATP alone serves as a
switch. ATNLSA1-4_D12 described in Example 7-4 is an antibody that
binds to ATP/adenosine. As shown in FIG. 19, ATP/adenosine is
thought to be fit between the antibody and its target antigen, and
thus the antibody comprises an antibody variable region that comes
in contact with the target antigen. Thus, the present inventors
conceived that synthetic antibody libraries that can isolate
ATP/adenosine switch antibodies whose binding activity to arbitrary
antigens is altered depending on the presence of ATP/adenosine
could be constructed by collecting, as a library, antibody variable
region segments that are capable of establishing contact with a
target antigen and maintaining ATP/adenosine binding.
[0806] The crystal structure of the complex between ATP and
ATP/adenosine antibody ATNLSA1-4_D12 obtained from a human antibody
library as described in Example 7-4 was analyzed. The result of
crystal structure analysis revealed the mode of adenosine (or ATP)
recognition by the antibody as well as identification of amino acid
residues that are considered not to be substantially involved in
adenosine (or ATP) binding in the antibody variable region. Amino
acid residues that have been identified to be closely involved in
the adenosine (ATP) binding are Ser52, Ser52a, Arg53, Gly96,
Leu100a, and Trp100c (Kabat numbering) in the heavy chain.
[0807] In designing such a library, positions that meet at least
one of the conditions described below were selected as suitable for
the library construction.
[0808] Condition 1: a position that is not closely involved in ATP
binding, or if involved in the binding, a position having an amino
acid other than the wild-type sequence that does not inhibit the
ATP binding;
[0809] Condition 2: a position that is polymorphic to a certain
extent in terms of amino acid occurrence frequency; and
[0810] Condition 3: a position that is not essential for the
formation of canonical structure.
In regions contained in both heavy chain and light chain of the
ATNLSA1-4_D12 sequence and that meet the conditions described
above, amino acids in the CDR1 and CDR2 regions that have an
occurrence frequency of 2% or more in the germ line, as well as
amino acids in the CDR3 region that have an occurrence frequency of
1% or more in the germ line were comprehensively substituted. These
substitutions were combined to construct multiple variants of
ATNLSA1-4_D12.
[0811] Alteration sites in the heavy chain (in the Table, positions
indicated by "Kabat" according to Kabat numbering), as well as
amino acids before alteration (in the table, amino acids referred
to as "natural sequence") at the sites and amino acids after
alteration (in the table, amino acids referred to as "altered amino
acids") are shown in Table 9.
TABLE-US-00044 TABLE 9 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 57 58 96
97 99 100 100a Wild type sequence T Y N N I N G R G D L Altered A A
A A A A A A A amino C acid E E D D D D D D D G G G G F F F F I I H
H H H K K K K K M M L L N N N N N N N Q P S S S S S S S S S S R R R
R R T T T T T W W V V V V V Y Y Y Y Y Y Y
[0812] Alteration sites in the light chain (in the Table, positions
indicated by "Kabat" according to Kabat numbering), as well as
amino acids before alteration (in the table, amino acids referred
to as "natural sequence") at the sites and amino acids after
alteration (in the table, amino acids referred to as "altered amino
acids") are shown in Table 10.
TABLE-US-00045 TABLE 10 LCDR1 LCDR2 LCDR3 Kabat 26 27 27a 27b 27c
28 29 31 32 50 51 52 53 54 55 89 90 91 92 93 94 95a 96 97 Wild type
sequence T S S D V G G N Y E V S K R P S S Y A G S N V V Altered A
A A A A A A A A A A A A amino C acid E E E E E D D D D D D D D D D
D D D D D G G G G G G G G G F F F F F I I I I I I H H H H H K K K K
K K K K M M L L L L L L L N N N N N N N N N N N N Q Q Q Q Q Q P P P
S S S S S S S S S S S S R R R R R R R R T T T T T T T T T T T T T T
T W W W V V V V V V Y Y Y Y Y Y Y Y Y
[0813] Each variant expressed and purified by the method described
in Example 7-1 was assayed for its ATP and adenosine binding by the
same method as described in Example 7-6 using Biacore. Based on the
assay result, the affinity of each variant for ATP was calculated
as a KD value. Sites in the heavy chain, where alteration does not
reduce the ATP-binding ability to less than 1/5 of the binding
ability of ATNLSA1-4_D12 (specifically, where the KD value is lower
than 42.5 .mu.mol/l), and sites in the light chain where the
ATP-binding ability is larger than that of ATNLSA1-4_D12
(specifically, where the KD value is smaller than 8.5 .mu.mol/l),
were assessed to be plausible for alteration. Amino acids
substituted at those sites were judged to be appropriate for
inclusion in the library (flexible residues to be introduced into
library).
[0814] Based on the assessment result on the ATP-binding ability of
each variant, the ATP-binding ability was predicted to be reduced
by collecting each site to construct a library. Thus, substitutions
were introduced at sites close to positions that are expected to be
involved in ATP binding, and various variants resulting from
combination of these substitutions were comprehensively assessed to
test whether it is possible to identify alterations which are
expected to have effect of augmenting the ATP-binding ability. Such
alteration sites (positions indicated by "Kabat" according to Kabat
numbering in the Table), and amino acids before alteration (amino
acids referred to as "wild type sequence" in the table) and amino
acids after alteration (amino acids referred to as "altered amino
acids" in the table) at the sites are shown in Table 11.
TABLE-US-00046 TABLE 11 HCDR1 HCDR2 HCDR3 LCDR3 Kabat 33 50 56 95
98 100b 95 WiLd type sequence T S Y F K N N Altered A A A A A A A A
amino C acid E E E E E E E D D D D D D D G G G G G G G F F F F F F
F I I I I I I I I H H H H H H H K K K K K K K M M M M M M M M L L L
L L L L L N N N N Q Q Q Q Q Q Q P P P P P P P S S S S S S R R R R R
R R T T T T T T W W W W W W W W V V V V V V V V Y Y Y Y Y Y Y
[0815] Each variant expressed and purified by the method described
in Example 7-1 was assayed for its ATP and adenosine binding by the
same assay method using Biacore as described in Example 7-6. The
assay result showed that the ATP and adenosine binding was expected
to be augmented by alterations at positions 56 and 100 and such
according to Kabat numbering (for example, amino acid alteration
such as Tyr56His and Asn100bLeu). It was determined that amino
acids substituted at the sites could be included in a library
(flexible residues to be introduced into a library).
[0816] In the CDR regions of ATNLSA1-4_D12, amino acid repertoires
containing amino acids selected by the above-described variant
analysis as suitable to be included in a library (flexible amino
acid residues to be introduced into a library) and amino acids
before alteration of the amino acids (specifically, amino acids
included in the natural sequence of ATNLSA1-4_D12), and sites
comprising such repertoires were designed to construct a library
for preparation of ATP/adenosine switch antibodies. The library was
constructed in such a manner that in an amino acid repertoire the
amino acid occurrence frequency is the same for every amino acid
(for example, when there are ten types of amino acids in an amino
acid repertoire, each amino acid occurs at 10%,).
[0817] Sites comprising amino acid repertoires in the heavy chain
(positions indicated by "Kabat" according to Kabat numbering in the
Table) and amino acid repertoires at the sites are shown in Table
12. Sites comprising amino acid repertoires in the light chain
(positions indicated by "Kabat" according to Kabat numbering in the
Table) and amino acid repertoires at the sites are shown in Table
13.
TABLE-US-00047 TABLE 12 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 56 57
58 59 95 97 98 99 100 100a 100b Wild type sequence T Y N N Y I N Y
F R K G D L N Altered A 17% 25% 11% 5% amino C acid E 9% 5% D 13%
9% 5% G 33% 17% 13% 11% 9% 5% F 33% 13% 50% 9% 5% I 25% 11% 5% H
33% 50% 50% 13% 11% 9% 5% 17% K 25% 11% 33% 9% 5% M 11% 5% 17% L
50% 11% 33% 5% 17% 50% N 50% 17% 13% 9% 5% 50% Q 9% 5% P 5% S 33%
17% 13% 5% R 17% 25% 11% 33% 9% 5% 17% T 33% 17% 13% 5% W 9% 5% 17%
V 11% 5% Y 33% 50% 13% 50% 50% 9% 5% 17%
TABLE-US-00048 TABLE 13 LCDR1 LCDR2 LCDR3 Kabat 27a 29 50 51 54 90
91 92 93 94 95 95a 96 97 Wild type sequence S S E V R S Y A G S N N
V V Altered A 17% 17% 14% 13% 6% 17% amino C acid E 14% 17% 6% D
17% 14% 14% 13% 6% 11% G 17% 14% 25% 14% 13% 6% 11% 17% F 17% 6% I
14% 13% 6% 11% H 6% 11% K 14% 50% 14% 6% M 6% 17% L 25% 6% 11% 33%
17% N 25% 14% 13% 6% 11% Q 14% 6% 11% P 6% 33% S 50% 17% 14% 25%
17% 14% 13% 6% 11% 17% R 50% 14% 17% 13% 6% T 50% 17% 25% 25% 14%
17% 14% 13% 6% W 14% 6% V 25% 25% 14% 33% 17% Y 14% 14% 17% 14% 6%
11%
[0818] The result of sequence analysis suggests that the framework
of ATNLSA1-4_D12 was derived from germ line VH3-21. Then, for the
purpose of improving antibody stability, the framework sequence of
ATNLSA1-4_D12 was restored to the germ line sequence VH3-21 by
introducing into the framework sequence of ATNLSA1-4_D12,
alterations Gln01Glu, Gln05Val, Asp10Gly, Asn30Ser, Leu48Val, and
Asn58Tyr (numerals represent Kabat numbers). ATNLSA1-4_D12 variants
expressed and purified by the method described in Example 7-1 were
measured for their Tm by DSC. DSC measurement was carried out by a
method known to those skilled in the art. Tm of the variant which
results from adding these alterations to ATNLSA1-4_D12 was markedly
improved from 74.37.degree. C. to 81.44.degree. C., and
stabilization of the structure was observed. It is sometimes
preferable to use highly stable frameworks for antibody libraries,
and thus a framework sequence to which alterations described above
had been added was used as the framework sequence of a library. The
framework used for the library is shown in Table 14.
TABLE-US-00049 TABLE 14 Framework SEQ ID NO: Sequence Heavy chain
framework 1 56 EVQLVESGGGLVKPGGFLRLSCAASGFTFS Heavy chain framework
2 57 WVRQAPGKGLEWVS Heavy chain framework 3 58
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR Heavy chain framework 4 59
WGQGTLVTVSS Light chain framework 1 60 QSALTQPPSASGSPGQTVTISC Light
chain framework 2 61 SWYQQHPGKAPKLMIY Light chain framework 3 62
GVPDRFSGSKSGNTASLTVSGLQAEDEADYFC Light chain framework 4 63
FGGGTKLTVL
[0819] Genes were synthesized to comprise respective sequences in a
library designed as described above (DNA2.0), and the gene library
was amplified with primers that are capable of amplifying VH and VL
respectively, by using a collection (library) of the respective
genes as a template. The sequences of primers used for VL
amplification are shown in SEQ ID NOs: 102 and 103, while the
sequences of primers used for VH amplification are shown in SEQ ID
NOs: 104 and 105. The amplified rationally designed gene library of
the heavy-chain and light-chain variable regions of human antibody
was inserted into an appropriate phagemid vector carrying both a
human IgG-derived CH1 sequence and a human IgG-derived light chain
constant region sequence. The phagemid vector was introduced into
E. coli by electroporation to construct a rationally designed
library which presents Fab domains containing a human antibody
variable region-constant region, and from which one can isolate
antibodies that are capable of binding to antigens via adenosine or
ATP as a switch. Such a rationally designed library which is
constituted with various H chains and L chains that have adenosine-
or ATP-binding activity is expected to be useful as a library
containing human antibodies that, with the adenosine (or ATP) is
fit in between antibody and antigen as shown in FIG. 19, can
efficiently obtain adenosine/ATP switch antibodies against any
arbitrary antigen. Furthermore, as described above, since
ATNLSA1-4_D12 binds not only to adenosine and ATP but also to ADP,
it was predicted to have binding activity to AMP and cAMP which are
structurally similar to ATP, ADP, and adenosine. This suggests that
such libraries are useful for isolating switch antibodies whose
binding activity to arbitrary target antigens is altered depending
on the presence of any one or more small molecules of ATP, ADP,
AMP, cAMP, and adenosine.
Example 9
Construction of an Immune Library to Obtain Adenosine/ATP/Adenosine
Switch Antibodies Containing Anti-ATP/Adenosine Antibody
Repertoires
[0820] Multiple phage-display libraries of rabbit antibodies which
present Fab domains comprising rabbit antibody sequences were
constructed using as a template mRNA collected from a B cell
population selected using MACS and FACS as described in Example
5-4, which expresses adenosine-PEG-biotin-binding antibodies. The
construction method was carried out by referring to Rader (Methods
Mol. Biol. (2009) 525, 101-28).
[0821] More specifically, cDNA was prepared by reverse
transcription using as a template mRNA collected from 600,000 cells
of the above-described B cells which were selected from nine
immunized rabbits. Using the cDNA as a template, the heavy chain
variable region sequence and the light chain variable
region-constant region sequence were amplified by PCR using the
primers shown in Table 15 under adequate conditions.
TABLE-US-00050 TABLE 15 SEQ Primer name ID NO: Sequence primer 1 64
TATTACTCGCGGCCCAGCCGGCCATGGCAGCC WTCGANWTGACCCAGACT primer 2 65
TATTACTCGCGGCCCAGCCGGCCATGGCAGCC TATGATNTGACCCAGACT primer 3 66
TATTACTCGCGGCCCAGCCGGCCATGGCAGCB CAAGTGCTGACCCAGACT primer 4 67
TATTACTCGCGGCCCAGCCGGCCATGGCAGCC MTYGTGATGACCCAGACT primer 5 68
TATTACTCGCGGCCCAGCCGGCCATGGCAGCC GCCGTGCTGACCCAGACT primer 6 69
TATTACTCGCGGCCCAGCCGGCCATGGCGGCT GACATTGTGATGACCCAG primer 7 70
TATTACTCGCGGCCCAGCCGGCCATGGCCGCC GAYRTYGTGATGACCCAG primer 8 71
CTCTTCTAGAACGCGTCTAAGCGTCACCCCTA TTGAAGCTC primer 9 72
TATTACTCGCGGCCCAGCCGGCCATGGCGCAG CYYGTGCTGACTCAGTCGCCCTC primer 10
73 CTCTTCTAGAACGCGTCTAAGCTTCTGCAGGG GCCAGGCTCTTC primer 11 74
TTCCGCCTCGGCGCTAGCCCAGGAGCAGSTGG WGGAGTCC primer 12 75
TTCCGCCTCGGCGCTAGCCCAGTCNNTGGAGG AGTCCGGG primer 13 76
TTCCGCCTCGGCGCTAGCCCAGTCGNNGGAGG AGTCCGGG primer 14 77
TTCCGCCTCGGCGCTAGCCCAGCAGCAGCTGG WGGAGTCC
[0822] A combination of an amplified library of rabbit antibody
heavy chain variable region genes and a library of rabbit antibody
light chain variable region-constant region genes was inserted into
an appropriate phagemid vector carrying a rabbit IgG-derived CH1
sequence. The phagemid vector was introduced into E. coli by
electroporation to construct a phage-display library of rabbit
antibodies (hereinafter, an antibody library from
adenosine-immunized rabbits) which presents Fab domains containing
a rabbit antibody variable region-constant region, and from which
one can isolate antibodies that are capable of binding to antigens
via adenosine or ATP as a switch. Such an adenosine immune library
which is constituted by various H chains and L chains that exhibit
adenosine binding property is expected to be useful as an immune
library, with adenosine (or ATP) is sandwiched in between antibody
and antigen as shown in FIG. 20, that can isolate adenosine/ATP
switch antibodies against any arbitrary antigen.
Example 10
Acquisition of Antibodies that Bind to Antigens in the Presence of
Adenosine and ATP from Antibody Library Using Phage Display
Techniques
[0823] (10-1) Acquisition of Antibodies that Bind to Antigens in
the Presence of Small Molecules from Library Using a Mixture of
Adenosine and ATP
[0824] Antibodies that exhibit antigen-binding activity in the
presence of adenosine and/or ATP were obtained from the constructed
phage-display library of antibodies from adenosine-immunized
rabbits and the phage-display library of rationally designed
antibodies. To obtain antibodies, phages displaying antibodies that
exhibit the ability to bind to antigens captured by beads in the
presence of adenosine and ATP were collected, and then the phages
were collected in eluate from the beads in the absence of adenosine
and ATP.
[0825] Phages were produced in E. coli containing the phagemid
vector constructed for phage display. To the culture medium of E.
coli in which phage production was carried out, 2.5 M NaCl/10% PEG
was added to precipitate phages. The precipitated phage fraction
was diluted with TBS to prepare a library suspension. Then, BSA was
added at a final concentration of 4% to the phage library
suspension. Panning was performed using antigen-immobilized
magnetic beads. The magnetic beads used were NeutrAvidin coated
beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin
coated beads (Dynabeads M-280 Streptavidin).
[0826] 500 pmol of biotin-labeled antigen and a final concentration
of 1 mM ATP-Na and adenosine were each added to the prepared phage
library suspension. The phage library suspension was contacted with
the antigen, adenosine, and ATP at room temperature for 60 minutes.
The BSA-blocked magnetic beads were added to the phage library
suspension, and the antigen-phage complex was allowed to bind to
the magnetic beads at room temperature for 15 minutes. The beads
were washed once with ATP- and adenosine-dissolved TBS. Then, the
beads were combined with 0.5 ml of 1 mg/ml trypsin. Immediately
after the beads at room temperature were suspended for 15 minutes,
a phage suspension was collected from the isolated beads using a
magnetic stand. The collected phage suspension was added to 10 ml
of E. coli cells of strain ER2738 at the logarithmic growth phase
(OD600=0.4 to 0.7). The E. coli was incubated at 37.degree. C. with
gentle stirring for one hour to be infected by phage. The infected
E. coli was seeded in a 225 mm.times.225 mm plate. Then, phages
were collected from the culture medium of the seeded E. coli to
prepare a liquid stock of phage library.
[0827] The first round of panning was carried out to collect phages
that are capable of antigen binding in the presence of adenosine
and ATP, while the second and subsequent rounds of panning were
performed to enrich phages that are capable of antigen binding only
in the presence of adenosine and ATP. Specifically, 40 pmol of
biotin-labeled antigen and a final concentration of 1 mM adenosine
and ATP were each added to the prepared phage library suspension.
Thus, the phage library was contacted with antigen, adenosine, and
ATP for 60 minutes at room temperature. BSA-blocked magnetic beads
were added, and the antigen-phage complex was allowed to bind to
the magnetic beads for 15 minutes at room temperature. The beads
were washed with 1 ml of adenosine and ATP-dissolved TBST
(hereinafter referred to as (adenosine+ATP)/TBST), adenosine, and
adenosine and ATP-dissolved TBS (hereinafter referred to as
(adenosine+ATP)/TBS). Then, the beads were combined with 0.5 ml of
TBS. Immediately after the beads were suspended at room
temperature, a phage suspension was collected from the isolated
beads using a magnetic stand. After this treatment was repeated,
the two separately eluted phage suspensions were combined together.
The pIII protein (helper phage-derived protein pIII) that does not
display Fab was cleaved off from phages by adding 5 .mu.l of 100
mg/ml trypsin to the collected phage suspension to eliminate the
ability of phages that do not display Fab to infect E. coli. The
phages collected from the trypsinized phage suspension were added
to 10 ml of E. coli cells of strain ER2738 at the logarithmic
growth phase (OD600=0.4 to 0.7). The E. coli was incubated at
37.degree. C. with gentle stirring for one hour to be infected by
phage. The infected E. coli was seeded in a 225 mm.times.225 mm
plate. Then, phages were collected from the culture medium of the
seeded E. coli to prepare a phage library suspension. Panning was
performed three times to isolate antibodies that have
antigen-binding activity in the presence of adenosine and ATP.
(10-2) Acquisition of Antibodies that Bind to Antigens in the
Presence of Adenosine and ATP from Antibody Library Using a
Negative Selection Method
[0828] A phage-display library of antibodies constructed from
rabbits immunized with adenosine or a phage-display library of
rationally designed antibodies was screened for antibodies that
exhibit antigen-binding activity in the presence of adenosine
and/or ATP. As a first step of screening, the phage-display
antibody library was contacted with biotin-labeled
antigen-streptavidin in the absence of adenosine and ATP to
eliminate phages displaying antibodies that have antigen-binding
activity even in the absence of adenosine and ATP. Then, panning
was performed in the same manner in the presence of adenosine and
ATP to screen for antibodies that exhibit antigen-binding activity
in the presence of adenosine and ATP.
[0829] Phages were produced in E. coli containing the constructed
phage-display phagemid. To the culture medium of E. coli in which
phage production took place, 2.5 M NaCl/10% PEG was added to
precipitate phages. The precipitated phage fraction was diluted
with TBS to prepare a library suspension. Then, BSA was added at a
final concentration of 4% to the phage library suspension. Panning
was performed using antigen-immobilized magnetic beads. The
magnetic beads used were NeutrAvidin coated beads (Sera-Mag
SpeedBeads NeutrAvidin-coated) and Streptavidin coated beads
(Dynabeads M-280 Streptavidin).
[0830] Together with 250 pmol of biotin-labeled antigen, a mixture
of adenosine and ATP was added at a final concentration of 1 mM to
the prepared phage library suspension. Thus, the phage library
suspension was contacted with the antigen, adenosine, and ATP for
60 minutes at room temperature. Then, BSA-blocked magnetic beads
were added to the phage library suspension, and allowed to bind to
the antigen-phage complex at room temperature for 15 minutes. The
beads were washed once with (adenosine+ATP)/TBS. Then, the beads
were combined with 0.5 ml of 1 mg/ml trypsin solution. Immediately
after the beads were suspended at room temperature for 15 minutes,
a phage suspension was collected from the isolated beads using a
magnetic stand. The collected phage suspension was added to 10 ml
of E. coli cells of strain ER2738 at the logarithmic growth phase
(OD600=0.4 to 0.7). The E. coli was incubated at 37.degree. C. with
gentle stirring for one hour to be infected by phage. The infected
E. coli was seeded in a 225 mm.times.225 mm plate. Then, phages
were collected from the culture medium of the seeded E. coli to
prepare a liquid stock of phage library.
[0831] The first round of panning was carried out to collect phages
that are capable of binding in the presence of adenosine and ATP,
while the second and subsequent rounds of panning were performed to
enrich phages that are capable of antigen binding only in the
presence of adenosine and ATP. Specifically, 250 pmol of
biotinylated antigen was added to BSA-blocked Sera-Mag NeutrAvidin
beads, and allowed to bind at room temperature for 15 minutes. The
beads were washed three times with TBS. The phage library
suspension subjected to BSA blocking was added to the washed beads,
and allowed to bind at room temperature for one hour. Phages that
did not bind to the antigens or beads were collected by isolating
the beads using a magnetic stand. Forty pmol of biotin-labeled
antigen, and a final concentration of 1 mM adenosine and ATP were
each added to the collected phages. Thus, the phage library was
contacted with the antigen, adenosine, and ATP for 60 minutes at
room temperature. Then, BSA-blocked magnetic beads were added to
the mixture of the labeled antigen, adenosine, ATP, and phage
library, and allowed to bind to the antigen-phage complex for 15
minutes at room temperature. The beads were washed with 1 ml of
(adenosine+ATP)/TBST and (adenosine+ATP)/TBS. Then, 0.5 ml of 1
mg/ml trypsin solution was added to the mixture. After the mixed
suspension was stirred at room temperature for 20 minutes, phages
were collected from the beads that had been separated using a
magnetic stand. The collected phages were added to 10 ml of E. coli
cells of strain ER2738 at the logarithmic growth phase (OD600=0.4
to 0.7). The E. coli was incubated at 37.degree. C. with gentle
stirring for one hour to be infected by phage. The infected E. coli
was seeded in a 225 mm.times.225 mm plate. Panning was performed
three times to isolate antibodies that have antigen-binding
activity in the presence of adenosine and ATP.
(10-3) Acquisition of Antibodies that Bind to Antigens in the
Presence of Adenosine and ATP from Antibody Library Using an
Alternating Panning Method
[0832] A phage-display antibody library constructed from rabbits
immunized with adenosine or a phage-display library of rationally
designed antibodies is screened for antibodies that exhibit
antigen-binding activity in the presence of adenosine and/or ATP.
As a first step of screening, the phage-display antibody library is
contacted with biotinylated adenosine and ATP-NeutrAvidin in the
presence of non-labeled antigens to collect a phage-display library
of antibodies that bind to adenosine and/or ATP in the presence of
the antigen. Then, the phage-display antibody library is contacted
with biotinylated antigen-streptavidin in the presence of adenosine
and ATP to collect antibodies that bind to the antigen in the
presence of adenosine and ATP. Thus, screening is carried out for
antibodies that have antigen-binding activity in the presence of
adenosine and ATP by performing panning in the alternating manner
described above.
[0833] Phages are produced in E. coli containing the phagemid
vector constructed for phage display. To the culture medium of E.
coli in which phage production is took place, 2.5 M NaCl/10% PEG is
added to precipitate phages. The precipitated phage fraction is
diluted with TBS to prepare a library suspension. Then, BSA is
added at a final concentration of 4% to the phage library
suspension. Panning is performed using antigen-immobilized magnetic
beads. The magnetic beads used are NeutrAvidin coated beads
(Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin coated
beads (Dynabeads M-280 Streptavidin).
[0834] Together with 1000 pmol of non-labeled antigen, 250 pmol of
biotinylated ATP, 2'-Adenosine-PEG-Biotin, and
5'-Adenosine-PEG-Biotin are added to the prepared phage library
suspension. Thus, the phage library suspension is contacted with
the antigen, adenosine, and ATP at room temperature for 60 minutes.
Then, BSA-blocked magnetic beads are added to the phage library
suspension, and the complex of phage with the antigen, and
adenosine and/or ATP is allowed to bind to the magnetic beads at
room temperature for 15 minutes. The beads are washed once with TBS
containing 1000 pmol of the antigen. Then, the beads are combined
with 0.5 ml of 1 mg/ml trypsin solution. Immediately after the
beads are suspended at room temperature for 15 minutes, a phage
suspension is collected from the isolated beads using a magnetic
stand. The collected phage suspension is added to 10 ml of E. coli
cells of strain ER2738 at the logarithmic growth phase (OD600=0.4
to 0.7). The E. coli is incubated at 37.degree. C. with gentle
stirring for one hour to be infected by phage. The infected E. coli
are plated onto a 225 mm.times.225 mm plate. Then, phages are
collected from the culture medium of the seeded E. coli to prepare
a phage library suspension.
[0835] A second round of panning is performed to enrich phages that
are capable of binding to the biotinylated antigen in the presence
of adenosine and ATP. Specifically, 40 pmol of biotinylated antigen
and a final concentration of 1 mM adenosine and ATP are added to
the prepared phage library solution. Thus, the phage library
suspension is contacted with the antigen, as well as adenosine and
ATP for 60 minutes at room temperature. Then, BSA-blocked magnetic
beads are added to the phage library solution, and the complex of
phage with the antigen as well as adenosine and/or ATP is allowed
to bind to the magnetic beads at room temperature for 15 minutes.
The beads are washed three times with TBST containing adenosine and
ATP at a final concentration of 1 mM, and twice with TBS containing
adenosine and ATP at a final concentration of 1 mM. Then, the beads
are combined with 0.5 ml of 1 mg/ml trypsin solution. Immediately
after the beads are suspended at room temperature for 15 minutes, a
phage solution is collected from the isolated beads using a
magnetic stand. The collected phage solution is added to 10 ml of
E. coli cells of strain ER2738 at the logarithmic growth phase
(OD600=0.4 to 0.7). The E. coli is incubated at 37.degree. C. with
gentle stirring for one hour to be infected by phage. The infected
E. coli is seeded in a 225 mm.times.225 mm plate. Then, phages are
collected from the culture medium of the seeded E. coli to prepare
a phage library solution.
[0836] At subsequent even-numbered rounds, panning is performed in
the same manner as the second-round panning. However, in the fourth
and subsequent rounds of panning, the number of bead washes with
(adenosine+ATP)/TBST and (adenosine+ATP)/TBS are both increased to
five times.
[0837] A third round of panning is performed to also enrich phages
that are capable of binding to biotinylated adenosine and ATP in
the presence of the antigen. Specifically, together with 250 pmol
of biotinylated ATP, 2'-adenosine-PEG-Biotin and
5'-adenosine-PEG-Biotin, 1000 pmol of non-labeled antigen is added
to the prepared phage library solution. Thus, the phage library
solution is contacted with the antigen, as well as adenosine and
ATP for 60 minutes at room temperature. Then, BSA-blocked magnetic
beads are added to the phage library solution, and the complex of
phage with the antigen as well as adenosine and/or ATP is allowed
to bind to the magnetic beads at room temperature for 15 minutes.
The beads are washed three times with TBST containing 1000 pmol of
the antigen, and twice with TBS containing 1000 pmol of the
antigen. Then, the beads are combined with 0.5 ml of a 1 mg/ml
trypsin solution. Immediately after the beads are suspended at room
temperature for 15 minutes, a phage solution is collected from the
isolated beads using a magnetic stand. The collected phage solution
is added to 10 ml of E. coli cells of strain ER2738 at the
logarithmic growth phase (OD600=0.4 to 0.7). The E. coli is
incubated at 37.degree. C. with gentle stirring for one hour to be
infected by phage. The infected E. coli is seeded in a 225
mm.times.225 mm plate. Then, phages are collected from the culture
medium of the seeded E. coli to prepare a phage library
solution.
[0838] At subsequent odd-numbered rounds, panning is performed in
the same manner as the third-round panning. However, in the fourth
and subsequent rounds of panning, the number of bead washes with
TBST containing the antigen and TBS containing the antigen is both
increased to five times. Alternatively, regardless of whether it is
an odd- or even-numbered round, panning in the third and subsequent
rounds is always performed in the same manner as the third-round
panning. However, in the fourth and subsequent rounds of panning,
the number of bead washes with TBST containing antigen and TBS
containing antigen is both increased to five times.
(10-4) Assessment of Binding Activity in the Presence or Absence of
Adenosine and/or ATP by Phage ELISA
[0839] Phage-containing culture supernatants were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained by the method
described above. The collected culture supernatants were treated by
ultrafiltration using NucleoFast 96 (MACHERY-NAGEL). 100 .mu.l of
the culture supernatants were added to each well of NucleoFast 96,
and it was centrifuged (4500 g for 45 minutes) to remove flow
through. After addition of 100 .mu.l of H.sub.2O, the NucleoFast 96
was washed by centrifugation (4500 g for 30 minutes). Finally, 100
.mu.l of TBS was added, and the NucleoFast 96 was allowed to stand
for five minutes at room temperature. A phage suspension was
collected from the supernatant in each well of the NucleoFast
96.
[0840] The purified phages, to which TBS or (adenosine+ATP)/TBS was
added, were subjected to ELISA by the following procedure. A
StreptaWell 96 microtiter plate (Roche) was coated overnight with
100 .mu.l of TBS containing biotin-labeled antigen. After the
antigen was removed by washing each well of the plate with TBST,
the wells were blocked with 250 .mu.l of 2% skim milk/TBS for one
hour or more. 2% skim milk/TBS was removed, and then the prepared,
purified phages were added to each well. The plate was allowed to
stand at 37.degree. C. for one hour to allow binding of
antibody-displaying phages to the antigen in each well in the
presence or absence of adenosine and/or ATP. After washing with
TBST or (adenosine+ATP)/TBST, HRP-conjugated anti-M13 antibody
(Amersham Pharmacia Biotech) diluted with TBS or
(adenosine+ATP)/TBS was added to each well. The plate was incubated
for one hour. Following washes with TBST or (adenosine+ATP)/TBST,
the TMB single solution (ZYMED) was added to each well. The
chromogenic reaction in the solution of each well was terminated by
adding sulfuric acid. Then, the developed color was assessed by
measuring absorbance at 450 nm. The result revealed that for three
types of antigens: human IL6, human IL6 receptor, and HAS (human
serum albumin), there were multiple types of antibodies bound in
the presence of small molecules. Antibodies that bind in the
presence of ATP were also obtained from the human naive antibody
library. Meanwhile, switch antibodies to human IL6, human IL6
receptor, and HAS could be obtained with greater efficiency. The
result of phage ELISA is shown in Table 16.
TABLE-US-00051 TABLE 16 human IL6 human IL6R HSA Number of panning
4 3 4 4 Number of clones subjected 96 96 96 96 to ELISA Number of
positive clones 35 23 64 52 (S/N ratio > 10) Nutter of dependent
clones 18 22 64 50 (SM +/- ratio > 2) Number of dependent clone
2 17 35 5 sequences
(10-5) Assessment for Binding Ability of Switch Antibodies Whose
Antigen-Binding Activity is Altered Depending on the Presence of
Adenosine and ATP, and Sequence Analysis
[0841] Genes were amplified using specific primers (SEQ ID NOs: 111
and 112) from clones that had been assessed to have antigen-binding
activity in the presence of adenosine or ATP based on the phage
ELISA result described in (10-4). The nucleotide sequences of the
genes were analyzed, and the result showed that multiple antibodies
that bind to antigens, human IL6, HSA, and human IL6R, and have
sequences different from one another were obtained. The amino acid
sequences of I6DL2C1-4.sub.--076 (antibody to human IL6),
HSDL3C5-4.sub.--015 (antibody to HSA), and 6RAD2C1-4.sub.--011 and
6RAD2C1-4.sub.--076 (antibodies to human IL-6R) are shown in Table
17.
TABLE-US-00052 TABLE 17 Heavy chain Light chain Clone name SEQ ID
NO SEQ ID NO I6DL2C5-4_076 SEQ ID NO: 78 SEQ ID NO: 79
HSDL3C5-4_015 SEQ ID NO: 80 SEQ ID NO: 81 6RAD2C1-4_011 SEQ ID NO:
82 SEQ ID NO: 83 6RAD2C1-4_076 SEQ ID NO: 84 SEQ ID NO: 85
(10-6) Identification of Small Molecules Required for Antigen
Binding of the Obtained Antibodies
[0842] Each of the obtained antibodies I6DL2C1-4.sub.--076,
HSDL3C5-4.sub.--015, 6RAD2C1-4.sub.--011, and 6RAD2C1-4.sub.--076
was subjected to ELISA. The small molecules used were 1 mM ATP,
adenosine, and a mixture thereof. The antigens used were
biotin-labeled human IL6, human IL6R, and HSA.
[0843] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of TBS
containing a biotin-labeled antigen. Following TBST wash to remove
the unbound biotin-labeled antigen from the plate, each well was
blocked with 250 .mu.l of 2% skim milk/TBS for one hour or more.
After 2% skim milk/TBS was removed from each well, 50 .mu.l of the
antibody-displaying phage was added to the plate. The plate was
allowed to stand at room temperature for one hour to allow binding
of each phage to the biotin-labeled antigen in each well in the
presence or absence of ATP and/or adenosine. After washing with
TBST with or without ATP and/or adenosine, an HRP-conjugated
anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS or
(adenosine and/or ATP)/TBS was added to each well. The plate was
incubated for one hour. Following wash with TBST with or without
each small molecule, the TMB single solution (ZYMED) was added to
each well. The chromogenic reaction in the solution of each well
was terminated by adding sulfuric acid. Then, the developed color
was assessed by measuring absorbance at 450 nm (FIGS. 25, 26, and
27).
Example 11
Binding Activity of Antibodies Obtained as Described in Example 2
to Human IL-6 in the Presence of Amino Acid Metabolites Other than
Kynurenine
[0844] Antibody I6NMSC1-3_A11 obtained as described in Example 2-4,
which binds to human IL-6 in the presence of small molecules, is an
antibody that binds to human IL-6 in the presence of kynurenine as
described in Example 3-2. Kynurenine is a tryptophan metabolite,
which is converted to anthranilic acid by kynureninase; to
3-hydroxykynurenine by kynurenine 3-hydroxylase; and to kynurenic
acid by kynurenine aminotransferase (Stefan Lob et. al., Nat Rev
Cancer. (2009) 9 (6), 445-452). Amino acid metabolites such as
tryptophan metabolites were assessed as to whether they are
appropriate as a non-limiting embodiment of cancer tissue-specific
compounds of the present invention, particularly cancer
cells-specific metabolites of the present invention.
[0845] I6NMSC1-3_A11 described in Example 3-2 which has
antigen-binding activity in the presence of kynurenine, a known
anti-human IL-6 antibody CLB8-F1, and GC413 as a negative control
were subjected to ELISA under the seven conditions described Table
18. Meanwhile, each amino acid and metabolites thereof were
appropriately prepared at the concentrations shown in Table 18
using the buffers indicated in Table 4. The antigen used was
biotin-labeled human IL-6.
TABLE-US-00053 TABLE 18 Condition Small molecule Concentration 1
Kynurenine 100 .mu.M 2 Tryptophan 100 .mu.M 3 Phenylalanine 100
.mu.M 4 Anthranilic acid 100 .mu.M 5 3-Hydroxykynurenine 100 .mu.M
6 Kynurenic acid 100 .mu.M 7 -- --
[0846] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing a biotin-labeled antigen. After washing with Wash buffer
to remove the unbound antigen from the plate, each well was blocked
for one hour or more with 250 .mu.l of Blocking Buffer. After
Blocking Buffer was removed from each well, the purified IgGs were
prepared to 2.5 .mu.g/ml in Sample Buffer containing small
molecules at the final concentrations shown in Table 18, and each
was aliquoted at 100 .mu.l into the plate. The plate was allowed to
stand at room temperature for one hour to allow binding of each IgG
to the antigen in each well. After washing with Wash Buffer
containing amino acids or amino acid metabolites at the final
concentrations shown in Table 18, an HRP-conjugated anti-human IgG
antibody (BIOSOURCE) diluted with Sample Buffer containing the same
amino acids and amino acid metabolites was added to each well. The
plate was incubated for one hour. Following wash with Wash Buffer
containing each amino acid or amino acid metabolite, the TMB single
solution (ZYMED) was added to each well. The chromogenic reaction
in the solution of each well was terminated by adding sulfuric
acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The compositions of buffers used are shown in
Table 4.
[0847] The measurement result is shown in FIG. 21. The absorbance
for CLB8-F1 was constant regardless of the type and presence of
small molecules. Meanwhile, the absorbance for I6NMSC1-3_A11 was
markedly lower under condition 7 (in the absence of small
molecules) as compared to under condition 1 (kynurenine solution).
Furthermore, the absorbance for I6NMSC1-3_A11 was high under
condition 2 (tryptophan solution) and condition 5
(3-hydroxykynurenine solution) as well as under condition 1. This
shows that I6NMSC1-3_A11 is an antibody that binds to human IL-6 as
an antigen not only in the presence of kynurenine, but also in the
presence of an amino acid (tryptophan) as a kynurenine precursor or
in the presence of a kynurenine metabolite.
[0848] This finding suggests that the same method can be used to
obtain antibodies that bind to an antigen of interest not only in
the presence of a single type of amino acid metabolite but also in
the presence of multiple different types of amino acids or amino
acid metabolites.
Example 12
Acquisition of Antibodies that Bind to Human IL-6 in the Presence
of Small Molecules from Human Antibody Library Using Phage-Display
Technique
[0849] (12-1) Acquisition of Antibodies that Bind to Human IL-6 in
the Presence of Small Molecules from the Library Using Bead Panning
or a Negative Selection Method
[0850] By the same method described in 2-2 and 2-3, the
phage-display library of naive human antibodies constructed as
described in Example 2-1 was screened for antibodies that exhibit
antigen-binding activity in the presence of small molecules.
(12-2) Assessment of Binding Activity in the Presence of Small
Molecules by Phage ELISA
[0851] Culture supernatants containing phages were obtained from
single colonies of E. coli obtained by the same method described in
Example 2-4. The phages were subjected to ELISA. By carrying out
phage ELISA using the 768 isolated clones, clones
"I6NMSC1-3.sub.--#03" and "I6NMSC1-3.sub.--#17", which exhibited
binding activity to human IL-6 as an antigen in the presence of
small molecule cocktail, were newly obtained.
(12-3) Expression and Purification of Antibodies that Bind to Human
IL-6
[0852] Genes were amplified from clones I6NMSC1-3.sub.--#03 and
I6NMSC1-3.sub.--#17 that had been assessed to have antigen-binding
activity in the presence of SC by phage ELISA, using specific
primers (SEQ ID NOs: 110 and 112); and their nucleotide sequences
were analyzed. The heavy-chain and light-chain sequences of
I6NMSC1-3.sub.--#03 are the sequences of SEQ ID NOs: 50 and 51,
respectively. Meanwhile, the heavy-chain and light-chain sequences
of I6NMSC1-3.sub.--#17 are the sequences of SEQ ID NOs: 52 and 53,
respectively. The gene sequence encoding the variable region of
I6NMSC1-3.sub.--#17 was inserted into an animal expression plasmid
for human IgG1/Lambda, while the gene sequence encoding the
variable region of I6NMSC1-3.sub.--#03, a known anti-human IL-6
antibody CLB8-F1 (the heavy chain and light chain sequences are
shown in SEQ ID NOs: 32 and 33, respectively), or an anti-human
glypican-3 antibody GC413 (the heavy chain and light chain
sequences are shown in SEQ ID NOs: 34 and 35, respectively) as a
negative control was inserted into an animal expression plasmid for
human IgG1/Kappa. The expressed antibodies were purified by the
method described in Example 3.
(12-4) Identification of Small Molecules Necessary for the Binding
of Antibody I6NMSC1-3.sub.--#03 to Human IL-6
[0853] I6NMSC1-3.sub.--#03 was subjected to ELISA under the nine
conditions described in Table 3. Meanwhile, each small molecule was
appropriately prepared at the concentrations shown in Table 3 using
the buffers indicated in Table 4. The antigen used was
biotin-labeled human IL-6.
[0854] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing the biotin-labeled antigen. After washing with Wash
buffer to remove the unbound antigen from the plate, each well was
blocked for one hour or more with 250 .mu.l of Blocking Buffer.
After removing Blocking Buffer from each well, the purified IgGs
were prepared to 2.5 .mu.g/ml in Sample Buffer containing small
molecules at the final concentrations shown in Table 3, and each
was aliquoted at 100 .mu.l into the plate. The plate was allowed to
stand at room temperature for one hour to allow binding of each IgG
to the antigen in each well. After washing with Wash Buffer
containing small molecules at the final concentrations shown in
Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)
diluted with Sample Buffer containing the same small molecules was
added to each well. The plate was incubated for one hour. Following
wash with Wash Buffer containing each small molecule, the TMB
single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The composition of the buffer used is shown
in Table 4.
[0855] The measurement result is shown in FIG. 22. The result
showed that the absorbance for I6NMSC1-3.sub.--#03 was markedly
lower under condition 9 (without small molecules) as compared to
that under condition 8 (the complete small molecule cocktail
solution). Similar to the result of phage ELISA, this result
confirmed that I6NMSC1-3.sub.--#03 has the property that its
antigen binding is altered depending on the presence or absence of
small molecules. Furthermore, the absorbance for
I6NMSC1-3.sub.--#03 was comparable between condition 7 (100 .mu.M
kynurenine) and condition 8; however, the absorbance was lower
under other conditions. This demonstrated that, like I6NMSC1-3_A11
described in Example 3, I6NMSC1-3.sub.--#03 was an antibody that
binds to human IL-6 as an antigen in the presence of kynurenine.
I6NMSC1-3.sub.--#03 has an amino acid sequence different from
I6NMSC1-3_A11, which demonstrates that the method described above
can be used to isolate different types of antibodies that bind to
antigens in the presence of small molecules.
(12-5) Identification of Small Molecules Necessary for the Binding
of Antibody I6NMSC1-3.sub.--#17 to Human IL-6
[0856] Three types of antibodies: obtained I6NMSC1-3.sub.--#17,
control CLB8-F1, and negative control GC413 were subjected to ELISA
under the nine conditions described in Table 3. Each small molecule
was prepared to an appropriate concentration shown in Table 3 using
the buffers shown in Table 4. The antigen used was biotin-labeled
human IL-6.
[0857] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing the biotin-labeled antigen. After washing with Wash
buffer to remove the unbound antigen from the plate, each well was
blocked for one hour or more with 250 .mu.l of Blocking Buffer.
After Blocking Buffer was removed from each well, the purified IgGs
were prepared to 0.15 .mu.g/ml in Sample Buffer containing small
molecules at the final concentrations shown in Table 3, and each
was aliquoted at 100 .mu.l into the plate. The plate was allowed to
stand at room temperature for one hour to allow binding of each IgG
to the antigen in each well. After washing with Wash Buffer
containing small molecules at the final concentrations shown in
Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)
diluted with Sample Buffer containing the same small molecules was
added to each well. The plate was incubated for one hour. Following
wash with Wash Buffer containing each small molecule, the TMB
single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The composition of the buffer used is shown
in Table 4.
[0858] The measurement result is shown in FIG. 23. The result
showed that the absorbance for CLB8-F1 was the same regardless of
the type and presence or absence of small molecule, whereas the
absorbance for I6NMSC1-3.sub.--#17 was markedly lower under
condition 9 (without small molecules) as compared to that under
condition 8 (the complete small molecule cocktail solution).
Similar to the result of phage ELISA, this result confirmed that
I6NMSC1-3.sub.--#17 has the property that its antigen binding is
altered depending on the presence or absence of small molecules.
Furthermore, the absorbance for I6NMSC1-3.sub.--#17 under condition
1 (1 mM ATP-Na) and condition 5 (1 mM succinic acid) was comparable
to that under condition 8; however, the absorbance was lower under
other conditions. This result suggests that I6NMSC1-3.sub.--#17 is
an antibody that binds to human IL-6 as an antigen in the presence
of either ATP-Na or succinic acid. ATP is known to be released from
cancer cells. Succinic acid is also known to be accumulated inside
and outside of cells in a cancer cell-specific manner. The
phenomenon that even in aerobic environments cancer cells
metabolize in a glycolysis-dependent manner rather than by
oxidative phosphorylation is known as Warburg effect. Both
glycolysis and oxidative phosphorylation are chronically limited in
ischemic-type cancers because of chronic insufficiency of blood
flow, and thus such cancers acquire energy in poorer conditions.
Ischemic-type cancers are known to perform energy metabolism
depending on fumarate respiration, resulting in accumulation of
succinic acid as a fumarate metabolite (Cancer Res. (2009) 69 (11),
4918-4925).
[0859] This demonstrated that by using such method, it is possible
to obtain antibodies that bind to antigens in the presence of a
small molecule other than kynurenine. Also, this showed that it is
possible to obtain antibodies that bind to antigens in the presence
of small molecules such as ATP-Na and succinic acid which share a
common feature that they have multiple negative charges but are
structurally different from each other.
Example 13
Acquisition of Antibodies that Bind to Human Serum Albumin
(Hereinafter Also Referred to as HSA) in the Presence of Small
Molecules by Phage-Display Techniques from Human Antibody
Library
[0860] (13-1) Acquisition of Antibodies that Bind to HSA in the
Presence of Small Molecules from Library by Bead Panning
[0861] The phage-display library of naive human antibodies
constructed as described in Example 2 was screened for antibodies
that exhibit HSA-binding activity in the presence of small
molecules, specifically, by collecting phages displaying antibodies
that in the presence of small molecules exhibit binding activity to
HSA captured by beads. Phages were collected from a phage
suspension eluted from the beads in the absence of small molecules.
In this preparation method, biotin-labeled HSA was used as the
antigen.
[0862] Phages produced in E. coli containing the phagemid vector
constructed for phage display were purified by a conventional
method. Then, a phage library suspension was prepared through
dialysis against TBS. Next, skim milk was added to the phage
library suspension at a final concentration of 3%. Panning was
performed using antigen-immobilized magnetic beads. The magnetic
beads used were NeutrAvidin coated beads (Sera-Mag SpeedBeads
NeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280
Streptavidin).
[0863] For efficient acquisition of small-molecule switch
antibodies that are dependent on small molecules which can serve as
a switch in cancer tissues, panning was performed to enrich
antibodies that bind to antigens in the presence of a mixture of
such small molecules (adenosine, adenosine triphosphate (adenosine
5'-triphosphate (ATP)), inosine, kynurenine, prostaglandin E2
(PGE2), succinic acid, and lactic acid (hereinafter referred to as
small molecule cocktail (SC)) but do not bind to antigens in the
absence of SC.
[0864] Specifically, together with 250 pmol of the biotin-labeled
antigen, SC containing adenosine triphosphate sodium salt (ATP-Na),
adenosine, inosine, succinic acid, and lactic acid at a final
concentration of 1 mM, prostaglandin E2 (PGE2) at a final
concentration of 1 .mu.M, and kynurenine at a final concentration
of 100 .mu.M, which had been adjusted to be pH 7.4 with NaOH, was
added and contacted with the prepared phage library suspension for
60 minutes at room temperature. Then, skim milk-blocked magnetic
beads were added to the phage library suspension, and the
antigen-phage complex was allowed to bind to the magnetic beads at
room temperature for 15 minutes. The beads were washed once with
SC/TBS (TBS containing SC). Then, the beads were combined with 0.5
ml of a 1 mg/ml trypsin solution. Immediately after the beads were
suspended at room temperature for 15 minutes, a phage suspension
was collected from the isolated beads using a magnetic stand. The
collected phage suspension was added to 10 ml of E. coli cells of
strain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7).
The E. coli was incubated at 37.degree. C. for one hour under
gentle stirring to be infected by phage. The infected E. coli was
seeded in a 225 mm.times.225 mm plate. Then, phages were collected
from the culture medium of the seeded E. coli to prepare a liquid
stock of phage library.
[0865] The first round of panning was carried out to collect phages
that are capable of binding in the presence of small molecules,
while the second and subsequent rounds of panning were performed to
enrich phages that are capable of antigen binding in the presence
of small molecules. Specifically, 40 pmol of the biotin-labeled
antigen, SC, and NaOH were added to the prepared phage library
suspension. Thus, the phage library was contacted with the antigen
and small molecules for 60 minutes at room temperature. Skim
milk-blocked magnetic beads were added and allowed to bind the
antigen-phage complex for 15 minutes at room temperature. The beads
were washed with 1 ml of SC/TBST and SC/TBS. Then, the beads were
combined with 0.5 ml of TBS. Immediately after the beads were
suspended at room temperature, a phage suspension was collected
from the isolated beads using a magnetic stand. After repeating
this treatment, the two separately eluted phage suspensions were
combined together. Then, the resultant beads were combined with 0.5
ml of TBS, and stirred at room temperature for five minutes. A
phage suspension was collected from the isolated beads using a
magnetic stand. The pIII protein (helper phage-derived protein
pIII) that does not display Fab was cleaved off from phages by
adding 5 .mu.l of 100 mg/ml trypsin to the collected phage
suspension to eliminate the ability of phages that do not display
Fab to infect E. coli. The phages collected from the trypsinized
phage suspension were added to 10 ml of E. coli cells of strain
ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.
coli was incubated with gentle stirring at 37.degree. C. for one
hour to be infected by phage. The infected E. coli was seeded in a
225 mm.times.225 mm plate. The two types of infected E. coli
obtained through two rounds of panning were mixed in equal amount
at this time point. Then, phages were collected from the culture
medium of the seeded E. coli to prepare a phage library suspension.
Panning was performed three times to obtain antibodies that have
antigen-binding activity in the presence of small molecules.
(13-2) Acquisition of Antibodies that Bind to HSA in the Presence
of Small Molecules from the Library Using a Negative Selection
Method
[0866] The constructed phage-display library of naive human
antibodies was screened for antibodies that exhibit HSA-binding
activity in the presence of small molecules. As a first step of
screening, the phage-display library of naive human antibodies was
contacted with biotin-labeled antigen-streptavidin in the absence
of small molecules to eliminate phages displaying antibodies that
have HSA-binding activity even in the absence of small molecules.
Then, panning was performed in the presence of small molecules in
the same manner. Thus, screening was carried out for antibodies
that have HSA-binding activity in the presence of small molecules.
Biotin-labeled HSA was used as the antigen.
[0867] Phages were produced in E. coli containing the phagemid
vector constructed for phage display. The produced phages were
purified by a conventional method, and then a phage library
suspension was prepared by dialyzing the phages against TBS. Then,
skim milk was added at a final concentration of 3% to the phage
library suspension. The magnetic beads used were NeutrAvidin coated
beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin
coated beads (Dynabeads M-280 Streptavidin). Panning was performed
using biotin-labeled HSA immobilized on magnetic beads.
[0868] Together with 250 pmol of biotin-labeled HSA, SC containing
ATP-Na, adenosine, inosine, succinic acid, and lactic acid at a
final concentration of 1 mM, PGE2 at a final concentration of 1
.mu.M, and kynurenine at a final concentration of 100 .mu.M, which
had been adjusted to be pH 7.4 with NaOH, was added and contacted
with the prepared phage library solution for 60 minutes at room
temperature. Then, skim milk-blocked magnetic beads were added to
the phage library solution, and allowed to bind to the complex of
phage with biotin-labeled HSA for 15 minutes at room temperature.
The beads were washed once with SC/TBS. Then, the beads were
combined with 0.5 ml of a 1 mg/ml trypsin solution. Immediately
after the beads were suspended at room temperature for 15 minutes,
a phage suspension was collected from the isolated beads using a
magnetic stand. The collected phage suspension was added to 10 ml
of E. coli cells of strain ER2738 at the logarithmic growth phase
(OD600=0.4 to 0.7). The E. coli was incubated with gentle stirring
at 37.degree. C. for one hour to be infected by phage. The infected
E. coli was seeded in a 225 mm.times.225 mm plate. Then, phages
were collected from the culture medium of the seeded E. coli to
prepare a phage library solution.
[0869] The first round of panning was carried out to collect phages
that are capable of binding in the presence of small molecules,
while the second and subsequent rounds of panning were performed to
enrich phages that are capable of binding to biotin-labeled HSA in
the presence of small molecules. Specifically, 250 pmol of
biotin-labeled HSA was added to skim milk-blocked Sera-Mag
NeutrAvidin beads and allowed to bind for 15 minutes at room
temperature. The beads were washed three times with TBS; and a skim
milk-blocked phage library solution was added to the beads, and
allowed to bind at room temperature for one hour. The beads were
isolated using a magnetic stand to collect phages that did not bind
to biotin-labeled HSA or the beads. Forty pmol of biotin-labeled
HSA, SC, and NaOH were added to the collected phages. Thus, the
phage library was contacted with biotin-labeled HSA and small
molecules in SC for 60 minutes at room temperature. Then, skim
milk-blocked magnetic beads were added to the mixture of
biotin-labeled HSA, SC, and phage library, and the complex of
biotin-labeled HSA and phage was allowed to bind to the magnetic
beads for 15 minutes at room temperature. The beads were washed
with 1 ml of SC/TBST and SC/TBS. Then, 0.5 ml of a 1 mg/ml trypsin
solution was added to the mixture. After the mixed solution was
stirred for 20 minutes at room temperature, phages were collected
from the beads separated using a magnetic stand. The collected
phages were added to 10 ml of E. coli cells of strain ER2738 at the
logarithmic growth phase (OD600=0.4 to 0.7). The E. coli was
incubated with gentle stirring at 37.degree. C. for one hour to be
infected by phage. The infected E. coli was seeded in a 225
mm.times.225 mm plate. Panning was performed three times to obtain
antibodies that have binding activity to biotin-labeled HSA in the
presence of small molecules.
(13-3) Assessment of Binding Activity in the Presence of Small
Molecules by Phage ELISA
[0870] Culture supernatants containing phages were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained by the method
described above. The collected culture supernatants were treated by
ultrafiltration using NucleoFast 96 (MACHEREY-NAGEL). 100 .mu.l of
the culture supernatants were added to each well, and the
NucleoFast 96 was centrifuged (4500 g for 45 minutes) to remove
flow-through. One hundred .mu.l of H.sub.2O was added to each well,
and again the NucleoFast 96 was centrifuged (4500 g for 30 minutes)
for washing. After 100 .mu.l of TBS was added, the NucleoFast 96
was allowed to stand for five minutes at room temperature. Finally,
phage solution contained in the supernatant of each well was
collected.
[0871] The purified phages, to which TBS or SC/TBS was added, were
subjected to ELISA by the following procedure. A StreptaWell 96
microtiter plate (Roche) was coated overnight with 100 .mu.l of TBS
containing biotin-labeled HSA. After biotin-labeled HSA was removed
by washing each well of the plate with TBST, the wells were blocked
with 250 .mu.l of 2% skim milk/TBS for one hour or more. 2% skim
milk/TBS was removed, and then the prepared, purified phages were
added to each well. The plate was allowed to stand at room
temperature for one hour to allow binding of antibody-displaying
phages to biotin-labeled HSA in each well in the presence or
absence of SC. After washing with TBST or SC/TBST, an
HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech)
diluted with TBS or SC/TBS was added to each well. The plate was
incubated for one hour. Following wash with TBST or SC/TBST, the
TMB single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm.
[0872] Clone HSNMSC1-4.sub.--#22, which has binding activity to HSA
as antigen in the presence of the small molecule cocktail, was
obtained by carrying out phage ELISA using the 782 isolated
clones.
(13-4) Expression and Purification of HSA-Binding Antibodies
[0873] Genes were amplified from clone HSNMSC1-4.sub.--#22 which
had been assessed to have binding activity to biotin-labeled HSA in
the presence of SC by the phage ELISA described in (13-3), using
specific primers (SEQ ID NOs: 110 and 112). The nucleotide
sequences of the genes were analyzed (the heavy chain and light
chain sequences are represented by SEQ ID NOs: 54 and 55,
respectively). Genes encoding the variable regions of
HSNMSC1-4.sub.--#22 were inserted into an animal expression plasmid
for human IgG1/Lambda. Meanwhile, genes encoding the variable
regions of the negative control anti-human glypican-3 antibody
GC413 (the heavy chain and light chain are represented by SEQ ID
NOs: 34 and 35, respectively) were inserted into an animal
expression plasmid for human IgG1/Kappa. The expressed antibodies
were purified by the method described in Example 3.
(13-5) Identification of Small Molecules Necessary for Binding of
the Obtained Antibodies to HSA
[0874] Two types of antibodies obtained: HSNMSC1-4.sub.--#22 and
GC413, were subjected to ELISA under the nine conditions described
in Table 3. Meanwhile, each small molecule was appropriately
prepared at the concentrations shown in Table 3 using the buffers
indicated in Table 19. Biotin-labeled HSA was used as the
antigen.
TABLE-US-00054 TABLE 19 Wash buffer 10 mM ACES, 150 mM NaCl, 0.05%
Tween20, pH7.4 Blocking Buffer 10 mM ACES, 150 mM NaCl, 2% Skim
Milk, pH7.4 Sample Buffer 10 mM ACES, 150 mM NaCl, Small molecule,
pH7.4
[0875] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing biotin-labeled HSA. After washing with Wash buffer to
remove unbound biotin-labeled HSA from the plate, each well was
blocked for one hour or more with 250 .mu.l of Blocking Buffer.
After Blocking Buffer was removed from each well, the purified IgGs
were prepared to 2.5 .mu.g/ml in Sample Buffer containing small
molecules at the final concentrations shown in Table 3, and each
was aliquoted at 100 .mu.l into the plate. The plate was allowed to
stand at room temperature for one hour to allow binding of each IgG
to biotin-labeled HSA in each well. After washing with Wash Buffer
containing small molecules at the final concentrations shown in
Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)
diluted with Sample Buffer containing the same small molecules was
added to each well. The plate was incubated for one hour. Following
wash with Wash Buffer containing each small molecule, the TMB
single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The composition of the buffer used is shown
in Table 19.
[0876] The measurement result is shown in FIG. 24. The result
showed that the absorbance for HSNMSC1-4.sub.--#22 was markedly
lower under condition 9 (without small molecules) as compared to
that under condition 8 (the complete small molecule cocktail
solution). Similar to phage ELISA, this result confirmed that
HSNMSC1-4.sub.--#22 has the property that its antigen binding is
altered depending on the presence or absence of small molecules.
Meanwhile, the absorbance for HSNMSC1-4.sub.--#22 was the same
between condition 2 (1 mM adenosine) and condition 8, but it was
markedly lower under other conditions. This result demonstrated
that HSNMSC1-4.sub.--#22 is an antibody that binds to HSA as an
antigen in the presence of adenosine. Thus, it was demonstrated
that such method can be used to isolate antibodies that bind to
antigens in the presence of small molecules other than
kynurenine.
Example 14
Acquisition of Antibodies that Bind to Human IL-6 Receptor (hIL-6R)
in the Presence of Small Molecules from Human Antibody Library
Using Phage Display Techniques
[0877] (14-1) Acquisition of Antibodies that Bind to hIL-6R in the
Presence of Small Molecules from the Library of Naive Human
Antibodies by Bead Panning
[0878] The phage-display library of naive human antibodies
constructed as described in Example 2 was screened for antibodies
that exhibit hIL-6R-binding activity in the presence of small
molecules, specifically, by collecting phages displaying antibodies
that in the presence of small molecules exhibit binding activity to
hIL-6R captured by beads. Phages were collected from a phage eluate
eluted from the beads in the absence of small molecules. In this
preparation method, biotin-labeled hIL-6R was used as the
antigen.
[0879] Phages produced in E. coli containing the phagemid vector
constructed for phage display were purified by a conventional
method. Then, a phage library solution was prepared by dialyzing
the phages against TBS. Next, BSA was added at a final
concentration of 4% to the phage library solution. Panning was
performed using antigen-immobilized magnetic beads. The magnetic
beads used were NeutrAvidin coated beads (Sera-Mag SpeedBeads
NeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280
Streptavidin).
[0880] For efficient acquisition of small molecule switch
antibodies that are dependent on small molecules which can serve as
a switch in cancer tissues, panning described in (2-2) was
performed to enrich antibodies that bind to antigens in the
presence of SC but do not bind to antigens in the absence of
SC.
[0881] Specifically, together with 250 pmol of biotin-labeled
antigen, SC prepared as described in (2-2) was added and contacted
with the prepared phage library solution at room temperature for 60
minutes. Then, the phage library solution was added to BSA-blocked
magnetic beads, and the antigen-phage complex was allowed to bind
to the magnetic beads for 15 minutes at room temperature. The beads
were washed once with SC/TBS (TBS containing SC). Then, the beads
were combined with 0.5 ml of a 1 mg/ml trypsin solution.
Immediately after the beads were suspended at room temperature for
15 minutes, the phage solution was collected from the isolated
beads using a magnetic stand. The collected phage solution was
added to 10 ml of E. coli cells of strain ER2738 at the logarithmic
growth phase (OD600=0.4 to 0.7). The E. coli was incubated with
gentle stirring at 37.degree. C. for one hour to be infected by
phage. The infected E. coli was seeded in a 225 mm.times.225 mm
plate. Then, phages were collected from the culture medium of the
seeded E. coli to prepare a liquid stock of phage library.
[0882] Panning was performed as described in (2-2), except adding
10 .mu.l of 100 mg/ml trypsin to cleave the pIII protein (helper
phage-derived pIII protein) from phages that do not display Fab in
order to eliminate the ability of the phages that do not display
Fab to infect E. coli.
(14-2) Acquisition of Antibodies that Bind to hIL-6R in the
Presence of Small Molecules from the Naive Human Antibody Library
Using a Negative Selection Method
[0883] The constructed phage-display library of naive human
antibodies was screened for antibodies that exhibit hIL-6R-binding
activity in the presence of small molecules. As a first step of
screening, the phage-display library of naive human antibodies was
contacted with biotin-labeled antigen-streptavidin in the absence
of small molecules to eliminate phages displaying antibodies that
have hIL-6R-binding activity even in the absence of small
molecules. Then, panning was performed in the presence of small
molecules in the same manner to screen for antibodies that have
hIL-6R-binding activity in the presence of small molecules. The
antigen used was biotin-labeled hIL-6R. Then, a phage library
solution was prepared by the method described in (2-3) using
biotin-labeled hIL-6R as an antigen.
(14-3) Assessment of Binding Activity in the Presence of Small
Molecules by Phage ELISA
[0884] Culture supernatants containing phages were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained as described in
(14-2). Phages purified by the method described in (2-4) were
subjected to ELISA by the following procedure. A StreptaWell 96
microtiter plate (Roche) was coated overnight with 100 .mu.l of TBS
containing biotin-labeled hIL-6R. After biotin-labeled hIL-6R was
removed by washing each well of the plate with TBST, the wells were
blocked with 250 .mu.l of 2% skim milk/TBS for one hour or more. 2%
skim milk/TBS was removed, and then the prepared, purified phages
were added to each well. The plate was allowed to stand at room
temperature for one hour to allow binding of antibody-displaying
phages to biotin-labeled hIL-6R in each well in the presence or
absence of SC. After washing with TBST or SC/TBST, an
HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech)
diluted with TBS or SC/TBS was added to each well. The plate was
incubated for one hour. Following wash with TBST or SC/TBST, the
TMB single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm.
[0885] Clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02, which have binding
activity to hIL-6R as an antigen in the presence of a small
molecule cocktail, were obtained by carrying out phage ELISA using
960 isolated clones.
(14-4) Expression and Purification of Antibodies that Bind to
hIL-6R
[0886] Genes were amplified using specific primers (SEQ ID NOs: 110
and 112) from clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02, which had
been assessed to have binding activity to biotin-labeled hIL-6R in
the presence of SC by the phage ELISA described in (14-3). The
nucleotide sequences of the genes were analyzed (6RNMSC1-2_F02: the
heavy chain and light chain sequences are shown in SEQ ID NOs: 86
and 87, respectively; 6RNMSC1-3_G02: the heavy chain and light
chain sequences are shown in SEQ ID NOs: 88 and 89, respectively).
Genes encoding the variable regions of 6RNMSC1-2_F02 and
6RNMSC1-3_G02, and those of the negative control anti-human
glypican-3 antibody GC413 (the heavy chain and light chain are SEQ
ID NOs: 34 and 35, respectively) were inserted into an animal
expression plasmid for human IgG1/Kappa. The expressed antibodies
were purified by the method described in Example 3.
(14-5) Identification of Small Molecules Necessary for Binding of
the Obtained Antibodies to Hil-6R
[0887] Three types of antibodies obtained: 6RNMSC1-2_F02 and
6RNMSC1-3_G02, and GC413 were subjected to ELISA under the nine
conditions described in Table 3. Meanwhile, each small molecule was
appropriately prepared at the concentrations shown in Table 3 using
the buffers indicated in Table 19. The antigen used was
biotin-labeled hIL-6R.
[0888] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing biotin-labeled hIL-6R. After washing with Wash buffer to
remove unbound biotin-labeled hIL-6R from the plate, each well was
blocked for one hour or more with 250 .mu.l of Blocking Buffer.
After Blocking Buffer was removed from each well, the purified IgGs
were prepared to 2.5 .mu.g/ml in Sample Buffer containing small
molecules at the final concentrations shown in Table 3, and each
was aliquoted at 100 .mu.l into the plate. The plate was allowed to
stand at room temperature for one hour to allow binding of each IgG
to biotin-labeled hIL-6R in each well. After washing with Wash
Buffer containing small molecules at the final concentrations shown
in Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)
diluted with Sample Buffer containing the same small molecules was
added to each well. The plate was incubated for one hour. Following
wash with Wash Buffer containing each small molecule, the TMB
single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The composition of the buffer used is shown
in Table 19.
[0889] The measurement result is shown in FIGS. 28 and 29. When
6RNMSC1-2_F02 or 6RNMSC1-3G02 was used, the absorbance was markedly
lower under condition 9 (in the absence of small molecules) as
compared to under condition 8 (the complete small molecule cocktail
solution). This result confirmed that 6RNMSC1-2_F02 and 6RNMSC1-3
G02 have the property that their antigen binding is altered
depending on the presence or absence of small molecules. Meanwhile,
when 6RNMSC1-2_F02 was used, the absorbance was the same between
condition 7 (100 .mu.M kynurenine) and condition 8, but the
absorbance was markedly lower under other conditions, which shows
that 6RNMSC1-2_F02 is an antibody that binds to hIL-6R as an
antigen in the presence of kynurenine (FIG. 28). Furthermore, when
6RNMSC1-3G02 was used, the absorbance was the same between
condition 1 (1 mM ATP-Na) and condition 8, but the absorbance was
markedly lower under other conditions, showing that 6RNMSC1-3G02 is
an antibody that binds to hIL-6R as an antigen in the presence of
ATP (FIG. 29). It was thus demonstrated that the method described
above can be used to isolate at one time multiple antibodies whose
antigen-binding activity is altered in the presence of a different
small molecule.
Example 15
Characterization of Antibody 6RNMSC1-2_F02
[0890] (15-1) ELISA Assessment of hIL6R-Binding Activity in the
Presence of Amino Acids and Amino Acid Metabolites Other than
Kynurenine
[0891] Antibody 6RNMSC1-2_F02 obtained as described in Example 14,
which binds to hIL-6R in the presence of small molecules, is an
antibody that binds to hIL-6R in the presence of kynurenine. Amino
acid metabolites such as tryptophan metabolites described in
Example 11 were assessed as to whether they are preferable as a
non-limiting embodiment of cancer tissue-specific compounds,
particularly cancer cell-specific metabolites, for use in the
present invention.
[0892] 6RNMSC1-2_F02 described in Example 14, which has
antigen-binding activity in the presence of kynurenine, and
negative control GC413 were subjected to ELISA under the seven
conditions described in Table 18. Meanwhile, each small molecule
was appropriately prepared at the concentrations shown in Table 18
using the buffers indicated in Table 4. The antigen used was
biotin-labeled hIL-6R. ELISA was carried out using the method
described in Example 11.
[0893] The measurement result is shown in FIG. 30. When
6RNMSC1-2_F02 was used, the absorbance was markedly lower under
condition 7 (in the absence of small molecules) as compared to
under condition 1 (kynurenine solution). Similarly, 6RNMSC1-2_F02
showed the same high absorbance under condition 5
(3-hydroxykynurenine solution) as under condition 1, suggesting
that it is an antibody that binds to hIL-6R as an antigen not only
in the presence of kynurenine but also in the presence of a
kynurenine metabolite. Furthermore, the absorbance for
6RNMSC1-2_F02 was markedly lower under other conditions, suggesting
that 6RNMSC1-2_F02 is an antibody that does not bind to hIL-6R as
an antigen in the presence of tryptophan, a kynurenine precursor.
The expression level of IDO, an enzyme that metabolizes tryptophan
to produce kynurenine, is elevated in cancer microenvironment.
Thus, antibodies that bind to antigens in the presence of
kynurenine or its metabolite but not in the presence of tryptophan
are expected to be important as antibodies that bind to antigens
only in cancer microenvironment. This suggests that the same method
can be used to obtain antibodies that bind to an antigen of
interest not only in the presence of a single amino acid metabolite
but also in the presence of multiple, structurally different amino
acid metabolites.
(15-2) Assessment of Kynurenine for its Effect on Human IL6
Receptor Binding by Surface Plasmon Resonance
[0894] Biacore T200 (GE Healthcare) was used to analyze the
interaction of 6RNMSC1-2_F02 with human IL6 receptor (IL-6R) in
antigen-antibody reaction. Sensor chip CM5 (GE Healthcare) was
immobilized with an appropriate amount of protein A (Invitrogen) by
amine coupling. The antibody of interest was captured by the chip
to allow interaction with IL-6R as an antigen. The running buffer
used was 20 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH
7.4. The interaction with the antigen IL-6R was assayed at
25.degree. C. The buffers used to dilute IL-6R were the running
buffer itself, and a buffer prepared by adding 100 .mu.mol/l
kynurenine to the running buffer, and in addition a buffer prepared
as a control by adding 10 mmol/l ATP to the running buffer.
[0895] A diluted IL-6R solution and a running buffer as a blank
were injected at a flow rate of 10 .mu.l/min for one minute to
allow interaction of IL-6R with 6RNMSC1-2_F02 captured on the
sensor chip. Then, the running buffer was injected at a flow rate
of 10 .mu.l/min for one minute. After observation of IL-6R
dissociation from the antibody, 10 mmol/l glycine-HCl (pH 1.5) was
injected at a flow rate of 30 .mu.l/min for 30 seconds to
regenerate the sensor chip. The dissociation constant K.sub.D (M)
of 6RNMSC1-2_F02 was calculated for IL-6R based on the association
rate constant ka (1/Ms) and dissociation rate constant kd (1/s),
both of which are kinetic parameters calculated from the sensorgram
obtained by the measurement. Each parameter was calculated using
the Biacore T200 Evaluation Software (GE Healthcare).
[0896] Sensorgrams obtained by this measurement for the interaction
between 6RNMSC1-2_F02 and 1 .mu.mol/l IL-6R in the presence of 100
.mu.mol/l kynurenine and in the presence or absence of 10 mmol/l
ATP are shown in FIG. 31. As shown in FIG. 31, 6RNMSC1-2_F02 binds
to IL-6R in the presence of 100 .mu.mol/l kynurenine; however, its
IL-6R binding was not detectable in the absence of kynurenine. This
demonstrates that 6RNMSC1-2_F02 has the property that it binds to
IL-6R via kynurenine as a switch. Meanwhile, the dissociation
constant K.sub.D of 6RNMSC1-2_F02 was 1.5 .mu.mol/l in the presence
of 100 .mu.mol/l kynurenine.
(15-3) Effect of Kynurenine as a Switch on Dissociation of
Antibodies from IL-6R
[0897] Biacore T200 (GE Healthcare) was used to evaluate whether
6RNMSC1-2_F02 which binds to IL-6R in the presence of kynurenine
dissociates in a kynurenine concentration-dependent manner in the
presence of kynurenine. The running buffers used were 20 mmol/l
ACES, 150 mmol/lNaCl, 0.05% (w/v) Tween20, pH 7.4, and 20 mmol/l
ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4, 100 .mu.mol/l
kynurenine, and assay was carried out at 25.degree. C. IL-6R was
immobilized onto Sensor chip CM5 by amine coupling; and
6RNMSC1-2_F02 was diluted to 5 .mu.g/ml with 20 mmol/lACES, 150
mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4, containing 100 .mu.mol/l
kynurenine, and it was interacted as an analyte for 180 seconds.
Then, the dissociation of IL-6R was monitored under each running
buffer condition. In order to compare the degree of dissociation
between the respective running buffer conditions, the values were
normalized by taking as 100 the amount of 6RNMSC1-2_F02 bound to
IL-6R in the presence of 100 .mu.mol/l kynurenine and compared. A
sensorgram that represents the interaction between 6RNMSC1-2_F02
and IL-6R after normalization is shown in FIG. 32. The result shown
in FIG. 32 demonstrates that 6RNMSC1-2_F02 has the property that it
binds to IL-6R in the presence of kynurenine and then rapidly
dissociates from IL-6R in the absence of kynurenine. Specifically,
the kynurenine-mediated regulation on binding of the antibody to
IL-6R was demonstrated to be reversible.
(15-4) Assessment of Kynurenine Concentration Effect on IL-6R
Binding
[0898] Next, Biacore T200 (GE Healthcare) was used to assess the
effect of kynurenine concentration on the antigen-antibody reaction
between 6RNMSC1-2_F02 and IL-6R. The running buffer used was 20
mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. The
antigen-antibody reaction between 6RNMSC1-2_F02 and human IL-6R was
assayed at 25.degree. C. IL-6R was immobilized onto sensor chip CM5
by amine coupling; and 6RNMSC1-2_F02 was diluted to 1 .mu.g/ml with
20 mmol/lACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4
containing various concentrations of kynurenine, and it was allowed
to interact as an analyte for 180 seconds to observe changes in the
amount of binding. The result is shown in FIG. 33. This result
demonstrated that the higher the concentration of kynurenine
serving as a switch, the greater the amount of 6RNMSC1-2_F02 bound
to IL-6R.
[0899] Meanwhile, since IL-6R is immobilized on a sensor chip in
this assay system, 6RNMSC1-2_F02 is thought to bind in a divalent
manner. In such an assay system where 6RNMSC1-2_F02 recognizes
IL-6R in a divalent manner, the amount of 6RNMSC1-2_F02 bound to
IL-6R was also observed to increase with a higher kynurenine
concentration. This result demonstrated that 6RNMSC1-2_F02 has the
property that it binds to IL-6R via kynurenine as a switch also in
divalent binding.
[0900] These results demonstrated that 6RNMSC1-2_F02 is an antibody
that binds to IL-6R in the presence of kynurenine with kynurenine
as a switch, but is dissociated from IL-6R in the absence of
kynurenine. Furthermore, it was confirmed that it is possible to
have full ON/OFF control of 6RNMSC1-2_F02 so that it does not
demonstrate IL-6R-binding activity in the absence of kynurenine.
The switch function was expected to be achieved in the manner such
as shown in FIG. 2.
(15-5) Effect of Kynurenine on the ADCC Activity of
6RNMSC1-2_F02
[0901] The genes encoding the variable regions of 6RNMSC1-2_F02
determined as described in Example 14 were inserted into an animal
expression plasmid for human IgG1/Kappa, which comprises an
antibody heavy-chain constant region comprising the sequence of SEQ
ID NO: 90 and a light-chain kappa constant region sequence
comprising the sequence of SEQ ID NO: 91. The respective genes
encoding the variable regions of a known anti-human IL-6R antibody,
MRA (the heavy chain and light chain sequences are shown in SEQ ID
NOs: 92 and 93, respectively), were also inserted into the animal
expression plasmid for human IgG1/Kappa, which has the
above-described constant regions (SEQ ID NOs: 90 and 91).
Antibodies were expressed using the method described below.
FreeStyle 293-F (Invitrogen) which was derived from human fetal
kidney cells were suspended at a cell density of
1.33.times.10.sup.6 cells/ml in the FreeStyle 293 Expression Medium
(Invitrogen), and aliquoted at 3 ml into each well of a 6-well
plate. The plasmid DNA was transfected into the cells by
lipofection. From the culture supernatants after four days of
culture in a CO.sub.2 incubator (37.degree. C., 8% CO.sub.2, 90
rpm), antibodies were purified by a method known to those skilled
in the art using rProtein A Sepharose.TM. Fast Flow (Amersham
Biosciences). Absorbance of the purified antibody solutions was
measured at 280 nm using a spectrophotometer. From values obtained
by the measurement, the concentrations of purified antibodies were
calculated using an extinction coefficient determined by the PACE
method (Protein Science (1995) 4, 2411-2423).
[0902] 6RNMSC1-2_F02 antibody which has an antigen-binding activity
in the presence of kynurenine as described in Example 14 was
assessed for its binding to soluble hIL-6R. As a first step in
assessing the ADCC activity of 6RNMSC1-2_F02 to hIL-6R-expressing
cells, 6RNMSC1-2_F02 was evaluated as to whether it has the ability
to bind to membrane-type hIL-6R expressed in hIL-6R-expressing
cells. Specifically, the binding of 6RNMSC1-2_F02 to cells of the
BaF/hIL-6R line (WO2012/073992) was assayed and analyzed using a
flow cytometer. An appropriate number of BaF/hIL-6R cells were
prepared and blocked with PBS containing 2% FBS on ice for one hour
or more. Supernatant was removed from the blocked cells by
centrifugation, and 100 .mu.l of 6RNMSC1-2_F02 or the control
antibody MRA (the heavy chain and light chain sequences are shown
in SEQ ID NOs: 92 and 93, respectively) was added under two
conditions: in the presence or absence of a final concentration of
kynurenine at 100 .mu.M. In this step, the antibodies were
contacted with cell-membrane hIL-6R on ice for 30 minutes. The
antibody-cell complex was washed with Wash Buffer containing
kynurenine or with Wash Buffer that does not contain kynurenine.
Then, in the presence or absence of kynurenine, the complex was
contacted with a secondary antibody (Beckman Coulter IM1627) that
recognizes the antibody constant region. After 30 minutes of
incubation with the antibody on ice, the cells were again washed
with Wash Buffer and then re-suspended in PBS/2% FBS. The binding
of 6RNMSC1-2_F02 to the prepared cells was assayed and analyzed
using the BD FACS cant II Flow Cytometer (BD).
[0903] The assay result was shown in FIG. 34. With the control
antibody MRA, fluorescence was detectable regardless of the
presence or absence of kynurenine. In contrast, for 6RNMSC1-2_F02,
fluorescence shift was observed for the first time in the presence
of 100 .mu.M kynurenine; however, fluoresce was not detectable in
the absence of kynurenine. This demonstrates that 6RNMSC1-2_F02 is
an antibody that has the ability to bind to hIL-6R expressed on
cell membrane in the presence of kynurenine.
[0904] In general, natural antibodies bind at their Fab directly to
antigens on target cells and their Fc binds to Fc.gamma.R on
effector cells, resulting in induction of cytotoxic activity (ADCC
activity) of effector cells against the target cells. In this
context, whether ADCC activity is exerted against hIL-6R-expressing
cells upon binding of 6RNMSC1-2_F02 to hIL-6R in the presence of
kynurenine was assessed by the method described below.
[0905] Variant 6RNMSC1-2_F02 with increased effector activity (the
heavy chain and light chain sequences are shown in SEQ ID NOs: 94
and 91, respectively) was used. At various concentrations of
6RNMSC1-2_F02, ADCC activity against hIL-6R-expressing cells was
assayed in the presence or absence of kynurenine according to the
method described in Reference Example 1. The assay result is shown
in FIG. 35.
[0906] The assay result confirmed that in the presence of
kynurenine, ADCC activity against hIL-6R-expressing cells was
induced by 6RNMSC1-2_F02 in an antibody concentration-dependent
manner. The finding demonstrates that the antigen binding of the
antibody via kynurenine induces ADCC activity against
antigen-expressing cells and, in terms of the function via
antitumor activity such as ADCC activity, antibodies that bind to
antigens in the presence of small molecules as a switch can also be
regulated by the presence of small molecules serving as a
switch.
[0907] Furthermore, there is a difference in the kynurenine
concentration between normal and tumor tissues, and thus it is
preferable that the ADCC activity of an antibody is exerted against
antigen-expressing tumor cells only at the kynurenine concentration
in tumor tissues, while at the normal-tissue kynurenine
concentration, the ADCC activity is impaired or not exerted. Then,
6RNMSC1-2_F02 was assessed for ADCC activity against
hIL-6R-expressing cells at various kynurenine concentrations
according to the method described in Reference Example 2. The assay
result is shown in FIG. 36. The assay result demonstrated that ADCC
activity against hIL-6R-expressing cells was included with
6RNMSC1-2_F02 in a kynurenine concentration-dependent manner.
Furthermore, in contrast to the approximately 10% ADCC activity at
4 to 6 .mu.M which is considered to be the kynurenine concentration
in normal tissues, the ADCC activity at 30 to 40 .mu.M which is
considered to be the kynurenine concentration in tumor tissues was
about 25%.
[0908] Based on these results, with 6RNMSC1-2_F02, the ADCC
activity against hIL-6R-expressing cells was weak in normal tissues
where the kynurenine concentration is low; and in tumor tissues
where the concentration is high, the ADCC activity of 6RNMSC1-2_F02
against hIL-6R-expressing cells was greater. This suggests that by
administering an antibody that uses kynurenine as a switch, the
cytotoxicity to normal tissues expressing a target antigen can be
reduced without impairing the pharmaceutical effect against tumor
tissues expressing the target antigen.
(15-6) Assessment of the Obtained Antibody for its Binding Activity
to hIL-6R in Mouse Serum by Igg ELISA
[0909] Antibody 6RNMSC1-2_F02 obtained as described in Example 14,
which binds to hIL-6R in the presence of small molecules, is an
antibody that binds to hIL-6R in the presence of kynurenine. So
far, 6RNMSC1-2_F02 has been assessed for its antigen-binding
ability in buffers such as PBS and TBS. Many unknown small
molecules including amino acids are considered to exist in mouse
serum, and one cannot rule out the possibility that such small
molecules affect the antigen binding of 6RNMSC1-2_F02. Thus,
6RNMSC1-2_F02 was assessed for its antigen-binding ability in mouse
serum.
[0910] As described in Example 14, antibody 6RNMSC1-2_F02 which has
antigen-binding activity in the presence of kynurenine, and the
known anti-hIL-6R antibody MRA were subjected to ELISA under the
two conditions described in Table 20. The antigen used was
biotin-labeled hIL-6R.
TABLE-US-00055 TABLE 20 Condition Buffer composition 1 Mouse serum
2 Mouse serum, 100 .mu.M Kynurenine
[0911] First, a StreptaWell 96 microtiter plate (Roche) was coated
at room temperature for one hour or more with 100 .mu.l of PBS
containing the biotin-labeled antigen. After washing with Wash
buffer to remove unbound antigen from the plate, each well was
blocked for one hour or more with 250 .mu.l of Blocking Buffer.
After Blocking Buffer was removed from each well, each of the
purified IgGs was prepared to 2.5 .mu.g/ml under condition 2 shown
in Table 20, and aliquoted at 100 .mu.l into the plate. The plate
was allowed to stand at room temperature for one hour to allow
binding of each IgG to the antigen in each well. After washing with
Wash Buffer containing 100 .mu.M kynurenine, an HRP-conjugated
anti-human IgG antibody (BIOSOURCE) diluted with Sample Buffer
containing kynurenine was added to each well. The plate was
incubated for one hour. Following wash with Wash Buffer containing
kynurenine, the TMB single solution (ZYMED) was added to each well.
The chromogenic reaction in the solution of each well was
terminated by adding sulfuric acid. Then, the developed color was
assessed by measuring absorbance at 450 nm.
[0912] The measurement result is shown in FIG. 37. With MRA, the
absorbance was the same regardless of the presence or absence of
kynurenine. In contrast, when 6RNMSC1-2_F02 was used, the
absorbance was markedly lower under condition 1 (mouse serum
without kynurenine) as compared to under condition 2 (mouse serum
in the presence of kynurenine) shown in Table 20. This suggests
that 6RNMSC1-2_F02 is an antibody that binds to hIL-6R as an
antigen in the presence of kynurenine without being affected by
unknown small molecules in mouse serum.
Example 16
Acquisition of Antibodies that Bind to Antigens in the Absence of
Adenosine or ATP from Antibody Library Using Phage-Display
Techniques
[0913] (16-1) Acquisition of Antibodies Whose Antigen Binding is
Inhibited in the Presence of Small Molecules from a Library Using a
Mixture of Adenosine and ATP
[0914] Antibodies that bind to target antigens in the presence of
small molecules serving as a switch were obtained as described in
Examples above. In the experiment described in this Example, the
present inventors attempted to obtain antibodies that bind to
target antigens in the absence of small molecules.
[0915] Antibodies that exhibit antigen-binding activity in the
absence of adenosine and/or ATP but whose binding ability is
impaired in the presence of adenosine and/or ATP were obtained from
a constructed phage-display library of rationally designed
antibodies. As a first step to isolate antibodies, a phage-display
library of antibodies was contacted with biotinylated adenosine and
ATP-NeutrAvidin to collect a phage-display library of antibodies
that bind to adenosine and/or ATP. Then, the phage-display antibody
library was contacted with biotinylated antigen-streptavidin in the
absence of adenosine and ATP to collect antibodies that bind to
antigens in the absence of adenosine and ATP. Panning was performed
in the alternating manner described above to screen for antibodies
that have binding activity to both antigen and adenosine and/or
ATP. In the presence of adenosine and ATP, the antigen binding of
antibodies with such properties was expected to be inhibited by
binding of adenosine and/or ATP to the antibodies.
[0916] Phages were produced in E. coli containing the phagemid
vector constructed for phage display. To the culture medium of E.
coli in which phage production was carried out, 2.5 M NaCl/10% PEG
was added to precipitate phages. The precipitated phage fraction
was diluted with TBS to prepare a phage library solution. Then, BSA
was added at a final concentration of 4% to the phage library
solution. Panning was performed using antigen-immobilized magnetic
beads. The magnetic beads used were NeutrAvidin-coated beads
(Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin-coated
beads (Dynabeads M-280 Streptavidin).
[0917] 500 pmol of biotinylated ATP, 2'-adenosine-PEG-Biotin, and
5'-adenosine-PEG-Biotin were added to the prepared phage library
solution. Thus, the phage library solution was contacted with
adenosine and ATP for 60 minutes at room temperature. Then,
BSA-blocked magnetic beads were added to the phage library solution
and the complex of phage with adenosine and/or ATP was allowed to
bind to the magnetic beads at room temperature for 15 minutes. The
beads were washed once with TBS, and then 0.5 ml of a 1 mg/ml
trypsin solution was added to the beads.
[0918] Immediately after the beads were suspended at room
temperature for 15 minutes, a phage solution was collected from the
isolated beads using a magnetic stand. The collected phage solution
was added to 10 ml of E. coli cells of strain ER2738 at the
logarithmic growth phase (OD600=0.4 to 0.7). The E. coli was
incubated with gentle stirring at 37.degree. C. for one hour to be
infected by phage. The infected E. coli was seeded in a 225
mm.times.225 mm plate. Then, phages were collected from the culture
medium of the seeded E. coli to prepare a phage library
solution.
[0919] The second round of panning was performed to enrich phages
capable of binding to the biotinylated antigen in the absence of
adenosine and ATP. Specifically, 250 pmol of biotinylated antigen
was added to the prepared phage library solution. Thus, the phage
library solution was contacted with the antigen for 60 minutes at
room temperature. Then, BSA-blocked magnetic beads were added to
the phage library solution, and the antigen-phage complex was
allowed to bind to the magnetic beads at room temperature for 15
minutes. The beads were washed twice with TBST and once with TBS.
Then, 0.5 ml of a 1 mg/ml trypsin solution was added to the beads.
Immediately after the beads were suspended at room temperature for
15 minutes, a phage solution was collected from the isolated beads
using a magnetic stand. The collected phage solution was added to
10 ml of E. coli cells of strain ER2738 at the logarithmic growth
phase (OD600=0.4 to 0.7). The E. coli was incubated with gentle
stirring at 37.degree. C. for one hour to be infected by phage. The
infected E. coli was seeded in a 225 mm.times.225 mm plate. Then,
phages were collected from the culture medium of the seeded E. coli
to prepare a phage library solution.
[0920] At subsequent odd-numbered rounds, panning was performed in
the same manner as the first-round panning. However, the number of
bead washes with TBST and TBS was increased to three times and
twice, respectively.
[0921] At subsequent even-numbered rounds, panning was performed in
the same manner as the second-round panning. However, in the fourth
and subsequent rounds of panning, the biotinylated antigen was
reduced to 40 pmol, and the number of bead washes with TBST and TBS
was increased to three times and twice, respectively.
(16-2) Assessment of Binding Activity in the Presence of Small
Molecules by Phage ELISA
[0922] Culture supernatants containing phages were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained by the method
described above. The collected culture supernatants were
ultrafiltrated using NucleoFast 96 (MACHEREY-NAGEL). 100 .mu.l of
the culture supernatants were added to each well, and the
NucleoFast 96 was centrifuged (4500 g for 45 minutes) to remove
flow-through. 100 .mu.l of H.sub.2O was added to each well, and
again the NucleoFast 96 was washed by centrifugation (4500 g for 30
minutes). After 100 .mu.l of TBS was added, the NucleoFast 96 was
allowed to stand at room temperature for five minutes. Finally, a
phage solution was collected from the supernatant in each well.
[0923] Purified phages, to which TBS, or ATP and adenosine/TBS had
been added, were subjected to ELISA by the following procedure. A
StreptaWell 96 microtiter plate (Roche) was coated overnight with
100 .mu.l of TBS containing a biotin-labeled antigen. After the
antigen was removed from each well of the plate by washing with
TBST, the wells were blocked with 250 .mu.l of 2% skim milk/TBS for
one hour or more. 2% skim milk/TBS was removed, and then the
prepared, purified phages were added to each well. The plate was
allowed to stand at 37.degree. C. for one hour to allow binding of
antibody-displaying phages to antigens in each well in the presence
or absence of 10 mM adenosine and ATP. After washing with TBST or
10 mM (ATP and adenosine)/TBST, an HRP-conjugated anti-M13 antibody
(Amersham Pharmacia Biotech) diluted with TBS or 10 mM (ATP and
adenosine)/TBS was added to each well. The plate was incubated for
one hour. Following wash with TBST or 10 mM (ATP and
adenosine)/TBST, the TMB single solution (ZYMED) was added to each
well. The chromogenic reaction in the solution of each well was
terminated by adding sulfuric acid. Then, the developed color was
assessed by measuring absorbance at 450 nm.
[0924] Phage ELISA was carried out using 96 isolated clones to
obtain from the library of rationally designed antibodies, clone
"I6RLSA1-6.sub.--011", which had an antigen-binding activity to
human IL-6 in the absence of ATP and adenosine, clone
"HSADSA1-6.sub.--020", which had antigen-binding activity to human
serum albumin (HSA) in the absence of ATP and adenosine, as well as
clones "6RRLSA1-6.sub.--037" and "6RRLSA1-6.sub.--045", which had
an antigen-binding activity to human IL-6 receptor in the absence
of ATP and adenosine (FIGS. 38, 39, and 45).
(16-3) Sequence Analysis of Antibodies for which Adenosine and ATP
Serve as a Switch
[0925] Genes were amplified using specific primers (SEQ ID NOs: 111
and 112) from clones that had been assessed to have antigen-binding
activity in the absence of adenosine and ATP based on the result of
phage ELISA described in (16-2). The nucleotide sequences of the
genes were analyzed. Based on the analysis result, the amino acid
sequences are shown in Table 21 below.
TABLE-US-00056 TABLE 21 Heavy chain Light chain Clone name SEQ ID
NO SEQ ID NO I6RLSA1-6_011 SEQ ID NO: 95 SEQ ID NO: 96
HSADSA1-6_020 SEQ ID NO: 97 SEQ ID NO: 98 6RRLSA1-6_037 SEQ ID NO:
106 SEQ ID NO: 107 6RRLSA1-6_045 SEQ ID NO: 108 SEQ ID NO: 109
Example 17
Acquisition of Antibodies that Bind to Antigens in the Presence of
Adenosine and ATP from Antibody Library Using Multivalent
Phage-Display Technique
[0926] (17-1) Acquisition of Antibodies that Bind to Antigens in
the Presence of Small Molecules from Library Using Multivalent
Display
[0927] Multivalent display of antibodies on phage was used to
obtain antibodies that have an antigen-binding activity in the
presence of adenosine and/or ATP from a phage-display library of
rationally designed antibodies. In obtaining antibodies from a
library, the acquisition probability increases when the ratio of
the antigen-binding ability between in the presence and absence of
small molecules is greater. Thus, panning based on augmentation of
the apparent binding ability was performed to efficiently collect
antibodies with binding ability in the presence of small molecules.
More specifically, the apparent binding ability was augmented
through an avidity effect (effect of multivalent antigen binding)
by allowing phages to display antibodies in a multivalent manner.
First, a phage-display library of rationally designed antibodies
was contacted with biotinylated antigens in the presence of
adenosine and ATP to collect a phage-display library of antibodies
that bind to the antigens in the presence of adenosine and ATP.
Then, according to the method described in Rondot (Nat. Biotechnol.
(2001) 19, 75-78), E. coli was infected with the collected
phage-display library of antibodies, and then infected with helper
phages that are deficient in the gene encoding pIII to prepare a
multivalent phage-display library of antibodies where antibodies
are presented at all pIIIs. The multivalent phage-display library
of antibodies was contacted with biotinylated antigen-streptavidin
in the presence of adenosine and ATP. After the library was
collected, phages were eluted from the beads in the absence of
adenosine and ATP, and collected in the eluate. This cycle of phage
preparation and panning was carried out several times to screen for
antibodies that have antigen-binding activity only in the presence
of adenosine and/or ATP.
[0928] E. coli containing the constructed phage-display phagemid
were infected with helper phage M13KO7 and cultured overnight at
30.degree. C. to produce a monovalent phage-display library of
antibodies. After phage production, 2.5 M NaCl/10% PEG was added to
the culture medium of E. coli to precipitate phages. The
precipitated phage fraction was diluted with TBS to prepare a phage
library solution. Then, BSA was added at a final concentration of
4% to the phage library solution. Panning was performed using
antigen-immobilized magnetic beads. The magnetic beads used were
NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated)
and Streptavidin-coated beads (Dynabeads M-280 Streptavidin).
[0929] The phage library solution was contacted with an antigen,
adenosine, and ATP at room temperature for 60 minutes by adding 500
pmol of biotin-labeled human IgA-Fc (SEQ ID NO: 99) as the antigen,
and a final concentration of 1 mM ATP-Na and adenosine to the
prepared phage library solution. BSA-blocked magnetic beads were
added to the phage library solution, and the antigen-phage complex
was allowed to bind to the magnetic beads at room temperature for
15 minutes. The beads were washed once with TBS dissolved with ATP
and adenosine. Then, 0.5 ml of a 1 mg/ml trypsin solution was added
to the beads. Immediately after the beads were suspended at room
temperature for 15 minutes, a phage solution was collected from the
isolated beads using a magnetic stand. The collected phage solution
was added to 10 ml of E. coli cells of strain ER2738 at the
logarithmic growth phase (OD600=0.4 to 0.7). The E. coli was
incubated with gentle stirring at 37.degree. C. for one hour to be
infected by phage. The infected E. coli was seeded in a 225
mm.times.225 mm plate. Then, helper phage M13KO7 or M13KO7ApIII
(referred to as hyperphage) (PROGEN Biotechnik) was allowed to
infect the culture medium of the seeded E. coli, and phages were
collected from the supernatant of the culture incubated overnight
at 30.degree. C. to prepare a monovalent phage-display antibody
library or a multivalent phage-display antibody library solution,
respectively.
[0930] The first round of panning was carried out to collect phages
that are capable of antigen binding in the presence of adenosine
and ATP, while the second and subsequent rounds of panning were
performed to enrich phages that are capable of antigen binding only
in the presence of adenosine and ATP. Specifically, 250 pmol of the
biotin-labeled antigen, and a final concentration of 1 mM adenosine
and ATP were each added to the prepared phage library solution.
Thus, the phage library was contacted with the antigen, adenosine,
and ATP at room temperature for 60 minutes. BSA-blocked magnetic
beads were added, and allowed to bind to the phage-antigen complex
for 15 minutes at room temperature. The beads were washed with 1 ml
of TBST dissolved with adenosine and ATP (hereinafter referred to
as (adenosine+ATP)/TBST) and with TBS dissolved with adenosine and
ATP (hereinafter referred to as (adenosine+ATP)/TBS). Then, the 0.5
ml of TBS was added to the beads. Immediately after the beads were
suspended at room temperature, a phage solution was collected from
the isolated beads using a magnetic stand. After this treatment was
repeated, the two separately eluted phage solutions were combined.
The pIII protein (helper phage-derived protein pIII) that does not
display Fab was cleaved off from phages by adding 5 .mu.l of 100
mg/ml trypsin to the collected phage solution to eliminate the E.
coli-infecting ability of the phages that do not display Fab. The
phages collected from the trypsinized phage solution were added to
10 ml of E. coli cells of strain ER2738 at the logarithmic growth
phase (OD600=0.4 to 0.7). The E. coli was incubated with gentle
stirring at 37.degree. C. for one hour to be infected with phage.
The infected E. coli was seeded in a 225 mm.times.225 mm plate.
Then, in the same manner as used in the first round panning, phages
were collected from the culture medium of the seeded E. coli to
obtain a monovalent phage-display antibody library and a
multivalent phage-display antibody library solution. Panning was
performed three times to obtain antibodies that have
antigen-binding activity in the presence of adenosine and ATP.
Meanwhile, in the third and subsequent rounds of panning, the
biotinylated antigen was used at 40 pmol.
(17-2) Assessment of Binding Activity in the Presence of Adenosine
and/or ATP by Phage ELISA
[0931] Culture supernatants containing phages were collected
according to a conventional method (Methods Mol. Biol. (2002) 178,
133-145) from single colonies of E. coli obtained by the method
described above. The collected culture supernatants were
ultrafiltrated using NucleoFast 96 (MACHERY-NAGEL). 100 .mu.l of
the culture supernatants were added to each well of NucleoFast 96
and centrifuged (4500 g for 45 minutes) to remove flow-through.
After 100 .mu.l of H.sub.2O was added, the NucleoFast 96 was washed
by centrifugation (4500 g for 30 minutes). Finally, 100 .mu.l of
TBS was added, and the NucleoFast 96 was allowed to stand for five
minutes at room temperature. A phage solution contained in the
supernatant in each well was collected.
[0932] Purified phages, to which TBS or (adenosine+ATP)/TBS was
added, were subjected to ELISA by the following procedure. A
StreptaWell 96 microtiter plate (Roche) was coated overnight with
100 .mu.l of TBS containing a biotin-labeled antigen. After the
antigen was removed from each well of the plate by washing with
TBST, the wells were blocked with 250 .mu.l of 2% skim milk/TBS for
one hour or more. 2% skim milk/TBS was removed, and then the
prepared, purified phages were added to each well. The plate was
allowed to stand for one hour to allow binding of
antibody-displaying phages to the antigen in each well in the
presence or absence of adenosine and ATP. After washing with TBST
or (adenosine+ATP)/TBST, an HRP-conjugated anti-M13 antibody
(Amersham Pharmacia Biotech) diluted with TBS or
(adenosine+ATP)/TBS was added to each well. The plate was incubated
for one hour. Following wash with TBST or (adenosine+ATP)/TBST, the
TMB single solution (ZYMED) was added to each well. The chromogenic
reaction in the solution of each well was terminated by adding
sulfuric acid. Then, the developed color was assessed by measuring
absorbance at 450 nm. The result shows that a greater number of
antibodies that have binding activity in the presence of small
molecules were obtained from the multivalent phage-display antibody
library (FIGS. 40 and 41). This finding suggests that antibodies
that have binding activity in the presence of small molecules can
be obtained more efficiently by using the multivalent phage-display
antibody library method. The result of phage ELISA is shown in
Table 22 below.
TABLE-US-00057 TABLE 22 Monovalent Multivalent presentation
presentation Number of panning 4 4 Number of clones subjected to
ELISA 96 96 Number of positive clones (Absorbance > 0.1) 6 28
Number of dependent clones (SM +/- ratio > 1.3) 1 19 Number of
dependent clone sequences 1 5
(17-3) Assessment of the Binding Ability of Antibodies that Use
Adenosine and ATP as a Switch, and Sequence Analysis
[0933] Genes were amplified using specific primers (SEQ ID NOs: 111
and 112) from clones that had been assessed to have antigen-binding
activity in the presence of adenosine and ATP based on the result
of phage ELISA described in (17-2). The nucleotide sequences of the
genes were analyzed. As a result, clone "2IADL3C5-4.sub.--048
(heavy chain, SEQ ID NOs: 100; light chain, SEQ ID NO: 101)" which
exhibits antigen-binding activity in the presence of adenosine and
ATP was obtained (FIG. 42).
Example 18
Characterization of ATP/Adenosine-Dependent Antibodies Obtained
from Library
[0934] (18-1) Preparation of ATP/Adenosine-Dependent Antibodies
Obtained from Library
[0935] Genes were amplified using specific primers from clones
6RAD2C1-4.sub.--001, 6RAD2C1-4.sub.--005, 6RAD2C1-4.sub.--011,
6RAD2C1-4.sub.--026, 6RAD2C1-4.sub.--030, 6RAD2C1-4.sub.--042,
6RAD2C1-4.sub.--076, 6RDL3C1-4.sub.--085, and 6RDL3C5-4.sub.--011,
which were obtained as described in Example 10 and were assessed to
have binding activity to biotin-labeled hIL-6R (hI-L6) in the
presence of ATP or adenosine; and their nucleotide sequences were
analyzed (Table 23).
TABLE-US-00058 TABLE 23 Heavy chain Light chain Clone name SEQ ID
NO SEQ ID NO 6RAD2C1-4_001 SEQ ID NO: 113 SEQ ID NO: 114
6RAD2C1-4_005 SEQ ID NO: 115 SEQ ID NO: 116 6RAD2C1-4_011 SEQ ID
NO: 82 SEQ ID NO: 83 6RAD2C1-4_026 SEQ ID NO: 117 SEQ ID NO: 118
6RAD2C1-4_030 SEQ ID NO: 119 SEQ ID NO: 120 6RAD2C1-4_042 SEQ ID
NO: 121 SEQ ID NO: 122 6RAD2C1-4_076 SEQ ID NO: 84 SEQ ID NO: 85
6RDL3C1-4_085 SEQ ID NO: 123 SEQ ID NO: 124 6RDL3C5-4_011 SEQ ID
NO: 125 SEQ ID NO: 126
[0936] The variable regions of 6RAD2C1-4.sub.--001,
6RAD2C1-4.sub.--005, 6RAD2C1-4.sub.--011, 6RAD2C1-4.sub.--026,
6RAD2C1-4.sub.--030, 6RAD2C1-4.sub.--042, 6RAD2C1-4.sub.--076,
6RDL3C1-4.sub.--085, and 6RDL3C5-4.sub.--011 were inserted into an
animal expression plasmid for human IgG1/Kappa that has the
antibody heavy chain constant region of SEQ ID NO: 90 and the light
chain kappa constant region sequence of SEQ ID NO: 91. Antibodies
were expressed using the method described below. FreeStyle 293-F
(Invitrogen) which was derived from human fetal kidney cells were
suspended at a cell density of 1.33.times.10.sup.6 cells/ml in the
FreeStyle 293 Expression Medium (Invitrogen), and aliquoted at 3 ml
into each well of a 6-well plate. The plasmid DNA was transfected
into the cells by lipofection. From the culture supernatants after
four days of culture in a CO.sub.2 incubator (37.degree. C., 8%
CO.sub.2, 90 rpm), antibodies were purified by a method known to
those skilled in the art using rProtein A Sepharose.TM. Fast Flow
(Amersham Biosciences). Absorbance of the purified antibody
solutions was measured at 280 nm using a spectrophotometer. From
values obtained by the measurement, concentrations of purified
antibodies were calculated using an extinction coefficient
determined by the PACE method (Protein Science (1995) 4,
2411-2423).
(18-2) Assessment of the Effect of Various Small Molecules on Human
IL6 Receptor Binding by Surface Plasmon Resonance
[0937] Biacore T200 (GE Healthcare) was used to evaluate the effect
of various small molecules on the antigen-antibody reaction of
IL-6R with 9 ATP/adenosine-dependent antibodies
(6RAD2C1-4.sub.--001, 6RAD2C1-4.sub.--005, 6RAD2C1-4.sub.--011,
6RAD2C1-4.sub.--026, 6RAD2C1-4.sub.--030, 6RAD2C1-4.sub.--042,
6RAD2C1-4.sub.--076, 6RDL3C1-4.sub.--085, and 6RDL3C5-4.sub.--011)
obtained from the library. The running buffer was used: 20 mmol/l
ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. Assay was
carried out at 25.degree. C. IL-6R was immobilized onto sensor chip
CM5 by amine coupling; and the antibodies were allowed to interact
as analyte for 120 seconds, and changes in the amount of binding
were observed. For dilution of the antibodies, the running buffer
or the running buffer containing any one of ATP, ADP, AMP, cAMP,
and adenosine (ADO) were used. The final concentration of each
small molecule and the final concentration of each antibody were
adjusted to 1 mM and 1 .mu.M, respectively. Meanwhile, under the 1
mM ATP condition, assay was carried out with a series of stepwise
antibody concentrations. The dissociation constant K.sub.D (mol/L)
of each clone for IL-6R was calculated from a plot of equilibrium
value against antibody concentration. The parameters were
calculated using the Biacore T200 Evaluation Software (GE
Healthcare). The dissociation constant K.sub.D of each clone in the
presence of 1 mM ATP is shown in Table 24.
TABLE-US-00059 TABLE 24 Dissociation constant K.sub.D Clone name
(mol/L) 6RAD2C1-4_01 3.0E-07 6RAD2C1-4_05 3.4E-07 6RAD2C1-4_11
2.3E-07 6RAD2C1-4_26 2.1E-07 6RAD2C1-4_30 3.3E-07 6RAD2C1-4_42
2.5E-07 6RAD2C1-4_76 2.5E-07 6RDL3C1-4_85 3.9E-07 6RDL3C5-4_11
1.3E-07
[0938] The amount of each clone that binds to IL-6R as determined
by this assay in the presence or absence of 1 mM small molecules is
shown in FIG. 43. As shown in FIG. 43, each clone binds to IL-6R in
the presence of 1 mM ATP, but their IL-6R binding was not
detectable in the absence of ATP. This demonstrates that each clone
has the property that it binds to IL-6R via ATP as a switch. In
small molecules besides ATP, binding of all clones was observed in
the presence of ADP, and some clones were shown to bind in the
presence of AMP and cAMP.
IL-6R Binding was not Detectable in the Presence of ADO.
[0939] This demonstrates that antibodies that bind to target
antigens in the presence of any one or more of ATP, ADP, AMP, and
cAMP can be obtained by using rationally designed libraries. As
described in this Example, panning was carried out in the presence
of both ATP and ADO which bind to antibody ATNLSA1-4_D12 used as a
reference in designing the design libraries. The result showed that
antibodies that strongly bind to target antigens were isolated in
the presence of ATP which strongly binds to ATNLSA1-4_D12 but not
in the presence of ADO which binds more weakly to ATNLSA1-4_D12
than ATP. Antibodies that bind to antigens in a manner depending on
a desired small molecule alone can be obtained by isolating
antigen-binding antibodies by contacting antigens with libraries in
the presence of the small molecule alone. For example, antibodies
that bind in the presence of ADO can be efficiently obtained from
libraries by performing panning in the presence of ADO alone.
(18-3) Effect of ATP on the ADCC Activity of Obtained
Antibodies
[0940] The obtained antibodies 6RAD2C1-4.sub.--030 and
6RAD2C1-4.sub.--011 were assessed by the method described below to
test whether ADCC activity against hIL-6R-expressing cells is
mediated via hIL-6R binding of the antibodies in the presence of
adenosine triphosphate (ATP). This assessment was carried out using
variant 6RAD2C1-4.sub.--030 with increased effector activity
(antibody heavy chain variable region, SEQ ID NO: 119; antibody
light chain variable region, SEQ ID NO: 120; antibody heavy chain
constant region, SEQ ID NO: 90, antibody light chain constant
region, SEQ ID NO: 91) and variant 6RAD2C1-4.sub.--011 with
increased effector activity (antibody heavy chain variable region,
SEQ ID NO: 82; antibody light chain variable region, SEQ ID NO: 83;
antibody heavy chain constant region, SEQ ID NO: 90; antibody light
chain constant region, SEQ ID NO: 91) prepared as described in
Example 18-1, as well as a known anti-human IL-6R antibody MRA
(antibody heavy chain variable region, SEQ ID NO: 92; antibody
light chain variable region, SEQ ID NO: 92; antibody heavy chain
constant region, SEQ ID NO: 90; antibody light chain constant
region, SEQ ID NO: 91) prepared as described in Example 15-5. At
various antibody concentrations in the presence or absence of ATP,
6RAD2C1-4.sub.--030, 6RAD2C1-4.sub.--011, and MRA were assessed for
the ADCC activity to hIL-6R-expressing cells according to the
method described in Reference Example 3. The assay result is shown
in FIG. 44.
[0941] The assay result confirmed antibody concentration-dependent
ADCC activity by 6RAD2C1-4.sub.--030 and 6RAD2C1-4.sub.--011 in the
presence of ATP. This finding shows that ADCC activity is induced
against antigen-expressing cells by antigen-antibody binding
mediated by not only kynurenine but also ATP; and thus it is
revealed that for antibodies that bind to antigens in the presence
of small molecules as a switch, their function of antitumor
activity such as ADCC activity can also be regulated by the
presence of small molecules serving as a switch.
[0942] These results suggest strong induction of ADCC activity
against hIL-6R-expressing cells is expected in tumor tissues where
ATP concentration is high, and weak induction of ADCC activity in
normal tissues where ATP concentration is low. Based on the above,
by administering antibodies for which ATP serves as a switch, the
cytotoxicity against normal tissues expressing a target antigen can
be reduced without impairing the pharmaceutical effect on tumor
tissues expressing the target antigen.
Reference Example 1
ADCC Activity of Test Antibodies Using Human Peripheral Blood
Mononuclear Cells as Effector Cells
[0943] Antibodies that bind antigens in a kynurenine-dependent
manner were assessed for their ADCC activity against
antigen-expressing cells at different antibody concentrations
according to the method described below. Human peripheral blood
mononuclear cells (hereinafter referred to as human PBMC) were used
as effector cells to measure the ADCC activity of each test
antibody as follows.
(1) Preparation of Human PBMC Solution
[0944] Syringes pre-filled with 200 .mu.l of 1000 units/ml heparin
solution (Novo-Heparin 5000 units for Injection; Novo Nordisk) were
used to collect 50 ml of peripheral blood from healthy volunteers
(male adult) affiliated with Chugai Pharmaceutical Co. Ltd. The
peripheral blood was diluted two-fold with PBS(-), and divided into
four equal parts, each of which was transferred into a
pre-centrifuged leukocyte separation tube Leucosep (Greiner
Bio-One) containing 15 ml of Ficoll-Paque PLUS. The separation
tubes containing an aliquot of the peripheral blood were
centrifuged at 2150 rpm for ten minutes at room temperature. Then,
the resulting mononuclear cell fractions were collected from the
tubes. The cells in each fraction were washed once with RPMI-1640
(nacalai tesque) supplemented with 10% FBS (hereinafter referred to
as 10% FBS/RPMI), and then suspended at a cell density of
1.times.10.sup.7 cells/ml in 10% FBS/RPMI. The cell suspensions
were used as the human PBMC solution in subsequent experiments.
(2) Preparation of Target Cells
[0945] 0.74 MBq of Cr-51 was added to 3.times.10.sup.6 cells of
BaF/hIL6R (Mihara et al., (Int. Immunopharmacol. (2005) 5, 1731-40)
which is Ba/F3 cells expressing human IL-6 receptor. Then, the
cells were incubated in 5% CO.sub.2 incubator at 37.degree. C. for
1 hour. After washing 3 times with 10% FBS/RPMI, the cells were
suspended at a cell density of 2.times.10.sup.5 cells/ml in 10%
FBS/RPMI. The cell suspension was used as the target cells in
subsequent experiments.
(3) Preparation of Kynurenine Solution
[0946] L-Kynurenine (sigma) was diluted to 5 mM with PBS(-), and
then its concentration was adjusted to 400 .mu.M using 10%
FBS/RPMI. The solution was used as the kynurenine solution in
subsequent experiments.
(4) Chrome Release Assay (ADCC)
[0947] ADCC activity was assessed based on specific chrome release
rate determined by chrome release assay. First, antibody solutions
prepared at various concentrations (0, 0.04, 0.4, 4, and 40
.mu.g/ml) were added at 50 .mu.l to each well of a round-bottomed
96-well plate. Then, the target cells prepared as described in (2)
were seeded at 50 .mu.l (1.times.10.sup.4 cells/well) to the wells.
Furthermore, the kynurenine solution prepared as described in (3)
was added at 50 .mu.l to the wells, and the plate was allowed to
stand at room temperature for 15 minutes. Then, the human PBMC
solution prepared as described in (1) was added at 50 .mu.l to each
well (5.times.10.sup.5 cells/well). The plate was allowed to stand
in5% CO.sub.2 incubator at 37.degree. C. for 4 hours, followed by
centrifugation. 100 .mu.l of culture supernatant from each well of
the plate was measured for radioactivity using a gamma counter. The
specific chrome release rate was determined based on the equation
below.
Chrome release rate (%)=(A-C).times.100/(B-C)
[0948] In this equation, "A" represents mean radioactivity (cpm) of
100 .mu.l of culture supernatant in each well. "B" represents mean
radioactivity (cpm) of 100 .mu.l of culture supernatant in a well
containing target cells, 50 .mu.l of 4% NP-40 aqueous solution
(Nonidet P-40; Nacalai Tesques), and 100 .mu.l of 10% FBS/RPMI.
Furthermore, "C" represents mean radioactivity (cpm) of 100 .mu.l
of culture supernatant in a well containing target cells, 150 .mu.l
of 10% FBS/RPMI or 100 .mu.l of 10% FBS/RPMI, and 50 .mu.l of
kynurenine solution. The test was carried out in duplicate. The
mean specific chrome release rate (%) that reflects the ADCC
activity of each test antibody was calculated based on the assay
described above.
Reference Example 2
ADCC Activity of Each Test Antibody Using Human Peripheral Blood
Mononuclear Cells as Effector Cells
[0949] Antibodies that bind to antigens in a kynurenine-dependent
manner were assessed for their ADCC activity against
antigen-expressing cells at various kynurenine concentrations
according to the method described below. Using human peripheral
blood mononuclear cells as effector cells, the ADCC activity of
each test antibody was assayed as follows. Human PBMC solution and
target cells were prepared by the same method as described in
Reference Example 1.
(1) Preparation of Kynurenine Solutions
[0950] L-Kynurenine (sigma) was diluted to 5 mM with PBS(-), and
then its concentration was adjusted to 1200, 400, 133, 44, 14.8,
and 4.9 .mu.M using 10% FBS/RPMI. The solutions were used as
kynurenine solutions in subsequent experiments.
(2) Chrome Release Assay (ADCC)
[0951] ADCC activity was assessed based on specific chrome release
rate determined by chrome release assay. First, an antibody
solution prepared to 200 .mu.g/ml was added at 50 .mu.l to each
well of a round-bottomed 96-well plate. Then, the target cells
prepared as described above were seeded at 50 .mu.l
(1.times.10.sup.4 cells/well) to the wells. Furthermore, the
kynurenine solution prepared at each concentration as described in
(1) was added at 50 .mu.l to the wells, and the plate was allowed
to stand at room temperature for 15 minutes. Then, the human PBMC
solution prepared as described above was added at 50 .mu.l to each
well (5.times.10.sup.5 cells/well). The plate was allowed to stand
in 5% CO.sub.2 incubator at 37.degree. C. for 4 hours, followed by
centrifugation. 100 .mu.l of culture supernatant from each well of
the plate was measured for radioactivity using a gamma counter. The
specific chrome release rate was determined based on the equation
described in Reference Example 1.
Reference Example 3
ADCC Activity of Test Antibodies Using Human NK Cell Line NK92 as
Effector Cells
[0952] Antibodies that bind to antigens in an ATP-dependent manner
were assessed for their ADCC activity against antigen-expressing
cells at various antibody concentrations according to the method
described below. The ADCC activity of each test antibody was
assayed by using as effector cells NK92-CD16(V) resulting from
forced expression of human FcgRIIIa in human NK cell line NK92 as
follows.
(1) Preparation of NK92-CD16(V)
[0953] NK92-CD16(V) was suspended at a cell density of
1.times.10.sup.7 cells/ml in RPMI/10% FBS. The cell suspension was
used as an NK92-CD16(V) solution in subsequent experiments.
(2) Preparation of Target Cells
[0954] 0.74 MBq of Cr-51 was added to 3.times.10.sup.6 cells of
CHO/hIL6R which is CHO cells expressing human IL-6 receptor. Then,
the cells were incubated in 5% CO.sub.2 incubator at 37.degree. C.
for 1 hour. After washing 3 times with 10% FBS/RPMI, the cells were
suspended at a cell density of 2.times.10.sup.5 cells/ml in 10%
FBS/RPMI. The cell suspension was used as target cells in
subsequent experiments.
(3) Preparation of ATP Solution
[0955] ATP (sigma) was diluted to 100 mM with 10% FBS/RPMI, and
then its concentration was adjusted to 4 mM. The solution was used
as an ATP solution in subsequent experiments.
(4) Chrome Release Assay (ADCC)
[0956] ADCC activity was assessed based on the specific chrome
release rate determined by chrome release assay. First, antibody
solutions prepared to various concentrations (0, 0.04, 0.4, 4, or
40 .mu.g/ml) were each added at 50 .mu.l to a well of a
round-bottomed 96-well plate. Then, the target cells prepared as
described in (2) were seeded at 50 .mu.l (1.times.10.sup.4
cells/well) to the wells. Furthermore, the ATP solution prepared as
described in (3) was added at 50 .mu.l to the wells, and the plate
was allowed to stand at room temperature for 15 minutes. Then, the
NK92-CD16(V) solution prepared as described in (1) was added at 50
.mu.l to each well (5.times.10.sup.5 cells/well). The plate was
allowed to stand in 5% CO.sub.2 incubator at 37.degree. C. for 4
hours, followed by centrifugation. 100 .mu.l of culture supernatant
from each well of the plate was measured for radioactivity using a
gamma counter. The specific chrome release rate was determined
based on the equation described in Reference Example 1.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 126 <210> SEQ ID NO 1 <211> LENGTH: 468
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 1 Met 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 <210> SEQ ID NO 2 <211> LENGTH:
1407 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 atgctggccg tcggctgcgc gctgctggct gccctgctgg
ccgcgccggg agcggcgctg 60 gccccaaggc gctgccctgc gcaggaggtg
gcgagaggcg tgctgaccag tctgccagga 120 gacagcgtga ctctgacctg
cccgggggta gagccggaag acaatgccac tgttcactgg 180 gtgctcagga
agccggctgc aggctcccac cccagcagat gggctggcat gggaaggagg 240
ctgctgctga ggtcggtgca gctccacgac tctggaaact attcatgcta ccgggccggc
300 cgcccagctg ggactgtgca cttgctggtg gatgttcccc ccgaggagcc
ccagctctcc 360 tgcttccgga agagccccct cagcaatgtt gtttgtgagt
ggggtcctcg gagcacccca 420 tccctgacga caaaggctgt gctcttggtg
aggaagtttc agaacagtcc ggccgaagac 480 ttccaggagc cgtgccagta
ttcccaggag tcccagaagt tctcctgcca gttagcagtc 540 ccggagggag
acagctcttt ctacatagtg tccatgtgcg tcgccagtag tgtcgggagc 600
aagttcagca aaactcaaac ctttcagggt tgtggaatct tgcagcctga tccgcctgcc
660 aacatcacag tcactgccgt ggccagaaac ccccgctggc tcagtgtcac
ctggcaagac 720 ccccactcct ggaactcatc tttctacaga ctacggtttg
agctcagata tcgggctgaa 780 cggtcaaaga cattcacaac atggatggtc
aaggacctcc agcatcactg tgtcatccac 840 gacgcctgga gcggcctgag
gcacgtggtg cagcttcgtg cccaggagga gttcgggcaa 900 ggcgagtgga
gcgagtggag cccggaggcc atgggcacgc cttggacaga atccaggagt 960
cctccagctg agaacgaggt gtccaccccc atgcaggcac ttactactaa taaagacgat
1020 gataatattc tcttcagaga ttctgcaaat gcgacaagcc tcccagtgca
agattcttct 1080 tcagtaccac tgcccacatt cctggttgct ggagggagcc
tggccttcgg aacgctcctc 1140 tgcattgcca ttgttctgag gttcaagaag
acgtggaagc tgcgggctct gaaggaaggc 1200 aagacaagca tgcatccgcc
gtactctttg gggcagctgg tcccggagag gcctcgaccc 1260 accccagtgc
ttgttcctct catctcccca ccggtgtccc ccagcagcct ggggtctgac 1320
aatacctcga gccacaaccg accagatgcc agggacccac ggagccctta tgacatcagc
1380 aatacagact acttcttccc cagatag 1407 <210> SEQ ID NO 3
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 3 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly 1 5 10 15 Val His Ser <210> SEQ ID NO 4 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Clostridium
sp. <400> SEQUENCE: 4 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu
Arg Val Pro Lys Val Ser 1 5 10 15 Ala Ser His Leu Glu 20
<210> SEQ ID NO 5 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 5 Ala 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 <210> SEQ ID NO 6
<211> LENGTH: 326 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 6 Ala 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 <210> SEQ ID NO 7 <211> LENGTH: 377 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
7 Ala 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 <210> SEQ ID NO 8
<211> LENGTH: 327 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 8 Ala 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 <210> SEQ ID NO 9 <211> LENGTH: 1125
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1125) <400> SEQUENCE: 9 atg tgg ttc ttg aca
act ctg ctc ctt tgg gtt cca gtt gat ggg caa 48 Met 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 96 Val 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 144 Val
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
192 Pro 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 240 Thr 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 288 Gly 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 336 Gln 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 384 Val 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 432 Asp 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 480 Lys 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 528 Ser
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
576 Thr 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 624 Val 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 672 Thr 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 720 Leu 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 768 Thr 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 816 Leu 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 864 Ser 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 912 Val
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
960 Val 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 1008 Lys 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 1056 Val 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 1104 Cys 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 1125 Glu Pro Gln Gly Ala Thr 370 <210> SEQ ID NO
10 <211> LENGTH: 374 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 10 Met 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
<210> SEQ ID NO 11 <211> LENGTH: 951 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(951)
<400> SEQUENCE: 11 atg act atg gag acc caa atg tct cag aat
gta tgt ccc aga aac ctg 48 Met 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 96 Trp 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 144 Ser 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 192 Ile 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 240 Arg 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 288
Ile 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 336 Asp 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 384 Pro 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 432 His 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 480 Trp 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 528 Ser 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 576 Asn 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 624 Thr 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 672
Met 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 720 Thr 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 768 Arg 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 816 Gln 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 864 Leu 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 912 Thr 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 951 Thr Leu Pro Pro Asn Asp His
Val Asn Ser Asn Asn 305 310 315 <210> SEQ ID NO 12
<211> LENGTH: 316 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 12 Met 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 <210>
SEQ ID NO 13 <211> LENGTH: 876 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(876) <400>
SEQUENCE: 13 atg gga atc ctg tca ttc tta cct gtc ctt gcc act gag
agt gac tgg 48 Met 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 96 Ala 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 144 Ala 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 192 Lys 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 240 Asp 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 288 Ser 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 336 Pro
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
384 Cys 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 432 Leu 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 480 Gly 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 528 Val 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 576 Ser 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 624 Asp 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 672 Pro 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 720 Ile
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 768 Val 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 816 Thr 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 864 Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu
Glu Pro Asp Asp Gln 275 280 285 aac cgt att tag 876 Asn Arg Ile 290
<210> SEQ ID NO 14 <211> LENGTH: 291 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 14 Met
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 <210> SEQ ID NO 15 <211>
LENGTH: 765 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(765) <400> SEQUENCE: 15 atg tgg cag ctg ctc
ctc cca act gct ctg cta ctt cta gtt tca gct 48 Met 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 96 Gly 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 144 Gln
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
192 Gly 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 240 Ser 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 288 Val 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 336 Ser 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 384 Ala 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 432 His 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 480 Gly 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 528 Lys
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
576 Gly 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 624 Gly 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 672 Val 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 720 Leu 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 765 Lys Asp His Lys Phe Lys Trp Arg
Lys Asp Pro Gln Asp Lys 245 250 <210> SEQ ID NO 16
<211> LENGTH: 254 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 16 Met 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 <210> SEQ ID NO 17 <211>
LENGTH: 702 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(702) <400> SEQUENCE: 17 atg tgg cag ctg ctc
ctc cca act gct ctg cta ctt cta gtt tca gct 48 Met 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 96 Gly 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 144 Gln
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
192 Gly 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 240 Ser 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 288 Val 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 336 Ser 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 384 Ala 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 432 His 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 480 Gly 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 528 Lys
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
576 Gly 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 624 Gly 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 672 Val 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
702 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 <210> SEQ ID
NO 18 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Met 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 <210> SEQ ID NO 19 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 19 Gly Gly Gly Ser 1 <210> SEQ
ID NO 20 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 20 Ser Gly Gly Gly 1 <210> SEQ ID NO 21 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 21 Gly Gly
Gly Gly Ser 1 5 <210> SEQ ID NO 22 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 22 Ser Gly Gly Gly Gly 1
5 <210> SEQ ID NO 23 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 23 Gly Gly Gly Gly Gly Ser 1 5 <210>
SEQ ID NO 24 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 24 Ser Gly Gly Gly Gly Gly 1 5 <210>
SEQ ID NO 25 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 25 Gly Gly Gly Gly Gly Gly Ser 1 5
<210> SEQ ID NO 26 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 26 Ser Gly Gly Gly Gly Gly Gly 1 5
<210> SEQ ID NO 27 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Asp
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 100 105 <210> SEQ ID NO 28 <211>
LENGTH: 365 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 28 Met 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 <210> SEQ ID NO 29 <211> LENGTH:
119 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 29 Met 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 <210> SEQ ID
NO 30 <211> LENGTH: 451 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 30 Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe 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 Asp Ala Val Thr Gly Asp Asp Trp
Tyr Phe Asp Leu Trp Gly 100 105 110 Arg 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 Met 420
425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 435 440 445 Pro Gly Lys 450 <210> SEQ ID NO 31
<211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 31 Ser Tyr Glu Leu Thr Gln Pro
Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr
Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser
Asp His 85 90 95 Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185
190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> SEQ ID NO 32
<211> LENGTH: 447 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 32 Glu Val Gln Leu Val Glu Ser Gly Gly Lys Leu Leu Lys
Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Phe 20 25 30 Ala Met Ser Trp Phe Arg Gln Ser Pro
Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly
Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met
Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala
Arg Gly Leu Trp Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Ser 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 <210> SEQ ID
NO 33 <211> LENGTH: 213 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 33 Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Met Ser Ala
Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser
Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser
Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr 85 90 95 Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly
Glu Cys 210 <210> SEQ ID NO 34 <211> LENGTH: 443
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 34 Gln 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 Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile 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 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 Ala Ala 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 Ala 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
<210> SEQ ID NO 35 <211> LENGTH: 219 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 35 Asp 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 Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg 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 <210> SEQ
ID NO 36 <211> LENGTH: 453 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 36 Gln Val Gln Leu Val
Leu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Met
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Ile Asn Pro Gln Ser Gly Asp Thr His Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Val Ser Thr
Gly Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Gly Ser Asp Asp Thr Ala
Ile Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Leu Ile Thr Ala Ala Gly
Pro Pro Pro Phe Glu His 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 Gly Lys 450 <210> SEQ ID NO 37
<211> LENGTH: 213 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 37 Ser Tyr Glu Leu Thr Gln Pro
Pro Ser Met Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Thr Met Pro
Cys Ser Gly Asn Gly Leu Gly Asp Lys Phe Val 20 25 30 Tyr Trp Tyr
Gln Gln Lys Pro Gly His Ser Pro Val Ala Val Ile Tyr 35 40 45 Gln
Asp Ala Lys Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser 50 55
60 Asn Ser Gly Gly Ser Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala
65 70 75 80 Met Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Gly
Thr Ala 85 90 95 Val Phe Gly Thr Gly Thr Arg Leu Ser Ile Leu Gly
Gln Pro Lys Ala 100 105 110 Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr Pro Gly Ala 130 135 140 Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys Ala Gly Val 145 150 155 160 Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185
190 Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala
195 200 205 Pro Thr Glu Cys Ser 210 <210> SEQ ID NO 38
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Oryctolagus cuniculus <400> SEQUENCE: 38 Cys Gln Ser Val Glu
Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr 1 5 10 15 Pro Leu Thr
Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala
Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ile Ile Gly Ala Asp Ser Ser Thr Trp Tyr Pro Ser Trp Val Lys
50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe
Cys Ala Arg Gly 85 90 95 Arg Phe Val Gly Tyr Thr Asn Ala Phe Asp
Pro Trp Gly Pro Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 <210> SEQ ID NO 39 <211> LENGTH: 220
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 39 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln
Ser Ser Gln Ser Val Trp Asn Asn 20 25 30 Asn Tyr Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Phe Asp
Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly
Arg Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80
Gln Cys Glu Asp Ala Ala Thr Tyr Tyr Cys His Gly Ser Tyr Ala Asn 85
90 95 Ser Gly Trp Tyr Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val
Val 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220
<210> SEQ ID NO 40 <211> LENGTH: 452 <212> TYPE:
PRT <213> ORGANISM: Oryctolagus cuniculus <400>
SEQUENCE: 40 Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro Gly Thr 1 5 10 15 Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Glu Gly Leu Glu Tyr Ile 35 40 45 Gly Phe Ile Asn Thr Gly Gly
Ser Ser Tyr Tyr Ala Pro Trp Ala Ile 50 55 60 Gly Arg Leu Thr Ile
Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val 85 90 95 Lys
Ser Tyr Val Asn Ser Asn Gly Tyr Phe Ile Phe Ser Arg Leu Asp 100 105
110 Leu Trp Gly Gln Gly Thr Leu 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 Glu Glu Met 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 <210> SEQ ID NO 41 <211> LENGTH: 218 <212>
TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus <400>
SEQUENCE: 41 Ala Gln Val Leu Thr Gln Thr Ala Ser Ser Val Ser Ala
Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Lys
Ser Val Tyr Asn Asn 20 25 30 Asn Phe Leu Ser Trp Tyr Gln Gln Lys
Leu Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Tyr Ala Ser Thr
Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Asp
Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Ile
Pro Ile Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val 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 <210> SEQ ID NO 42
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Oryctolagus cuniculus <400> SEQUENCE: 42 Cys Gln Ser Val Glu
Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr 1 5 10 15 Pro Leu Thr
Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn 20 25 30 Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile Gly Gly Ser Gly Asp Thr Gly Tyr Ala Ser Trp Ala Asn
50 55 60 Gly Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu
Lys Met 65 70 75 80 Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe
Cys Val Arg His 85 90 95 Ser Val Gly Ala Ser Trp Trp Val Phe Asn
Ile Trp Gly Pro Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 <210> SEQ ID NO 43 <211> LENGTH: 219
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 43 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln
Ser Ser Gln Ser Val Tyr Ser Gly 20 25 30 Asn Phe Phe Ala Trp Phe
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Asp
Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Thr Tyr Tyr Asn 85
90 95 Ser Gly Trp Ser Asn Val Phe Gly Gly Gly Thr Glu Val Val Val
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 <210> SEQ
ID NO 44 <211> LENGTH: 443 <212> TYPE: PRT <213>
ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 44 Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Met Pro Gly Gly 1 5 10 15
Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr 20
25 30 Asn Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr
Ile 35 40 45 Gly Phe Ile Asp Ser Gly Gly Ser Ala Tyr Tyr Ala Asn
Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr
Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Gly Val Asn Val Asp Tyr Tyr
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 <210> SEQ ID NO 45 <211> LENGTH: 217 <212>
TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus <400>
SEQUENCE: 45 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala
Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln
Ser Val Tyr Ser Asn 20 25 30 Asn Tyr Leu Ala Trp Phe Gln Gln Lys
Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Ala Ser Asn
Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val 65 70 75 80 Val Cys Asp
Asp Ala Ala Ser Tyr Tyr Cys Gln Gly Tyr Tyr Tyr Gly 85 90 95 Gly
Ile Gly Pro 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 <210> SEQ ID NO 46
<211> LENGTH: 122 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 46 Gln Val Gln Leu Gln Glu Ser
Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Thr Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ser
Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asn 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 Phe Gly Arg Lys Gly Asp Leu Asn Trp Val
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 47 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 47 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu
Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ala Gly Ser 85
90 95 Asn Asn Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 48 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 48 Gln 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 Tyr Ser Ile Ser Asp 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 Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Ala Tyr Tyr Cys 85 90 95 Ala Arg Val Leu Ala Arg Ile Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 49 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 49 Asp 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 Gln Asp Ile Ser Ser Tyr 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
Glu 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 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 100 105
<210> SEQ ID NO 50 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 50 Glu
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 Ala Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe 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 Lys Gly Pro Leu Val
Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met 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 <210> SEQ ID NO 51
<211> LENGTH: 219 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 51 Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Leu Ser Leu Leu Asn Arg 20 25 30 Asp Gly Lys
Thr Phe Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Glu Val Ser Ser Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile
65 70 75 80 Asn Arg Val Glu Ala Asp Asp Ala Gly Val Tyr Tyr Cys Met
Gln Gly 85 90 95 Leu His Leu Pro Arg Thr Phe Gly Leu Arg Thr Lys
Val 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
<210> SEQ ID NO 52 <211> LENGTH: 450 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 52 Gln
Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe 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 Asp Arg Gly Met
Gly Gly Asp Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145
150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265
270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390
395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> SEQ ID NO
53 <211> LENGTH: 216 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 53 Gln Ser Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr His Cys Ala Ala Trp
Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170
175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
<210> SEQ ID NO 54 <211> LENGTH: 456 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 54 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Tyr Gly Phe Thr Phe Ser Arg Tyr
20 25 30 Gly Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Asn Asp Ala Ser Asn Gln Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ser
Glu Asp Thr Gly Leu Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Ser Ser
Pro Lys Thr Pro Thr Ser Thr Trp Ser Ser 100 105 110 Leu Glu Ser Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145
150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile 195 200 205 Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265
270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 355 360 365 Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390
395 400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys 450
455 <210> SEQ ID NO 55 <211> LENGTH: 217 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
55 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu 35 40 45 Met Ile His Asp Val Ser Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Thr
Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Thr Thr Pro Tyr
Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr
Glu Cys Ser 210 215 <210> SEQ ID NO 56 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Phe Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser 20 25 30 <210> SEQ ID NO 57
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 57 Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> SEQ ID NO 58
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 58 Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210>
SEQ ID NO 59 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 59 Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> SEQ ID NO 60
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 60 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser
Cys 20 <210> SEQ ID NO 61 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
61 Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr
1 5 10 15 <210> SEQ ID NO 62 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 62 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys
Ser Gly Asn Thr Ala Ser 1 5 10 15 Leu Thr Val Ser Gly Leu Gln Ala
Glu Asp Glu Ala Asp Tyr Phe Cys 20 25 30 <210> SEQ ID NO 63
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 63 Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 1 5 10 <210> SEQ ID NO 64 <211> LENGTH: 50
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 64 tattactcgc
ggcccagccg gccatggcag ccwtcganwt gacccagact 50 <210> SEQ ID
NO 65 <211> LENGTH: 50 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(39)..(39) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 65 tattactcgc ggcccagccg gccatggcag
cctatgatnt gacccagact 50 <210> SEQ ID NO 66 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 66
tattactcgc ggcccagccg gccatggcag cbcaagtgct gacccagact 50
<210> SEQ ID NO 67 <211> LENGTH: 50 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 67 tattactcgc ggcccagccg gccatggcag
ccmtygtgat gacccagact 50 <210> SEQ ID NO 68 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 68
tattactcgc ggcccagccg gccatggcag ccgccgtgct gacccagact 50
<210> SEQ ID NO 69 <211> LENGTH: 50 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 69 tattactcgc ggcccagccg gccatggcgg
ctgacattgt gatgacccag 50 <210> SEQ ID NO 70 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 70
tattactcgc ggcccagccg gccatggccg ccgayrtygt gatgacccag 50
<210> SEQ ID NO 71 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 71 ctcttctaga acgcgtctaa gcgtcacccc
tattgaagct c 41 <210> SEQ ID NO 72 <211> LENGTH: 55
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 72 tattactcgc ggcccagccg
gccatggcgc agcyygtgct gactcagtcg ccctc 55 <210> SEQ ID NO 73
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 73 ctcttctaga acgcgtctaa gcttctgcag gggccaggct cttc 44
<210> SEQ ID NO 74 <211> LENGTH: 40 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 74 ttccgcctcg gcgctagccc aggagcagst
ggwggagtcc 40 <210> SEQ ID NO 75 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (25)..(26) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 75 ttccgcctcg
gcgctagccc agtcnntgga ggagtccggg 40 <210> SEQ ID NO 76
<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 76 ttccgcctcg gcgctagccc agtcgnngga
ggagtccggg 40 <210> SEQ ID NO 77 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 77 ttccgcctcg gcgctagccc
agcagcagct ggwggagtcc 40 <210> SEQ ID NO 78 <211>
LENGTH: 122 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 78 Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Ser Ser Arg Ser Thr His Ile Phe 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 Tyr Gly Arg Arg Trp Ala Met Asn Trp Val
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 79 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly
Gly Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Lys Ser Tyr Ala 85
90 95 Glu Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 80 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 80 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn His Ile Asn 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 Tyr Gly His Arg Arg Asp Leu Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 81 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 81 Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gly Gly Ser Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Val Thr Tyr Ser Ile 85 90 95 Ala Asp Pro Leu Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO
82 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 82 Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Arg Ser Ser Tyr 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 Tyr Gly Ala Leu Gly Gln Met Asn
Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 83 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 83 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Ser
Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Arg Ser Ser Ser 85
90 95 Met Asn Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 84 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 84 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Ser Tyr Ile Asp 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 Tyr Gly Ala Leu Gly Gln Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 85 <211> LENGTH: 109 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 85 Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gln Thr Ser Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Ser Arg Ser Ile Lys 85 90 95 His Pro Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 <210> SEQ ID NO 86
<211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 86 Gln 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 Ala Pro Val Val Ala Arg Pro Arg Gly
Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 120 <210> SEQ ID NO 87 <211> LENGTH: 112
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Asp Ile Val Met Thr Gln Thr Pro Leu Ser
Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu
Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu
Ile His Glu Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ala Gly Thr Asn Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85
90 95 Thr Gln Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 <210> SEQ ID NO 88 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 88 Glu Val Gln Leu Val Gln Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Val Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe 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 Leu Val Pro Ser Ser Gly Tyr Pro Gly Arg Phe Asp Pro
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 89 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 89 Asn
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 Ser
20 25 30 Leu Asn Trp Tyr Gln Gln Ile 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 Ser Tyr Tyr Cys
Gln Gln Ser Tyr Ser Ala Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 <210> SEQ ID NO 90 <211>
LENGTH: 328 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 90 Ala 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 325 <210> SEQ ID NO
91 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 91 Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40
45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 105 <210> SEQ ID NO 92 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 92 Gln 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 115 <210> SEQ ID
NO 93 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 93 Asp 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 100 105 <210> SEQ ID NO 94 <211> LENGTH: 328
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 94 Ala 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 Asp 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 Glu 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 325 <210> SEQ
ID NO 95 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 95 Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Arg Ser Ser His Ile Ser 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 Phe Gly Arg Lys Tyr Arg Met Asn
Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 96 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 96 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp
Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Val Thr Gly Ile 85
90 95 Trp Ser Val Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 97 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn His Ala Thr 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 Phe Gly Lys Lys Gly Arg Tyr Leu Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 98 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Asp Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Tyr Val Ser Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Leu Thr Thr Asp Ser 85 90 95 Leu Asn Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO
99 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 99 Cys His Pro Arg Leu
Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu 1 5 10 15 Leu Gly Ser
Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp 20 25 30 Ala
Ser Gly Val Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala 35 40
45 Val Gln Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser
50 55 60 Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys
Thr Phe 65 70 75 80 Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro
Leu Thr Ala Thr 85 90 95 Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro
Glu Val His Leu Leu Pro 100 105 110 Pro Pro Ser Glu Glu Leu Ala Leu
Asn Glu Leu Val Thr Leu Thr Cys 115 120 125 Leu Ala Arg Gly Phe Ser
Pro Lys Asp Val Leu Val Arg Trp Leu Gln 130 135 140 Gly Ser Gln Glu
Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg 145 150 155 160 Gln
Glu Pro Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu 165 170
175 Arg Val Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met
180 185 190 Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr
Ile Asp 195 200 205 Arg Leu Ala Gly Lys Gly Gly Gly Gly Ser Gly Leu
Asn Asp Ile Phe 210 215 220 Glu Ala Gln Lys Ile Glu Trp His Glu 225
230 <210> SEQ ID NO 100 <211> LENGTH: 122 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
100 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Gly His 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Arg Ser Asn His 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 Tyr Gly
Arg Lys Phe Met Met Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 101
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 101 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser
Cys Thr Gly Thr Ser Thr Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gln Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Arg Ser
Ser Asn 85 90 95 Leu Ser Pro Gly Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 <210> SEQ ID NO 102 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 102 actcgcggcc
cagccggcca tggcg 25 <210> SEQ ID NO 103 <211> LENGTH:
27 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 103 taggacggtc
agcttggtac ctccgcc 27 <210> SEQ ID NO 104 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 104 gcgcagccgg cgctagcc
18 <210> SEQ ID NO 105 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 105 tgggcccttg gtcgacgc 18
<210> SEQ ID NO 106 <211> LENGTH: 122 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 106
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5
10 15 Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Arg Ser Gly His Arg His
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 Tyr Gly Lys
Lys Gly Asn Arg Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 107
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 107 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser
Cys Thr Gly Thr Ser Thr Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Ser Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Thr Thr
Tyr Ala 85 90 95 Lys Asn Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 <210> SEQ ID NO 108 <211> LENGTH:
122 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 108 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn His Arg Ser 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 Phe Gly Lys Arg Phe Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 109 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 109
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Thr
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Lys Thr Ser Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Ser Arg Arg Tyr Arg 85 90 95 Arg Ser Leu Ser Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 110 cgcaacgcaa ttaatgtgag 20 <210> SEQ ID NO 111
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 111 gcgtcacact ttgctatg 18 <210> SEQ ID NO 112
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 112 tgagttccac gacaccgtca c 21 <210> SEQ ID NO 113
<211> LENGTH: 122 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 113 Glu Val Gln Leu Val Glu Ser
Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Ser Ser Arg Ser Asn Tyr 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 Tyr Gly Arg Leu His Asp Arg Asn Trp Val
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 114 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 114 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Arg Ser Tyr Gly 85
90 95 Ala Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 115 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 115 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Thr His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Ser Tyr Ile His 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 Phe Gly Lys Leu Gly Glu Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 116 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 116
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Val Arg Ser Asp Gly 85 90 95 His Gly Pro Met Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 117 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 117 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ala His 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 Tyr Gly Arg Leu Gly Asp Arg
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 118 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 118 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Ala Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Lys
Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Leu Arg Ala Ala Thr 85
90 95 Thr Gly Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 119 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 119 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Gly Tyr Ala 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 Phe Gly Ala Leu Asn Thr Leu Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 120 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 120
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Thr Arg Thr Ala Asn 85 90 95 Ala Gly Val Leu Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 121 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 121 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Ser Tyr Ala Ser 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 Tyr Gly Arg Leu Asn Ser His
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 122 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 122 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Gly Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Arg Ala Thr Thr 85
90 95 Lys Gly Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 123 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 123 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn Tyr 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 Tyr Gly Arg Leu His Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 124 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 124
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Ser Arg Ser Tyr Gly 85 90 95 Ala Gly Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 125 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 125 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Gly Tyr Ile Phe 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 Phe Gly Lys Leu Asn His Met
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 126 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 126 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Thr Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Arg Ala Ile Thr 85
90 95 Arg Gly Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 126
<210> SEQ ID NO 1 <211> LENGTH: 468 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met
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
<210> SEQ ID NO 2 <211> LENGTH: 1407 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2
atgctggccg tcggctgcgc gctgctggct gccctgctgg ccgcgccggg agcggcgctg
60 gccccaaggc gctgccctgc gcaggaggtg gcgagaggcg tgctgaccag
tctgccagga 120 gacagcgtga ctctgacctg cccgggggta gagccggaag
acaatgccac tgttcactgg 180 gtgctcagga agccggctgc aggctcccac
cccagcagat gggctggcat gggaaggagg 240 ctgctgctga ggtcggtgca
gctccacgac tctggaaact attcatgcta ccgggccggc 300 cgcccagctg
ggactgtgca cttgctggtg gatgttcccc ccgaggagcc ccagctctcc 360
tgcttccgga agagccccct cagcaatgtt gtttgtgagt ggggtcctcg gagcacccca
420 tccctgacga caaaggctgt gctcttggtg aggaagtttc agaacagtcc
ggccgaagac 480 ttccaggagc cgtgccagta ttcccaggag tcccagaagt
tctcctgcca gttagcagtc 540 ccggagggag acagctcttt ctacatagtg
tccatgtgcg tcgccagtag tgtcgggagc 600 aagttcagca aaactcaaac
ctttcagggt tgtggaatct tgcagcctga tccgcctgcc 660 aacatcacag
tcactgccgt ggccagaaac ccccgctggc tcagtgtcac ctggcaagac 720
ccccactcct ggaactcatc tttctacaga ctacggtttg agctcagata tcgggctgaa
780 cggtcaaaga cattcacaac atggatggtc aaggacctcc agcatcactg
tgtcatccac 840 gacgcctgga gcggcctgag gcacgtggtg cagcttcgtg
cccaggagga gttcgggcaa 900 ggcgagtgga gcgagtggag cccggaggcc
atgggcacgc cttggacaga atccaggagt 960 cctccagctg agaacgaggt
gtccaccccc atgcaggcac ttactactaa taaagacgat 1020 gataatattc
tcttcagaga ttctgcaaat gcgacaagcc tcccagtgca agattcttct 1080
tcagtaccac tgcccacatt cctggttgct ggagggagcc tggccttcgg aacgctcctc
1140 tgcattgcca ttgttctgag gttcaagaag acgtggaagc tgcgggctct
gaaggaaggc 1200 aagacaagca tgcatccgcc gtactctttg gggcagctgg
tcccggagag gcctcgaccc 1260 accccagtgc ttgttcctct catctcccca
ccggtgtccc ccagcagcct ggggtctgac 1320 aatacctcga gccacaaccg
accagatgcc agggacccac ggagccctta tgacatcagc 1380 aatacagact
acttcttccc cagatag 1407 <210> SEQ ID NO 3 <211> LENGTH:
19 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 3 Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser
<210> SEQ ID NO 4 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Clostridium sp. <400> SEQUENCE: 4
Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5
10 15 Ala Ser His Leu Glu 20 <210> SEQ ID NO 5 <211>
LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 5 Ala 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 <210> SEQ ID NO 6 <211> LENGTH: 326
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 6 Ala 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 <210> SEQ ID NO 7
<211> LENGTH: 377 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 7 Ala 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 <210> SEQ ID NO 8 <211> LENGTH: 327 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 8 Ala 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 <210> SEQ ID NO 9
<211> LENGTH: 1125 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(1125) <400> SEQUENCE: 9 atg
tgg ttc ttg aca act ctg ctc ctt tgg gtt cca gtt gat ggg caa 48 Met
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 96
Val 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 144 Val 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 192 Pro 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 240 Thr 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 288 Gly 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 336 Gln 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 384 Val 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 432 Asp 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 480 Lys
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 528 Ser 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 576 Thr 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 624 Val 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 672 Thr 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 720 Leu 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 768 Thr 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 816 Leu 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 864
Ser 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 912 Val 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 960 Val 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 1008 Lys 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 1056 Val 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 1104 Cys 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 1125 Glu Pro Gln Gly Ala Thr 370
<210> SEQ ID NO 10 <211> LENGTH: 374 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10 Met
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 <210> SEQ ID NO 11 <211> LENGTH: 951
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(951) <400> SEQUENCE: 11 atg act atg gag acc
caa atg tct cag aat gta tgt ccc aga aac ctg 48 Met 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 96 Trp 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 144 Ser
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
192 Ile 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 240 Arg 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 288 Ile 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 336 Asp 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 384 Pro 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 432 His 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 480 Trp 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 528 Ser
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
576 Asn 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 624 Thr 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 672 Met 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 720 Thr 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 768 Arg 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 816 Gln 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 864
Leu 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 912 Thr 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
951 Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315
<210> SEQ ID NO 12 <211> LENGTH: 316 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 12 Met
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 <210> SEQ ID NO 13 <211> LENGTH: 876
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(876) <400> SEQUENCE: 13 atg gga atc ctg tca
ttc tta cct gtc ctt gcc act gag agt gac tgg 48 Met 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 96 Ala 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 144 Ala
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
192 Lys 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 240 Asp 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 288 Ser 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 336 Pro 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 384 Cys 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 432 Leu 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 480 Gly 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 528 Val
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
576 Ser 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 624 Asp 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 672 Pro 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 720 Ile 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 768 Val 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 816 Thr 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 864 Tyr Ser Leu
Leu Met His Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280 285 aac
cgt att tag 876 Asn Arg Ile 290 <210> SEQ ID NO 14
<211> LENGTH: 291 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 14 Met 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 <210> SEQ ID NO 15 <211> LENGTH: 765 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(765)
<400> SEQUENCE: 15 atg tgg cag ctg ctc ctc cca act gct ctg
cta ctt cta gtt tca gct 48 Met 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 96 Gly 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 144 Gln 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 192 Gly 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 240 Ser 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 288 Val 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 336 Ser 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 384 Ala 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 432 His 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 480 Gly 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
528 Lys 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 576 Gly 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 624 Gly 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 672 Val 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 720 Leu 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 765 Lys Asp His Lys Phe Lys
Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> SEQ ID NO 16
<211> LENGTH: 254 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 16 Met 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 <210> SEQ ID NO 17 <211>
LENGTH: 702 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(702) <400> SEQUENCE: 17 atg tgg cag ctg ctc
ctc cca act gct ctg cta ctt cta gtt tca gct 48 Met 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 96 Gly 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 144 Gln
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
192 Gly 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 240 Ser 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 288 Val 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 336 Ser 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 384 Ala 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 432 His 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 480 Gly 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 528 Lys
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
576 Gly 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 624 Gly 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 672 Val 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
702 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 <210> SEQ ID
NO 18 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Met 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 <210> SEQ ID NO 19 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 19 Gly Gly Gly Ser 1 <210> SEQ
ID NO 20 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 20 Ser Gly Gly Gly 1 <210> SEQ ID NO 21
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 21 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 22
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 22 Ser Gly Gly Gly Gly 1 5 <210> SEQ ID NO 23
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 23 Gly Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 24
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 24 Ser Gly Gly Gly Gly Gly 1 5 <210> SEQ ID NO 25
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 25 Gly Gly Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO
26 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 26 Ser Gly Gly Gly Gly Gly Gly 1 5 <210> SEQ ID NO
27 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 27 Asp 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 100 105 <210> SEQ ID NO 28 <211> LENGTH: 365
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 28 Met 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 <210> SEQ ID NO 29 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 29 Met 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 <210> SEQ ID
NO 30 <211> LENGTH: 451 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 30 Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe 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 Asp Ala Val Thr Gly Asp Asp Trp Tyr Phe Asp Leu Trp Gly 100
105 110 Arg 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 Met 420 425 430 His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys
450 <210> SEQ ID NO 31 <211> LENGTH: 214 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
31 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys
1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys
Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val
Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro
Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu
Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr
Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130
135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser
210 <210> SEQ ID NO 32 <211> LENGTH: 447 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 32 Glu Val Gln Leu Val Glu Ser Gly
Gly Lys Leu Leu Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Met Ser Trp
Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu
Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Gly Leu Trp Gly Tyr Tyr Ala Leu Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Ser 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 <210> SEQ ID NO 33 <211> LENGTH: 213 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: an artificially synthesized
sequence <400> SEQUENCE: 33 Gln Ile Val Leu Ile Gln Ser Pro
Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln
Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr
Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65
70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro
Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 34 <211> LENGTH: 443 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 34 Gln 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 Ile Arg
Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile 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 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 Ala Ala 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
Ala 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 <210> SEQ ID NO 35
<211> LENGTH: 219 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 35 Asp 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 Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg 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 <210> SEQ ID NO 36
<211> LENGTH: 453 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 36 Gln Val Gln Leu Val Leu Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Met Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Asn Pro Gln Ser Gly Asp Thr His Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Val Ser Thr Gly Tyr
65 70 75 80 Met Gln Leu Ser Ser Leu Gly Ser Asp Asp Thr Ala Ile Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ser Leu Ile Thr Ala Ala Gly Pro Pro
Pro Phe Glu His 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 Gly Lys
450 <210> SEQ ID NO 37 <211> LENGTH: 213 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
37 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Met Ser Val Ser Pro Gly Gln
1 5 10 15 Thr Ala Thr Met Pro Cys Ser Gly Asn Gly Leu Gly Asp Lys
Phe Val 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly His Ser Pro Val
Ala Val Ile Tyr 35 40 45 Gln Asp Ala Lys Arg Pro Ser Gly Val Pro
Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Gly Ser Thr Ala Thr
Leu Thr Ile Ser Gly Ala Gln Ala 65 70 75 80 Met Asp Glu Ala Asp Tyr
Tyr Cys Gln Ala Trp Asp Ser Gly Thr Ala 85 90 95 Val Phe Gly Thr
Gly Thr Arg Leu Ser Ile Leu Gly Gln Pro Lys Ala 100 105 110 Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 130
135 140 Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
Val 145 150 155 160 Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser His Arg Ser Tyr 180 185 190 Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Thr Glu Cys Ser 210
<210> SEQ ID NO 38 <211> LENGTH: 446 <212> TYPE:
PRT <213> ORGANISM: Oryctolagus cuniculus <400>
SEQUENCE: 38 Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro Gly Thr 1 5 10 15 Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile
Asp Leu Ser Asn Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Gly Ala Asp Ser
Ser Thr Trp Tyr Pro Ser Trp Val Lys 50 55 60 Gly Arg Phe Thr Ile
Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Arg
Phe Val Gly Tyr Thr Asn Ala Phe Asp Pro Trp Gly Pro Gly Thr 100 105
110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230
235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355
360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> SEQ ID NO
39 <211> LENGTH: 220 <212> TYPE: PRT <213>
ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 39 Ala Gln
Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15
Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Trp Asn Asn 20
25 30 Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys
Leu 35 40 45 Leu Ile Phe Asp Ala Ser Thr Leu Ala Ser Gly Val Pro
Ser Arg Phe 50 55 60 Ser Gly Arg Gly Ser Gly Thr Gln Phe Thr Leu
Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Glu Asp Ala Ala Thr Tyr Tyr
Cys His Gly Ser Tyr Ala Asn 85 90 95 Ser Gly Trp Tyr Asp Asn Ala
Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150
155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 220 <210> SEQ ID NO 40 <211>
LENGTH: 452 <212> TYPE: PRT <213> ORGANISM: Oryctolagus
cuniculus <400> SEQUENCE: 40 Cys Gln Ser Val Glu Glu Ser Gly
Gly Arg Leu Val Thr Pro Gly Thr 1 5 10 15 Pro Leu Thr Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Tyr Met Ser Trp
Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Tyr Ile 35 40 45 Gly Phe
Ile Asn Thr Gly Gly Ser Ser Tyr Tyr Ala Pro Trp Ala Ile 50 55 60
Gly Arg Leu Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile 65
70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
Arg Val 85 90 95 Lys Ser Tyr Val Asn Ser Asn Gly Tyr Phe Ile Phe
Ser Arg Leu Asp 100 105 110 Leu Trp Gly Gln Gly Thr Leu 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 Glu Glu Met 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 <210> SEQ ID
NO 41 <211> LENGTH: 218 <212> TYPE: PRT <213>
ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 41 Ala Gln
Val Leu Thr Gln Thr Ala Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15
Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Lys Ser Val Tyr Asn Asn 20
25 30 Asn Phe Leu Ser Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro Lys
Leu 35 40 45 Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Pro
Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu
Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Asp Asp Ala Ala Thr Tyr Tyr
Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Ile Pro Ile Asn Ala Phe Gly
Gly Gly Thr Glu Val Val Val 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 <210> SEQ ID NO 42 <211> LENGTH: 446
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 42 Cys Gln Ser Val Glu Glu Ser Gly Gly Arg
Leu Val Thr Pro Gly Thr 1 5 10 15 Pro Leu Thr Leu Thr Cys Thr Val
Ser Gly Ile Asp Leu Ser Ser Asn 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Gly
Gly Ser Gly Asp Thr Gly Tyr Ala Ser Trp Ala Asn 50 55 60 Gly Arg
Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80
Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Val Arg His 85
90 95 Ser Val Gly Ala Ser Trp Trp Val Phe Asn Ile Trp Gly Pro Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210>
SEQ ID NO 43 <211> LENGTH: 219 <212> TYPE: PRT
<213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE:
43 Ala Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly
1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr
Ser Gly 20 25 30 Asn Phe Phe Ala Trp Phe Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Thr Leu Ala Ser
Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Gln Gly Thr Tyr Tyr Asn 85 90 95 Ser Gly Trp Ser
Asn Val Phe Gly Gly Gly Thr Glu Val Val Val 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 <210> SEQ ID NO 44 <211>
LENGTH: 443 <212> TYPE: PRT <213> ORGANISM: Oryctolagus
cuniculus <400> SEQUENCE: 44 Cys Gln Ser Leu Glu Glu Ser Gly
Gly Arg Leu Val Met Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Asn Ile Gln Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Phe
Ile Asp Ser Gly Gly Ser Ala Tyr Tyr Ala Asn Trp Ala Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Met 65
70 75 80
Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly 85
90 95 Gly Val Asn Val Asp Tyr Tyr 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 <210> SEQ ID NO 45
<211> LENGTH: 217 <212> TYPE: PRT <213> ORGANISM:
Oryctolagus cuniculus <400> SEQUENCE: 45 Ala Gln Val Leu Thr
Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val
Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Ser Asn 20 25 30 Asn
Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40
45 Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Asp Val 65 70 75 80 Val Cys Asp Asp Ala Ala Ser Tyr Tyr Cys Gln Gly
Tyr Tyr Tyr Gly 85 90 95 Gly Ile Gly Pro 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
<210> SEQ ID NO 46 <211> LENGTH: 122 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 Gln
Val Gln Leu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Leu 35 40 45 Ser Ser Ile Ser Ser Arg Ser Asn Tyr Ile Asn 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 Phe Gly Arg Lys
Gly Asp Leu Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 47
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 47 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Ala
Gly Ser 85 90 95 Asn Asn Val Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 <210> SEQ ID NO 48 <211> LENGTH:
119 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 48 Gln 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 Tyr Ser Ile Ser Asp 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 Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Ala Tyr Tyr Cys 85 90 95 Ala Arg Val Leu Ala Arg Ile Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 49 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 49 Asp 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 Gln Asp Ile Ser Ser Tyr 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
Glu 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 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 100 105
<210> SEQ ID NO 50 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 50 Glu 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 Ala Met Ile Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe 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 Lys Gly Pro Leu Val Ser Asp Ala Phe Asp Ile Trp Gly Gln
Gly 100 105 110 Thr Met 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
<210> SEQ ID NO 51 <211> LENGTH: 219 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 51 Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10
15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Leu Ser Leu Leu Asn Arg
20 25 30 Asp Gly Lys Thr Phe Leu Tyr Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Ser Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Gln Ile 65 70 75 80 Asn Arg Val Glu Ala Asp Asp Ala
Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Leu His Leu Pro Arg Thr
Phe Gly Leu Arg Thr Lys Val 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 <210> SEQ ID NO 52 <211> LENGTH:
450 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 52 Gln Val Gln Leu Val Gln Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe 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 Asp Arg Gly Met Gly Gly Asp Ala Phe Asp Ile Trp Gly
Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210
215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330
335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly
Lys 450 <210> SEQ ID NO 53 <211> LENGTH: 216
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 53 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr His Cys Ala Ala Trp
Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170
175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
<210> SEQ ID NO 54 <211> LENGTH: 456 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 54 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Tyr Gly Phe Thr Phe Ser Arg Tyr
20 25 30 Gly Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Asn Asp Ala Ser Asn Gln Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ser
Glu Asp Thr Gly Leu Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Ser Ser
Pro Lys Thr Pro Thr Ser Thr Trp Ser Ser 100 105 110 Leu Glu Ser Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145
150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile 195 200 205 Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265
270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 355 360 365 Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390
395 400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys 450
455 <210> SEQ ID NO 55 <211> LENGTH: 217 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
55 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu 35 40 45 Met Ile His Asp Val Ser Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Thr
Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Thr Thr Pro Tyr
Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr
Glu Cys Ser 210 215 <210> SEQ ID NO 56 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Phe Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser 20 25 30 <210> SEQ ID NO 57
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 57 Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> SEQ ID NO 58
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 58 Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210>
SEQ ID NO 59 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 59 Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> SEQ ID NO 60
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 60 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser
Cys 20 <210> SEQ ID NO 61 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
61 Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr
1 5 10 15 <210> SEQ ID NO 62
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 62 Gly Val Pro Asp Arg Phe Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser 1 5 10 15 Leu Thr Val Ser Gly
Leu Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys 20 25 30 <210>
SEQ ID NO 63 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 63 Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> SEQ ID NO 64
<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(38)..(38) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 64 tattactcgc ggcccagccg gccatggcag
ccwtcganwt gacccagact 50 <210> SEQ ID NO 65 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (39)..(39) <223>
OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 65
tattactcgc ggcccagccg gccatggcag cctatgatnt gacccagact 50
<210> SEQ ID NO 66 <211> LENGTH: 50 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 66 tattactcgc ggcccagccg gccatggcag
cbcaagtgct gacccagact 50 <210> SEQ ID NO 67 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 67
tattactcgc ggcccagccg gccatggcag ccmtygtgat gacccagact 50
<210> SEQ ID NO 68 <211> LENGTH: 50 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 68 tattactcgc ggcccagccg gccatggcag
ccgccgtgct gacccagact 50 <210> SEQ ID NO 69 <211>
LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 69
tattactcgc ggcccagccg gccatggcgg ctgacattgt gatgacccag 50
<210> SEQ ID NO 70 <211> LENGTH: 50 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: an artificially synthesized sequence
<400> SEQUENCE: 70 tattactcgc ggcccagccg gccatggccg
ccgayrtygt gatgacccag 50 <210> SEQ ID NO 71 <211>
LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 71
ctcttctaga acgcgtctaa gcgtcacccc tattgaagct c 41 <210> SEQ ID
NO 72 <211> LENGTH: 55 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 72 tattactcgc ggcccagccg gccatggcgc agcyygtgct gactcagtcg
ccctc 55 <210> SEQ ID NO 73 <211> LENGTH: 44
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <400> SEQUENCE: 73 ctcttctaga acgcgtctaa
gcttctgcag gggccaggct cttc 44 <210> SEQ ID NO 74 <211>
LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 74
ttccgcctcg gcgctagccc aggagcagst ggwggagtcc 40 <210> SEQ ID
NO 75 <211> LENGTH: 40 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(25)..(26) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 75 ttccgcctcg gcgctagccc agtcnntgga
ggagtccggg 40 <210> SEQ ID NO 76 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: an artificially
synthesized sequence <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (26)..(27) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 76 ttccgcctcg
gcgctagccc agtcgnngga ggagtccggg 40 <210> SEQ ID NO 77
<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 77 ttccgcctcg gcgctagccc agcagcagct ggwggagtcc 40
<210> SEQ ID NO 78 <211> LENGTH: 122 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 78 Glu
Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10
15 Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly His
20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Ser Arg Ser Thr His Ile Phe 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 Tyr Gly Arg Arg
Trp Ala Met Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 79
<211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 79 Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Asp Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gly Gly Ser Lys Lys Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Leu Lys Ser Tyr Ala 85 90 95 Glu Gly Pro Met Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO
80 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 80 Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Arg Ser Asn His Ile Asn 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 Tyr Gly His Arg Arg Asp Leu Asn
Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 81 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly
Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Val Thr Tyr Ser Ile 85
90 95 Ala Asp Pro Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 82 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 82 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Ser Tyr 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 Tyr Gly Ala Leu Gly Gln Met Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 83 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 83 Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Ser Val Ser Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Ser Arg Ser Ser Ser 85 90 95 Met Asn Val Leu Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO
84 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 84 Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Arg Ser Ser Tyr Ile Asp 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 Tyr Gly Ala Leu Gly Gln Arg Asn
Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 85 <211> LENGTH: 109
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 85 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Arg Ser Ile Lys 85
90 95 His Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
<210> SEQ ID NO 86 <211> LENGTH: 123 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 86 Gln
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 Ala Pro Val
Val Ala Arg Pro Arg Gly Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 87
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 87 Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn
Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro
Arg Leu Leu Ile His Glu Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55
60
Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asn Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln Ala 85 90 95 Thr Gln Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 110 <210> SEQ ID NO 88 <211>
LENGTH: 122 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 88 Glu Val Gln Leu Val Gln Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val
Val Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe 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 Leu Val Pro Ser Ser Gly Tyr Pro Gly Arg
Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 89 <211> LENGTH: 106
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 89 Asn 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 Ser 20 25 30 Leu Asn Trp Tyr Gln Gln
Ile 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 Ser Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Trp Thr 85
90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210>
SEQ ID NO 90 <211> LENGTH: 328 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 Ala 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 325
<210> SEQ ID NO 91 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 91 Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10
15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105 <210> SEQ ID NO 92 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 92 Gln 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 115
<210> SEQ ID NO 93 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 93 Asp
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 100 105 <210> SEQ ID NO 94 <211>
LENGTH: 328 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: an
artificially synthesized sequence <400> SEQUENCE: 94 Ala 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
Asp 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 Glu 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 325 <210> SEQ ID
NO 95 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 95 Glu Val Gln Leu Val
Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Arg Ser Ser His Ile Ser 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 Phe Gly Arg Lys Tyr Arg Met Asn
Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 <210> SEQ ID NO 96 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 96 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp
Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Val Thr Gly Ile 85
90 95 Trp Ser Val Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 97 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn His Ala Thr 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 Phe Gly Lys Lys Gly Arg Tyr Leu Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 98 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Gln
Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5 10
15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Asp Tyr
20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Tyr Val Ser Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Leu Thr Thr Asp Ser 85 90 95 Leu Asn Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID NO
99 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 99 Cys His Pro Arg Leu
Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu 1 5 10 15 Leu Gly Ser
Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp 20 25 30 Ala
Ser Gly Val Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala 35 40
45 Val Gln Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser
50 55 60 Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys
Thr Phe 65 70 75 80 Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro
Leu Thr Ala Thr 85 90 95 Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro
Glu Val His Leu Leu Pro 100 105 110 Pro Pro Ser Glu Glu Leu Ala Leu
Asn Glu Leu Val Thr Leu Thr Cys 115 120 125 Leu Ala Arg Gly Phe Ser
Pro Lys Asp Val Leu Val Arg Trp Leu Gln 130 135 140 Gly Ser Gln Glu
Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg 145 150 155 160 Gln
Glu Pro Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu 165 170
175 Arg Val Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met
180 185 190 Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr
Ile Asp 195 200 205 Arg Leu Ala Gly Lys Gly Gly Gly Gly Ser Gly Leu
Asn Asp Ile Phe 210 215 220 Glu Ala Gln Lys Ile Glu Trp His Glu 225
230 <210> SEQ ID NO 100 <211> LENGTH: 122 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
100 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Gly His 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Arg Ser Asn His 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 Tyr Gly Arg Lys Phe Met Met Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 101 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 101
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Phe
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Asn Ser Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Leu Arg Ser Ser Asn 85 90 95 Leu Ser Pro Gly Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 102 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 102 actcgcggcc cagccggcca tggcg 25 <210> SEQ ID NO
103 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 103 taggacggtc agcttggtac ctccgcc 27 <210> SEQ ID
NO 104 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 104 gcgcagccgg cgctagcc 18 <210> SEQ ID NO 105
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 105 tgggcccttg gtcgacgc 18 <210> SEQ ID NO 106
<211> LENGTH: 122 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 106 Glu Val Gln Leu Val Glu Ser
Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Thr Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Ser Ser Arg Ser Gly His Arg His 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 Tyr Gly Lys Lys Gly Asn Arg Asn Trp Val
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 107 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 107 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Ser
Thr Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Thr Thr Tyr Ala 85
90 95 Lys Asn Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 108 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 108 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn His Arg Ser 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 Phe Gly Lys Arg Phe Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 109 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 109
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Thr
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Lys Thr Ser Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Ser Arg Arg Tyr Arg 85 90 95 Arg Ser Leu Ser Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 110 cgcaacgcaa ttaatgtgag 20 <210> SEQ ID NO 111
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 111 gcgtcacact ttgctatg 18 <210> SEQ ID NO 112
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: an artificially synthesized sequence <400>
SEQUENCE: 112 tgagttccac gacaccgtca c 21 <210> SEQ ID NO 113
<211> LENGTH: 122 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens
<400> SEQUENCE: 113 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn Tyr 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 Tyr Gly Arg Leu His Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 114 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 114
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Ser Arg Ser Tyr Gly 85 90 95 Ala Gly Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 115 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 115 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr His 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Ser Tyr Ile His 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 Phe Gly Lys Leu Gly Glu Arg
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 116 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 116 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Val Arg Ser Asp Gly 85
90 95 His Gly Pro Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 117 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 117 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn Tyr Ala His 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 Tyr Gly Arg Leu Gly Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 118 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 118
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Ala
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Lys Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Leu Arg Ala Ala Thr 85 90 95 Thr Gly Val Leu Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 119 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 119 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Gly Tyr Ala 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 Phe Gly Ala Leu Asn Thr Leu
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 120 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 120 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Asp Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Val Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Arg Thr Ala Asn 85
90 95 Ala Gly Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 121 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 121 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Ser Tyr Ala Ser 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 Tyr Gly Arg Leu Asn Ser His
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 122 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 122 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Phe Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Gly Ser Lys Lys Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Arg Ala Thr Thr 85
90 95 Lys Gly Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 <210> SEQ ID NO 123 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 123 Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly His 20 25 30 Thr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser
Ser Arg Ser Asn Tyr 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 Tyr Gly Arg Leu His Asp Arg Asn Trp Val Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 124 <211> LENGTH: 110 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 124
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln 1 5
10 15 Thr Val Thr Ile Ser Cys Thr Gly Thr Ser Thr Asp Val Gly Asp
Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gln Val Ser Lys Lys Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Phe Cys Ser Ser Arg Ser Tyr Gly 85 90 95 Ala Gly Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> SEQ ID
NO 125 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 125 Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Gly Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Ser Ser Arg Ser Gly Tyr Ile Phe 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 Phe Gly Lys Leu Asn His Met
Asn Trp Val Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 126 <211> LENGTH:
110 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 126 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala
Ser Gly Ser Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly
Thr Ser Thr Asp Val Gly Thr Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gln
Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Thr Arg Ala Ile Thr 85
90 95 Arg Gly Val Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110
* * * * *
References