U.S. patent application number 13/612615 was filed with the patent office on 2013-03-21 for human cgrp receptor binding proteins.
The applicant listed for this patent is Thomas C. Boone, David W. Brankow, Colin V. Gegg, JR., Shaw-Fen Sylvia Hu, Chadwick T. King, Hsieng Sen Lu, Licheng Shi, Cen Xu. Invention is credited to Thomas C. Boone, David W. Brankow, Colin V. Gegg, JR., Shaw-Fen Sylvia Hu, Chadwick T. King, Hsieng Sen Lu, Licheng Shi, Cen Xu.
Application Number | 20130071410 13/612615 |
Document ID | / |
Family ID | 41718325 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130071410 |
Kind Code |
A1 |
Boone; Thomas C. ; et
al. |
March 21, 2013 |
Human CGRP Receptor Binding Proteins
Abstract
Antigen binding proteins that bind to human CGRP receptor (CGRP
R) are provided. Nucleic acids encoding the antigen binding
protein, vectors, and cells encoding the same are also provided.
The antigen binding proteins can inhibit binding of CGRP R to CGRP,
and are useful in a number of CGRP R related, disorders including
the treatment and/or prevention of migraine headaches.
Inventors: |
Boone; Thomas C.; (Newbury
Park, CA) ; Brankow; David W.; (Northridge, CA)
; Gegg, JR.; Colin V.; (Newbury Park, CA) ; Hu;
Shaw-Fen Sylvia; (Thousand Oaks, CA) ; King; Chadwick
T.; (Vancouver, CA) ; Lu; Hsieng Sen;
(Westlake Village, CA) ; Shi; Licheng; (Newbury
Park, CA) ; Xu; Cen; (Newbury Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boone; Thomas C.
Brankow; David W.
Gegg, JR.; Colin V.
Hu; Shaw-Fen Sylvia
King; Chadwick T.
Lu; Hsieng Sen
Shi; Licheng
Xu; Cen |
Newbury Park
Northridge
Newbury Park
Thousand Oaks
Vancouver
Westlake Village
Newbury Park
Newbury Park |
CA
CA
CA
CA
CA
CA
CA |
US
US
US
US
CA
US
US
US |
|
|
Family ID: |
41718325 |
Appl. No.: |
13/612615 |
Filed: |
September 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12642711 |
Dec 18, 2009 |
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13612615 |
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61203569 |
Dec 23, 2008 |
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61264622 |
Nov 25, 2009 |
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Current U.S.
Class: |
424/172.1 ;
435/252.33; 435/254.2; 435/320.1; 435/331; 435/69.6; 530/387.3;
530/387.9; 530/388.22; 530/389.1; 536/23.53 |
Current CPC
Class: |
A61P 25/04 20180101;
C07K 2317/565 20130101; A61P 29/00 20180101; C07K 2317/76 20130101;
A61P 9/00 20180101; A61P 25/00 20180101; C07K 2317/92 20130101;
A61K 2039/505 20130101; C07K 2319/30 20130101; C07K 2317/21
20130101; A61K 39/39533 20130101; A61P 3/10 20180101; A61P 25/06
20180101; C07K 2317/32 20130101; C07K 16/2869 20130101; C07K
2317/56 20130101; A61K 39/395 20130101 |
Class at
Publication: |
424/172.1 ;
530/389.1; 530/387.9; 530/387.3; 536/23.53; 530/388.22; 435/320.1;
435/252.33; 435/254.2; 435/331; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/13 20060101 C12N015/13; C07H 21/04 20060101
C07H021/04; A61P 25/00 20060101 A61P025/00; C12P 21/02 20060101
C12P021/02; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 5/10 20060101
C12N005/10; C07K 16/28 20060101 C07K016/28; A61P 25/06 20060101
A61P025/06 |
Claims
1. An isolated antigen-binding protein, wherein the isolated
antigen binding protein selectively inhibits the human CGRP
receptor.
2. The isolated antigen binding protein of claim 1, wherein the
isolated antigen binding protein selectively inhibits the human
CGRP receptor with a selectivity ratio of 100 or more.
3. The isolated antigen binding protein of claim 2, wherein the
isolated antigen binding protein selectively inhibits the human
CGRP receptor with a selectivity ratio of 500 or more.
4. The isolated antigen binding protein of claim 1, wherein the
isolated antigen binding protein specifically binds to human CGRP R
with a K.sub.D.ltoreq.100 nM.
5. The isolated antigen binding protein of claim 4, wherein the
isolated antigen binding protein specifically binds to human CGRP R
with a K.sub.D.ltoreq.10 nM as determined using a FACS binding
assay.
6. The isolated antigen binding protein of claim 1, wherein the
isolated antigen binding protein has a Ki of less than 10 nM in a
CGRP binding competition assay.
7. The isolated. antigen binding protein of claim 6, wherein the
isolated antigen binding protein has a Ki of less than 1 nM in a
radiolabeled .sup.12I-CGRP binding competition assay to membranes
from cells expressing human CGRP R.
8. The isolated antigen-binding protein of claim 1, wherein the
isolated antigen binding protein competes for binding to human CGRP
R with a reference antibody, said reference antibody comprising (i)
a heavy chain variable region comprising a sequence selected from
the group consisting of SEQ ID NOs:161, 163, 164, 166 and 168; and
(ii) a light chain variable region comprising a sequence selected
from the group consisting of SEQ ID NOs: 140, 143, 146, 148 and
150.
9. The isolated antigen binding protein of claim 8, wherein the
reference antibody comprises (i) a heavy chain defined by a
sequence selected from the group consisting of SEQ ID NOs:32, 34,
35, 37 and 39; and (ii) a light chain defined by a sequence
selected from the group consisting of SEQ ID NOs: 15, 18, 21, 23
and 25.
10. The isolated antigen binding protein of claim 9, wherein the
reference antibody comprises a heavy chain and a light chain
defined by one of the following pairs of sequences: SEQ ID NO: 32
and SEQ ID NO: 15; SEQ ID NO: 34 and SEQ ID NO: 18; SEQ ID NO: 35
and SEQ ID NO: 21; SEQ ID NO: 37 and SEQ ID NO: 23; and SEQ ID NO:
39 and SEQ ID NO: 25.
11. An isolated antigen-binding protein comprising (A) one or more
heavy chain complementary determining regions (CDRHs) selected from
the group consisting of: (i) a CDRH1 having SEQ ID NO:134; (ii) a
CDRH2 having SEQ ID NO:135; (iii) a CDRH3 having SEQ ID NO:136; and
(iv) a CDRH of (i), (ii) or (iii) that contains one, two, three or
four amino acid substitutions, deletions or insertions; (B) one or
more light chain complementary determining regions (CDRLs) selected
from the group consisting of: (i) a CDRL1 selected from the group
consisting of SEQ ID NOs:107, 111 and 118; (ii) a CDRL2 selected
from the group consisting of SEQ ID NOs: 108, 112 and 119; (iii) a
CDRL3 selected from the group consisting of SEQ ID NOs: 109, 113
and 120; and optionally (iv) a CDRL of (i), (ii) and (iii) that
contains one, two, three or four amino acid substitutions,
deletions or insertions; or (C) one or more heavy chain CDRHs of
(A) and one or more light chain CDRLs of (B).
12. The isolated antigen binding protein of claim 11, wherein the
CDRHs are further selected from the group consisting of: (i) a
CDRH1 having SEQ ID NO:131; (ii) a CDRH2 having SEQ ID NO:132;
(iii) a CDRH3 having SEQ ID NO:133; and (iv) a CDRH of (i), (ii)
and (iii) that contains one, two or three amino acid substitutions,
deletions or insertions.
13. The isolated antigen binding protein of claim 12, wherein the
CDRHs are further selected from the group consisting of: (i) a
CDRH1 selected from the group consisting of SEQ ID NO:76, 88, 100,
121, 125 and 128; (ii) a CDRH2 selected from the group consisting
of SEQ ID NO: 89, 101, 122, 124, 126, and 129; (iii) a CDRH3
selected from the group consisting of SEQ ID NO: 78, 90, 102, 123,
127, and 130; and (iv) a CDRH of (i), (ii) and (iii) that contains
one, two or three amino acid substitutions, deletions or
insertions.
14. The isolated antigen binding protein of claim 13, wherein the
CDRHs are further selected from the group consisting of: (i) a
CDRH1 selected from the group consisting of SEQ ID NO: 73, 76, 79,
82, 85, 88, 92, 97, and 100; (ii) a CDRH2 selected from the group
consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98,
101, and 129; (iii) a CDRH3 selected from the group consisting of
SEQ ID NO: 75, 78, 81, 84, 87, 90, 96, 99, 102, and 123; and (iv) a
CDRH of (i), (ii) and (iii) that contains one, two or three amino
acid substitutions, deletions or insertions.
15. The isolated antigen binding protein of claim 11, wherein the
CDRLs are further selected from the group consisting of: (i) a
CDRL1 selected from the group consisting of SEQ ID NOs:107, 111 and
115; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs:
108, 112 and 116; (iii) a CDRL3 selected from the group consisting
of SEQ ID NOs: 109, 113 and 117; and (iv) a CDRL of (i), (ii) and
(iii) that contains one, two, three, or four amino acid
substitutions, deletions or insertions.
16. The isolated antigen binding protein of claim 15, wherein the
CDRLs are further selected from the group consisting of: (i) a
CDRL1 selected from the group consisting of SEQ ID NOs: 42, 45, 51,
57, 62, 69, 103, and 110; (ii) a CDRL2 selected, from the group
consisting of SEQ ID NOs: 43, 52, 55, 58, 63, 70, 104, 108, and
114; (iii) a CDRL3 selected from the group consisting of SEQ ID
NOs: 44, 47, 53, 56, 59, 64, 105, and 106; and (iv) a CDRL of (i),
(ii) and, (iii) that contains one, two or three amino acid
substitutions, deletions or insertions.
17. The isolated antigen binding protein of claim 16, wherein the
CDRLs are further selected from the group consisting of: (i) a
CDRL1 selected from the group consisting of SEQ ID NOs: 42, 45, 48,
51, 54, 57, 62, 65, 66, and 69; (ii) a CDRL2 selected from the
group consisting of SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61, 63, 67,
and 70; (iii) a CDRL3 selected from the group consisting of SEQ ID
NOs: 44, 47, 50, 53, 56, 59, 64, 68, 71, and 72; and (iv) a CDRL of
(i), (ii) and (iii) that contains one, two or three amino acid
substitutions, deletions or insertions.
18. The isolated antigen binding protein of claim 11, wherein the
isolated antigen-binding protein comprises at least one CDRH and at
least one CDRL.
19. The isolated antigen binding protein of claim 18, wherein the
isolated antigen-binding protein comprises at least two CDRH and at
least two CDRL.
20. The isolated antigen binding protein of claim 19, wherein the
isolated antigen-binding protein comprises a CDRH1, a CDRH2, a
CDRH3, a CDRL1, a CDRL2 and a CDRL3.
21. An isolated antigen-binding protein comprising a heavy chain
variable region (V.sub.H) sequence that has at least 90% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs:158-170.
22. An isolated antigen-binding protein comprising a light chain
variable region (V.sub.L) sequence that has at least 90% sequence
identity with an amino acid sequence selected from the group
consisting of SEQ ID NOs:137-153.
23. An isolated antigen-binding protein comprising a V.sub.H
sequence that has at least 90% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 158,
159, and 162-172, and a V.sub.L sequence that has at least 90%
sequence identity with an amino acid sequence selected from the
group consisting of SEQ ID NOs: 137, 138, 140-145, 148-151, and
153-157.
24. The isolated antigen binding protein of claim 1, wherein the
isolated antigen-binding protein is selected from the group
consisting of a monoclonal antibody, a Fab fragment, an Fab'
fragment, an F(ab').sub.2 fragment, an FV fragment, a diabody, and
a single chain antibody.
25. The isolated antigen binding protein of claim 24, wherein the
isolated antigen-binding protein is a monoclonal antibody selected
from the group consisting of a fully human antibody, a humanized
antibody and a chimeric antibody.
26. The isolated antigen binding protein of claim 25, wherein the
monoclonal antibody is an IgG1-, IgG2-, IgG3-, or IgG4-type
antibody.
27. The isolated antigen binding protein of claim 26, wherein the
monoclonal antibody is an IgG1 or IgG2 antibody,
28. An isolated nucleic acid polynucleotide that encodes an
antigen-binding protein of claim 1.
29. The isolated nucleic acid polynucleotide of claim 28, wherein
the polynucleotide comprises a sequence that is 80% or more
identical with a sequence selected from the group consisting of SEQ
ID NOs:175, 176, 178, 179, 180, 181, 182, 183, 186, 187, 188, 189,
191, 192, 193, 194, 195, 196, 197, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209 and 210.
30. The isolated nucleic acid polynucleotide of claim 28, wherein
the polynucleotide comprises a sequence that is 80% or more
identical with a sequence selected from the group consisting of SEQ
ID NOs:224-258.
31. The isolated nucleic acid polynucleotide of claim 28, wherein
the polynucleotide comprises a sequence capable of hybridizing
under stringent hybridization conditions with a sequence selected
from the group consisting of SEQ ID NOs:224-258.
32. An expression vector comprising an isolated polynucleotide of
claim 28.
33. A cell line transformed with expression vector of claim 32.
34. A method of making an antigen-binding protein of claim 1,
comprising preparing the antigen binding protein from a host cell
that secretes the antigen-binding protein.
35. The method of claim 34, wherein said antigen binding protein is
generated using an immunogen comprising soluble CGRP receptor.
36. The method of claim 35, wherein said soluble CGRP receptor is
obtained by co-expressing and purifying an N-terminal extracellular
domain (ECD) of human CRLR and an ECD of human RAMP1.
37. The method of claim 36, wherein said ECD of human CRLR
comprises SEQ ID NO: 6 and said ECD of RAMP1 comprises SEQ ID NO:
8.
38. A pharmaceutical composition comprising an antigen binding
protein of claim 1 and a pharmaceutically acceptable excipient.
39. A method for treating a condition associated with CGRP R in a
patient, comprising administering to a patient an effective amount
of an isolated antigen-binding protein of claim 1.
40. The method of claim 39, wherein the condition is headache.
41. The method of claim 40, wherein the condition is migraine.
42. The method of claim 41, wherein the method comprises
prophylactic treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
12/642,711 filed 18 Dec. 2009, pending, which claims benefit of
priority to U.S. Ser. No. 61/203,569, filed 23 Dec. 2008 and U.S.
Ser. No. 61/264,622, filed 25 Nov. 2009.
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Dec. 16,
2009, is named A-1472-US-CNT-TakenFromA-1472-US-NP091212.txt, and
is 293,258 bytes in size.
BACKGROUND
[0003] The calcitonin superfamily of peptides includes at least
five known members: calcitonin, amylin, adrenomedullin, and two
calcitonin gene-related peptides ("CGRP"), CGRP1 (also known as
ctCGRP, or CGRP) and CGRP2 (also known as .beta.CGRP). CGRP is a 37
amino acid vasoactive neuropeptide expressed in both the central
and peripheral nervous systems, and has been shown to be a potent
vasodilator in the periphery, where CGRP-containing neurons are
closely associated with blood vessels. CGRP-mediated vasodilatation
is also associated with neurogenic inflammation, as part of a
cascade of events that results in extravasation of plasma and
vasodialation of the microvasculature and is present in migraine.
Amylin also has specific binding sites in the CNS and is thought to
regulate gastric emptying and have a role in carbohydrate
metabolism. Adrenomedullin is a potent vasodilator. adrenomedullin
has specific receptors on astrocytes and its messenger RNA is
upregulated in CNS tissues that are subject to ischemia.
(Zimmermann, et al., Identification of adrenomedullin receptors in
cultured rat astrocytes and in neuroblastoma glioma hybrid cells
(NG108-15), Brain Res., 724:238-245 (1996); Wang et al., Discovery
of adrenomedullin in rat ischemic cortex and evidence for its role
in exacerbating focal brain ischemic damage, Proc. Natl. Acad. Sci.
USA, 92:11480-11484 (1995)).
[0004] Calcitonin is involved in the control of bone metabolism and
is also active in the central nervous system (CNS). The biological
activities of CGRP include the regulation of neuromuscular
junctions, of antigen presentation within the immune system, of
vascular tone and of sensory neurotransmission. (Poyner, D. R.,
Calcitonin gene-related peptide: multiple actions, multiple
receptors, Pharmacol. Ther., 56:23-51 (1992); Muff et al.,
Calcitonin, calcitonin gene related peptide, adrenomedullin and
amylin: homologous peptides, separate receptors and overlapping
biological actions, Eur. J. Endocrinol., 133: 17-20 (1995)). Three
calcitonin receptor stimulating peptides (CRSPs) have also been
identified in a number of mammalian species; the CRSPs may form a
new subfamily in the CGRP family. (Katafuchi, T and Minamino, N,
Structure and biological properties of three calcitonin
receptor-stimulating peptides, novel members of the calcitonin
gene-related peptide family, Peptides, 25(11):2039-2045
(2004)).
[0005] The calcitonin superfamily peptides act through
seven-transmembrane-domain G-protein-coupled receptors (GPCRs). The
calcitonin receptor ("CT", "CTR" or "CT receptor") and CGRP
receptors are type II ("family B") GPCRs, which family includes
other GPCRs that recognize regulatory peptides such as secretin,
glucagon and vasoactive intestinal polypeptide (VIP). The best
characterized splice variants of human calcitonin receptor differ
depending on the presence (formerly CTR.sub.II+ or CTR1 now known
as CT.sub.(b)) or absence (the major splice variant, formerly
CTR.sub.II- or CTR.sub.2, now known as CT.sub.(a)) of 16 amino
acids in the first intracellular loop. (Gorn et al., Expression of
two human skeletal calcitonin receptor isoforms cloned from a giant
cell tumor of bone: the first intracellular domain modulates ligand
binding and signal transduction, J. Clin. Invest., 95:2680-2691
(1995); Hay et al., Amylin receptors: molecular composition and
pharmacology, Biochem. Soc. Trans., 32:865-867 (2004); Poyner et
al., 2002). The existence of at least two CGRP receptor subtypes
had been proposed from differential antagonist affinities and
agonist potencies in a variety of in vivo and in vitro bioassays.
(Dennis et al., CGRP8-37, A calcitonin gene-related peptide
antagonist revealing calcitonin gene-related peptide receptor
heterogeneity in brain and periphery, J. Pharmacol. Exp. Ther.,
254:123-128 (1990); Dennis et al., Structure-activity profile of
calcitonin gene-related peptide in peripheral and brain tissues.
Evidence for multiplicity, J. Pharmacol. Exp. Ther., 251:718-725
(1989); Dumont et al., A potent and selective CGRP2 agonist,
[Cys(Et)2,7]hCGRP: comparison in prototypical CGRP.sub.1 and CGRP2
in vitro assays, Can. J. Physiol. Pharmacol., 75:671-676
(1997)).
[0006] The CGRP.sub.1 receptor subtype was found to be sensitive to
the antagonist fragment CGRP(8-37). (Chiba et al., Calcitonin
gene-related peptide receptor antagonist human CGRP-(8-37), Am. J.
Physiol., 256:E331-E335 (1989); Dennis et al. (1990); Mimeault et
al., Comparative affinities and antagonistic potencies of various
human calcitonin gene-related peptide fragments on calcitonin
gene-related peptide receptors in brain and periphery, J.
Pharmacol. Exp. Ther., 258:1084-1090 (1991)). By contrast, the
CGRP.sub.2 receptor was sensitive to linear human CGRP (hCGRP)
analogs, in which the cysteine residues at positions 2 and 7 were
derivatized (e.g., with acetoaminomethyl [Cys(ACM).sup.2,7] or
ethylamide [Cys(Et).sup.2,7]) but CGRP.sub.2 receptor was
insensitive to fragment CGRP(8-37). (Dennis et al. (1989); Dennis
et al. (1990); Dumont et al. (1997)).
[0007] Ligand specificity of calcitonin receptor and
calcitonin-like receptor ("CL", "CLR" "CRLR") depend on the
co-expression of members of a family of accessory proteins called
the receptor activity modifying proteins (RAMPs). The RAMP family
includes three polypeptides (RAMP1, RAMP2 and RAMP3) that act as
receptor modulators that determine the ligand specificity of
receptors for the calcitonin family members. RAMPs are type I
transmembrane proteins that share about 30% amino acid sequence
identity and a common predicted topology, with short cytoplasmic
C-termini, one trans-membrane domain and large extracellular
N-termini that are responsible for the specificity. (McLatchie et
al., (1998) RAMPs regulate the transport and ligand specificity of
the calcitonin-receptor-like receptor, Nature, 393:333-339; Fraser
et al., (1999) The amino terminus of receptor activity modifying
proteins is a critical determinant of glycosylation state and
ligand binding of calcitonin receptor-like receptor, Molecular
Pharmacology, 55:1054-1059).
[0008] In 1998, the CGRP.sub.1 receptor was identified as a
heterodimer composed of a novel single transmembrane domain
accessory protein, receptor activity-modifying protein 1 (RAMP1),
and CRLR, (McLatchie et al., supra). Cross-linking experiments
suggested the CGRP receptor consisted of a one-to-one
stoichiometric arrangement of CRLR and RAMP1 (Hilairet et al. JBC
276, 42182-42190 (2001)), more recent studies using several
methodologies such as BRET and BiFC revealed that the functional
CGRP receptor complex may be composed of asymmetric homo-oligomer
of CRLR and monomer of RAMP1 (Heroux et al. JBC 282, 31610-31620
(2007)).
[0009] A purified CRLR N-terminal domain has been shown to
specifically bind .sup.125I-CGRP (Chauhan et al. Biochemistry 44,
782 (2005)), confirming the important and direct interaction
between the CRLR with CGRP ligand. In particular, Leu 24 and Leu 34
of CRLR are believed to constitute the docking site of the
C-terminus Phe37 of CGRP (Banerjee et al. BMC Pharmacol. 6, 9
(2006)). Furthermore, Koller et al. (FEBS Lett. 531, 464-468
(2002)) obtained evidence that that the N-terminal 18 amino acid
residues of CRLR contributes the selective interaction with CGRP or
adrenomedullin, and Ittner et al (Biochemistry 44, 5749-5754
(2005)) suggested that the N-terminal amino acid residues 23-60 of
CRLR mediate association with RAMP1.
[0010] A structure-function analysis of RAMP1 identified residues
91-103, which correlate to "helix 3" (Simms et al. Biophys. J. 91,
662-669 (2006)), as potentially significant in interaction with
CRLR, and residues Trp74 and Phe92 as potentially interacting with
the CGRP ligand in connection with its binding to the CGRP receptor
complex. Ligand binding studies using a human/rat RAMP1 chimera
suggest that the binding site for certain small molecule inhibitors
of CGRP R (e.g., BIBN409613S), is located within a region which
includes amino acids 66-102 of RAMP1 (Mallee et al, JBC 277,
14294-14298 (2002)).
[0011] CRLR has 55% overall amino acid sequence identity with CTR,
although the transmembrane domains are almost 80% identical.
(McLatchie et al. (1998); Poyner et al., International union of
pharmacology, XXXII. The mammalian calcitonin gene-related
peptides, adrenomedullin, amylin and calcitonin receptors,
Pharmacol. Rev., 54:233-246 (2002)).
[0012] CRLR has been shown to form a high affinity receptor for
CGRP, when associated with RAMP1, or, to preferentially bind
adrenomedullin when associated with RAMP2 or RAMP3. (McLatchie et
al. (1998); Sexton et al., Receptor activity modifying proteins,
Cellular Signaling, 13:73-83 (2001); Conner et al., Interaction of
calcitonin-gene-related peptide with its receptors, Biochemical
Society Transactions 30(Part 4): 451-454 (2002)). The glycosylation
state of CRLR is associated with its pharmacology. RAMPs 1, 2, and
3 transport CRLR to the plasma membrane with similar efficiencies,
however RAMP1 presents CRLR as a terminally glycosylated, mature
glycoprotein and a CGRP receptor, whereas RAMPS 2 and 3 present
CRLR as an immature, core glycosylated adrenomedullin receptor
("AM" or "AMR" or "AM receptor". (Fraser et al. (1999)).
Characterization of the CRLR/RAMP2 and CRLR/RAMP3 receptors in
HEK293T cells by radioligand binding (.sup.125I-adrenomedullin as
radioligand), functional assay (cAMP measurement), or biochemical
analysis (SDS-polyacrylamide gel electrophoresis) revealed them to
be indistinguishable, even though RAMPs 2 and 3 share only 30%
amino acid sequence identity. (Fraser et al. 1999)). Differences
have been observed, however, in the pharmacology for CRLR expressed
with RAMP 2 versus RAMP 3. Both CGRP and CGRP8-37, as well as
adrenomedullin and the adrenomedullin-derived peptide AM 22-52, are
active at the RAMP 3 heterodimer, indicating that this complex may
act as both a CGRP and an AM receptor. (Howitt et al., British
Journal of Pharmacology, 140;477-486 (2003); Muff et al.,
Hypertens. Res., 26:S3-S8 (2003)). Co-expression of human CRLR with
rat RAMP1, and vice versa, suggested that the RAMP1 species
determined the pharmacological characteristics of the CRLR/RAMP1
complex with respect to several small molecule CGRP receptor
antagonists tested. (Mallee et al., Receptor Activity-Modifying
Protein 1 determines the species selectivity of non-peptide CGRP
receptor antagonists, J. Biol. Chem., 277(16):14294-14298 (2002)).
Unless associated with a RAMP, CRLR is not known to bind any
endogenous ligand; it is currently the only GPCR thought to behave
this way. (Conner et al., A key role for transmembrane prolines in
calcitonin receptor-like agonist binding and signaling:
implications for family B G-protein-coupled receptors, Molec.
Pharmacol., 67(1):20-31 (2005)).
[0013] Calcitonin receptor (CT) has also been demonstrated to form
heterodimeric complexes with RAMPs, which are known as amylin
receptors ("AMY", "AMY R" or "AMY receptor"). Generally, CT/RAMP1
receptors (referred to as "AMY.sub.1" or "AMY1") have high affinity
for salmon calcitonin, amylin and CGRP and lower affinity for
mammalian calcitonins. For CT/RAMP2 receptors ("AMY.sub.2" or
"AMY2") and CT/RAMP3 receptors ("AMY.sub.3" or "AMY3"), a similar
pattern is principally observed, although the affinity for CGRP is
lower and may not be significant at physiologically relevant ligand
concentrations. The precise receptor phenotype is dependent on cell
type and CTR splice variant (CT.sub.(a) or CT.sub.(b)),
particularly for RAMP2-generated amylin receptors. For example, a
pure population of osteoclast-like cells reportedly expressed
RAMP2, CTR, and CRLR, but not RAMP1 or RAMP3. (Hay et al. (2004);
Christopoulos et al., Multiple amylin receptors arise from receptor
activity-modifying protein interaction with the calcitonin receptor
gene product, Molecular Pharmacology, 56:235-242 (1999); Muff et
al., An amylin receptor is revealed following co-transfection of a
calcitonin receptor with receptor activity modifying proteins-1 or
-3, Endocrinology, 140:2924-2927 (1999); Sexton et al. (2001);
Leuthauser et al., Receptor-activity-modifying protein 1 forms
heterodimers with two G-protein-coupled receptors to define ligand
recognition, Biochem. J., 351:347-351 (2000); Tilakaratne et al.,
Amylin receptor phenotypes derived from human calcitonin
receptor/RAMP co-expression exhibit pharmacological differences
dependent on receptor isoform and host cell environment, J.
Pharmacol. Exp. Ther., 294:61-72 (2000); Nakamura et al.,
Osteoclast-like cells express receptor activity modifying protein
2: application of laser capture microdissection, J. Molec.
Endocrinol., 34:257-261 (2005)).
[0014] Table 1, below, summarizes the relationship of the receptor
components discussed above.
TABLE-US-00001 TABLE 1 Receptor CT (calcitonin Component CRLR (CL)
receptor) RAMP1 CGRP receptor AMY1 receptor RAMP2 AM1 receptor AMY2
receptor RAMP3 AM2 receptor AMY3 receptor
[0015] Therapeutic uses of CGRP antagonists have been proposed.
Noda et al. described the use of CGRP or CGRP derivatives for
inhibiting platelet aggregation and for the treatment or prevention
of arteriosclerosis or thrombosis. (EP 0385712 B1). Liu et al.
disclosed therapeutic agents that modulate the activity of CTR,
including vehicle-conjugated peptides such as calcitonin and human
.alpha.CGRP. (WO 01/83526 A2; U.S. 2002/0090646 A1). Vasoactive
CGRP peptide antagonists and their use in a method for inhibiting
CGRP binding to CGRP receptors were disclosed by Smith et al.; such
CGRP peptide antagonists were shown to inhibit CGRP binding to
coronary artery membranes and to relax capsaicin-treated pig
coronary arteries. (U.S. Pat. No. 6,268,474 B1; and U.S. Pat. No.
6,756,205 B2). Rist et al. disclosed peptide analogs with CGRP
receptor antagonist activity and their use in a drug for treatment
and prophylaxis of a variety of disorders. (DE 19732944 A1).
[0016] CGRP is a potent vasodilator that has been implicated in the
pathology of a number of vasomotor symptoms, such as all forms of
vascular headache, including migraines (with or without aura) and
cluster headache. Durham, N. Engl. J. Med. 350:1073-1075, 2004,
Migraine pathophysiology involves the activation of the trigeminal
ganglia, where CGRP is localized, and CGRP levels significantly
increase during a migraine attack. This in turn, promotes cranial
blood vessel dilation and neurogenic inflammation and
sensitization. (Doods, H., Curr. Opin. Investig. Drugs, 2:1261-1268
(2001)). Further, the serum levels of CGRP the external jugular
vein are elevated in patients during migraine headache. Goadsby et
al., Ann. Neurol. 28:183-7, 1990, Intravenous administration of
human ci-CGRP induced headache and migraine in patients suffering
from migraine without aura, supporting the view that CGRP has a
causative role in migraine (Lassen et al, Cephalalgia 22:54-61,
2002).
[0017] Migraine is a complex, common neurological condition that is
characterized by severe, episodic attacks of headache and
associated features, which may include nausea, vomiting,
sensitivity to light, sound or movement. In some patients, the
headache is preceded or accompanied by an aura. The headache pain
may be severe and may also be unilateral in certain patients.
Migraine attacks are disruptive to daily life. In US and Western
Europe, the overall prevalence of migraine sufferers is 11% of the
general population (6% males; 15-18% females). Furthermore, the
median frequency of attacks in an individual is 1.5/month. While
there are a number of treatments available to alleviate or reduce
symptoms, preventive therapy is recommended for those patients
having more than 3-4 attacks of migraine per month. Goadsby, et al.
New Engl. J. Med. 346(4): 257-275, 2002. Some migraine patients
have been treated with topiramate, an anticonvulsant that blocks
voltage-dependent sodium channels and certain glutamate receptors
(AMPA-kainate), potentiates GABA-A receptor activity, and blocks
carbonic anhydrase. The relatively recent success of serotonin
5HT-I B/ID and/or 5HT-1 a receptor agonists, such as sumatriptan,
in some patients has led researchers to propose a serotonergic
etiology of the disorder. Unfortunately, while some patients
respond well to this treatment, others are relatively resistant to
its effects.
[0018] Possible CGRP involvement in migraine has been the basis for
the development and testing of a number of compounds that inhibit
release of CGRP (e.g., sumatriptan), antagonize at the CGRP
receptor (e.g., dipeptide derivative BIBN4096BS (Boehrinwer
Ingelheim); CGRP(8-37)), or interact with one or more of
receptor-associated proteins, such as, RAMP1. Brain, S. et al.,
Trends in Pharmacological Sciences 23:51-53, 2002. Alpha-2
adrenoceptor subtypes and adenosine A1 receptors also control
(inhibit) CGRP release and trigeminal activation (Goadsby et al.,
Brain 125:1392-401, 2002). On the other hand, treatment with
compounds that exclusively inhibit neurogenic inflammation (e.g.,
tachykinin NKI receptor antagonists) or trigeminal activation
(e.g., 5HT10 receptor agonists) appears to be relatively
ineffective as acute treatments for migraine, leading some to
question whether inhibiting release of CGRP is the basis of
effective anti-migraine treatments. Arulmani et al., Eur. J.
Pharmacol. 500:315-330, 2004.
[0019] Although the precise pathophysiology of migraine is not yet
well understood, the therapeutic use of CGRP antagonists and
CGRP-targeting aptamers has been proposed for the treatment of
migraine and other disorders. (E.g., Olesen Calcitonin gene-related
peptide receptor antagonist BIBN 4096 BS for the acute treatment of
migraine, New Engl. J. Med., 350:1104-1110 (2004); Perspective:
CGRP-receptor antagonists--a fresh approach to migraine, New Engl.
J. Med., 350:1075 (2004); Vater et al., Short bioactive Spiegelmers
to migraine-associated calcitonin gene-related peptide rapidly
identified by a novel approach: tailored-SELEX, Nuc. Acids Res.,
31(21 e130):1-7 (2003); WO 96/03993). Further, a potent
small-molecule CGRP antagonist has been shown to relieve
moderate-to-severe migraine attacks, including migraine pain and
migraine-associated symptoms, in a recent Phase III clinical trial
(Connor, et al. Efficacy and Safety of telcagepant (MK-0974), a
Novel Oral CGRP Receptor Antagonist, for Acute Migraine Attacks.
Poster, European Headache and Migraine Trust International
Congress, London, England, September 2008).
[0020] CGRP may also be involved in chronic pain syndromes other
than migraine. In rodents, intrathecally delivered CGRP induces
severe pain, and CGRP levels are enhanced in a number of pain
models. In addition, CGRP antagonists partially block nociception
in acute pancreatitis in rodents (Wick, et al., (2006) Surgery,
Volume 139, Issue 2, Pages 197-201). Together, these observations
imply that a potent and selective CGRP receptor antagonist can be
an effective therapeutic for treatment of chronic pain, including
migraine.
SUMMARY
[0021] Isolated antibodies, antigen-binding fragments thereof and
other isolated antigen-binding proteins that bind CGRP R,
particularly primate CGRP R, e.g., human CGRP R, are described
herein. Such isolated antigen-binding proteins may selectively
inhibit primate CGRP R (as compared with primate AM1, AM2, CT or
amylin receptors) and may bind both the CRLR and RAMP1 components
of CGRP R. The CGRP R binding proteins were found to inhibit,
interfere with, or modulate at least one of the biological
responses related to CGRP R, and as such, are useful for
ameliorating the effects of CGRP R-related diseases or disorders.
Binding of certain antigen-binding proteins to CGRP R can,
therefore, have one or more of the following activities:
inhibiting, interfering with, or modulating CGRP R, inhibiting
vasodialation, decreasing neurogenic inflammation, and alleviating,
ameliorating, treating, preventing, or reducing symptoms of chronic
pain or migraine.
[0022] In one exemplary aspect, the isolated antigen-binding
proteins selectively inhibit human CGRP receptor (as compared with
the human AM1, AM2 or amylin receptors). In some embodiments, the
isolated antigen binding protein selectively inhibits the human
CGRP receptor with a selectivity ratio of 50 or more, 75 or more,
100 or more, 150 or more, 200 or more, 250 or more, 300 or more,
400 or more, 500 or more, 750 or more or 1,000 or more. The degree
of selective inhibition may be determined using any suitable
method, e.g., using cAMP assay as described in the Examples herein.
In some embodiments, the isolated antigen binding protein
specifically binds to both human CRLR and human RAMP1, and does not
specifically bind to human AM1, human AM2 or a human amylin
receptor (e.g., AMY1 or AMY2). For example, the isolated antigen
binding protein may specifically bind human CGRP R with a
K.sub.D.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, or .ltoreq.5
nM. In some embodiments, the isolated antigen binding protein
specifically binds to human CGRP R with a K.sub.D.ltoreq.100 nM,
.ltoreq.10 nM, or .ltoreq.5 nM as determined using a FACS binding
assay and analyzed, for example, using methods described in
Rathanaswami, et al., Biochemical and Biophysical Research
Communications 334 (2005) 1004-1013. In some embodiments, the
isolated antigen binding protein has a Ki of .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, nM or .ltoreq.0.1 nM in a CGRP binding
competition assay. In some embodiments, the isolated antigen
binding protein has a Ki of .ltoreq.100 nM, .ltoreq.50 nM,
.ltoreq.20 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.5 nM or
.ltoreq.0.1 nM in a radiolabeled .sup.125I-CGRP binding competition
assay to membranes from cells expressing human CGRP R, for example,
the assay described, in Example 5 herein.
[0023] In another exemplary aspect, the isolated antigen-binding
proteins compete for binding to human CGRP R, e.g., the
extracellular portion of CGRP R, with a reference antibody
comprising a heavy chain variable region comprising a sequence
selected from the group consisting of SEQ ID NO:158-170 and a light
chain variable region comprising a sequence selected from the group
consisting of SEQ ID NO:137-153. In some embodiments, binding
competition is assessed using a binning assays, e.g., using a
Biacore analysis, for example, as described in Example 7 herein. In
some embodiments, the isolated antigen binding protein competes for
binding to human CGRP R with a reference antibody, the reference
antibody comprising (i) a heavy chain variable region comprising a
sequence selected from the group consisting of SEQ ID NOs:161, 163,
164, 166 and 168; and (ii) a light chain variable region comprising
a sequence selected from the group consisting of SEQ ID NOs: 110,
143, 146, 148 and 150. In certain embodiments, the reference
antibody comprises (i) a heavy chain defined by a sequence selected
from the group consisting of SEQ ID NOs:32, 34, 35, 37 and 39; and
(ii) a light chain defined by a sequence selected from the group
consisting of SEQ ID NOs: 15, 18, 21, 23 and 25. In more specific
embodiments, the reference antibody comprises a heavy chain and a
light chain defined by one of the following pairs of sequences: (i)
SEQ ID NO: 32 and SEQ ID NO: 15; (ii) SEQ ID NO: 34 and SEQ ID NO:
18; (iii) SEQ ID NO: 35 and SEQ ID NO: 21; (iv) SEQ ID NO: 37 and
SEQ ID NO: 23; and (v) SEQ ID NO: 39 and SEQ ID NO: 25. In one such
embodiment, the reference antibody comprises a heavy chain
comprising SEQ ID NO: 32 and a light chain comprising SEQ ID NO:
15. In another such embodiment, the reference antibody comprises a
heavy chain comprising SEQ ID NO: 34 and a light chain comprising
SEQ ID NO: 18. In another such embodiment, the reference antibody
comprises a heavy chain comprising SEQ ID NO: 35 and a light chain
comprising SEQ ID NO: 21. In another such embodiment, the reference
antibody comprises a heavy chain comprising SEQ ID NO: 37 and a
light chain comprising SEQ ID NO: 23. In another such embodiment,
the reference antibody comprises a heavy chain comprising SEQ ID
NO: 39 and a light chain comprising SEQ ID NO: 25.
[0024] In certain embodiments, the isolated antigen-binding
proteins that compete for binding to human CGRP R also selectively
inhibit the human CGRP receptor, e.g., with a selectivity ratio of
100 or more, 250 or more, 500 or more, 750 or more, 1,000 or more,
2,500 or more, 5,000 or more or 10,000 or more, and such
selectivity may be determined, e.g., using a cAMP assay as
described in the Examples herein. In related embodiments, the
isolated antigen-binding proteins that compete for binding to human
CGRP R specifically binds to human CGRP R with a K.sub.D.ltoreq.1
.mu.M, .ltoreq.100 nM, .ltoreq.10 nM, or .ltoreq.5 nM, e.g., as
determined using a FACS binding assay and analyzed, for example,
using methods described in Rathanaswami, et al., Biochemical and
Biophysical Research Communications 334 (2005) 1004-1013. In
related embodiments, the isolated antigen-binding proteins that
compete for binding to human CGRP R have a Ki of .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.5 nM or <0.1 nM in a CGRP
binding competition assay, e.g., in a radiolabeled .sup.125I-CGRP
binding competition assay to membranes from cells expressing human
CGRP R, for example, the assay described in Example 5 herein.
[0025] In any of the above-mentioned embodiments, the isolated
antigen-binding protein that competes for binding to human CGRP R
may be, for example, a monoclonal antibody, a polyclonal antibody,
a recombinant antibody, a human (e.g., fully human) antibody, a
humanized antibody, a chimeric antibody, a multi-specific antibody,
or an antigen binding fragment thereof. Further, the antibody
fragment of the isolated antigen-binding protein that competes for
binding to human CGRP R can be a Fab fragment, and Fab' fragment,
an F(ab').sub.2 fragment, an Fv fragment, a diabody or a single
chain antibody molecule; and may be, for example, a human
monoclonal antibody, e.g., an IgG1-, IgG2-, IgG3-, or IgG4-type
antibody. In certain embodiments, the isolated antigen binding
proteins that compete for binding to human CGRP R may be
neutralizing antigen binding proteins.
[0026] In certain exemplary aspects, the isolated antigen-binding
proteins described, e.g., isolated antibodies or fragments thereof,
comprise (A) one or more heavy chain complementary determining
regions (CDRHs) selected from the group consisting of: (i) a CDRH1
having SEQ ID NO:134; (ii) CDRH2 having SEQ ID NO:135; (iii) CDRH3
having SEQ ID NO:136; and optionally (iv) a CDRH of (i), (ii) and
(iii) that contains one or more amino acid substitutions (e.g.,
conservative amino acid substitutions), deletions or insertions
that collectively total no more than four amino acids; (B) one or
more light chain complementary determining regions (CDRLs) selected
from the group consisting of: (i) a CDRL1 selected from the group
consisting of SEQ ID NOs:107, 111 and 118; (ii) a CDRL2 selected
from the group consisting of SEQ ID NOs: 108, 112 and 119; (iii) a
CDRL3 selected from the group consisting of SEQ ID NOs: 109, 113
and 120; and optionally (iv) a CDRL of (i), (ii) and (iii) that
contains one or more, e.g., one, two, three, four or more, amino
acid substitutions (e.g., conservative amino acid substitutions),
deletions or insertions that collectively total no more than four
amino acids; or (C) one or more heavy chain CDRHs of (A) and one or
more light chain CDRLs of (B).
[0027] In some embodiments, the CDRHs are further selected from the
group consisting of: (i) a CDRH1 having SEQ ID NO:131; (ii) a CDRH2
having SEQ ID NO:132; (iii) a CDRH3 having SEQ ID NO:133; and
optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or
more, e.g., one, two, three, four or more amino acid substitutions
(e.g., conservative amino acid substitutions), deletions or
insertions that collectively total no more than three amino acids.
In related embodiments, the CDRHs are further selected from the
group consisting of: (i) a CDRH1 selected from the group consisting
of SEQ ID NO:76, 88, 100, 121, 125 and 128; (ii) a CDRH2 selected
from the group consisting of SEQ ID NO: 89, 101, 122, 124, 126, and
129; (iii) a CDRH3 selected from the group consisting of SEQ ID NO:
78, 90, 102, 123, 127, and 130; and optionally (iv) a CDRH of (i),
(ii) and (iii) that contains one or more, e.g., one, two, three,
four or more amino acid substitutions (e.g., conservative, amino
acid substitutions), deletions or insertions that collectively
total no more than two amino acids. In other related embodiments,
the CDRHs are further selected from the group consisting of: (i) a
CDRH1 selected from the group consisting of SEQ ID NO: 73, 76, 79,
82, 85, 88, 92, 97, and 100; (ii) CDRH2 selected from the group
consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98,
101, and 129; (iii) a CDRH3 selected from the group consisting of
SEQ ID NO: 75, 78, 81, 84, 87, 90, 96, 99, 102, and 123; and
optionally (iv) a CDRH of (i), (ii) and (iii) that contains one or
more, e.g., one, two, three, four or more amino acid substitutions
(e.g., conservative amino acid substitutions), deletions or
insertions that collectively total no more than two amino
acids.
[0028] In some embodiments, the CDRLs are further selected from the
group consisting of: (i) a CDRL1 selected from the group consisting
of SEQ ID NOs:107, 111 and 115; (ii) a CDRL2 selected from the
group consisting of SEQ ID NOs: 108, 112 and 116; (iii) CDRL3
selected from the group consisting of SEQ ID NOs: 109, 113 and 117;
and optionally (iv) a CDRL of (i), (ii) and (iii) that contains one
or more, e.g., one, two, three, four or more amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In some embodiments, the amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions collectively total no more than three amino
acids per CDRL. In some embodiments, the amino acid substitutions,
deletions or insertions collectively total no more than two amino
acids per CDRL. In related embodiments, the CDRLs are further
selected from the group consisting of: (i) a CDRL1 selected from
the group consisting of SEQ ID NOs: 42, 45, 51, 57, 62, 69, 103,
and 110; (ii) a CDRL2 selected from the group consisting of SEQ ID
NOs: 43, 52, 55, 58, 63, 70, 104, 108, and 114; (iii) a CDRL3
selected from the group consisting of SEQ ID NOs: 44, 47, 53, 56,
59, 64, 105, and 106; and optionally (iv) a CDRL of (i), (ii) and
(iii) that contains one or more, e.g., one, two, three, four or
more amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions that collectively total no
more than two amino acids. In additional related embodiments, the
CDRLs are further selected from the group consisting of: (i) a
CDRL1 selected from the group consisting of SEQ ID NOs: 42, 45, 48,
51, 54, 57, 62, 65, 66, and 69; (ii) a CDRL2 selected from the
group consisting of SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61, 63, 67,
and 70; (iii) a CDRL3 selected from the group consisting of SEQ ID
NOs: 44, 47, 50, 53, 56, 59, 64, 68, 71, and 72; and optionally
(iv) a CDRL of (i), (ii) and (iii) that contains one or more, e.g.,
one, two, three, four or more amino acid substitutions (e.g.,
conservative amino acid substitutions), deletions or insertions. In
one embodiment, the total number of amino acid substitutions,
deletions or insertions is no more than two amino acids per CDR. In
another embodiment, the amino acid substitutions are conservative
substitutions.
[0029] In another embodiment, the isolated antigen-binding protein
comprises at least one or two CDRH of any of the above-mentioned
(A) and at least one or two CDRL of any of the above-mentioned (B).
In yet another embodiment, the isolated antigen-binding protein
comprises (i) at least three CDRH of any of the above-mentioned
(A), where the three CDRHs include CDRH1, a CDRH2 and a CDRH3, and
(ii) at least three CDRL of any of the above-mentioned (B), where
the three CDRLs include CDRL1, a CDRL2 and a CDRL3. In additional
embodiments, the isolated antigen binding proteins described above
comprise a first amino acid sequence comprising at least one CDRH
and a second amino acid sequence comprising at least one CDRL. In
one embodiment, the first and the second amino acid sequences are
covalently bonded to each other.
[0030] In another aspect, the isolated antigen-binding protein
includes a CDRH1, a CDRH2 and a CDRH3. In one embodiment, CDRH1
comprises SEQ ID NO:73, CDRH2 comprises SEQ ID NO:74 and CDRH3
comprises SEQ ID NO:75. In another embodiment, CDRH1 comprises SEQ
ID NO:76, CDRH.sup.2 comprises SEQ ID NO:77 and CDRH3 comprises SEQ
ID NO:78. In another embodiment, CDRH1 comprises SEQ ID NO:79,
CDRH2 comprises SEQ ID NO:80 and CDRH3 comprises SEQ ID NO:81. In
another embodiment, CDRH1 comprises SEQ ID NO:82, CDRH2 comprises
SEQ ID NO:83 and CDRH3 comprises SEQ ID NO:84. In another
embodiment, CDRH1 comprises SEQ ID NO:85, CDRH2 comprises SEQ ID
NO:86 and CDRH3 comprises SEQ ID NO:87. In another embodiment,
CDRH1 comprises SEQ ID NO:88, CDRH2 comprises SEQ ID NO:89 and
CDRH3 comprises SEQ ID NO:90. In another embodiment, CDRH1
comprises SEQ ID NO:76, CDRH2 comprises SEQ ID NO:91 and CDRH3
comprises SEQ ID NO:78. In another embodiment, CDRH1 comprises SEQ
ID NO:92, CDRH2 comprises SEQ ID NO:93 and CDRH3 comprises SEQ ID
NO:94. In another embodiment, CDRH1 comprises SEQ ID NO:76, CDRH2
comprises SEQ ID NO:95 and CDRH3 comprises SEQ ID NO:78. In another
embodiment, CDRH1 comprises SEQ ID NO:73, CDRH2 comprises SEQ ID
NO:74 and CDRH3 comprises SEQ ID NO:96. In another embodiment,
CDRH1 comprises SEQ ID NO:97, CDRH2 comprises SEQ ID NO:98 and
CDRH3 comprises SEQ ID NO:99. In another embodiment, CDRH1
comprises SEQ ID NO:100, CDRH2 comprises SEQ ID NO:101 and CDRH3
comprises SEQ ID NO:102.
[0031] In another aspect, the isolated antigen-binding protein
includes a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence.
In one embodiment, CDRL1 comprises SEQ ID NO:42, CDRL2 comprises
SEQ ID NO:43 and CDRL3 comprises SEQ ID NO:44. In another
embodiment, CDRL1 comprises SEQ ID NO:45, CDRL2 comprises SEQ ID
NO:46 and CDRL3 comprises SEQ ID NO:47. In another embodiment,
CDRL1 comprises SEQ ID NO:48, CDRL2 comprises SEQ ID NO:49 and
CDRL3 comprises SEQ ID NO:50. In another embodiment, CDRL1
comprises SEQ ID NO:51, CDRL2 comprises SEQ ID NO:52 and CDRL3
comprises SEQ ID NO:53. In another embodiment, CDRL1 comprises SEQ
ID NO:54, CDRL2 comprises SEQ ID NO:55 and CDRL3 comprises SEQ ID
NO:56. In another embodiment, CDRL1 comprises SEQ ID NO:57, CDRL2
comprises SEQ ID NO:58 and CDRL3 comprises SEQ ID NO:59. In another
embodiment, CDRL1 comprises SEQ ID NO:60, CDRL2 comprises SEQ ID
NO:55 and CDRL3 comprises SEQ ID NO:56. In another embodiment,
CDRL1 comprises SEQ ID NO:45, CDRL2 comprises SEQ ID NO:61 and
CDRL3 comprises SEQ ID NO:47. In another embodiment, CDRL1
comprises SEQ ID NO:62, CDRL2 comprises SEQ ID NO:63 and CDRL3
comprises SEQ ID NO:64. In another embodiment, CDRL1 comprises SEQ
ID NO:65, CDRL2 comprises SEQ ID NO:55 and CDRL3 comprises SEQ ID
NO:56. In another embodiment, CDRL1 comprises SEQ ID NO:66, CDRL2
comprises SEQ ID NO:67 and CDRL3 comprises SEQ ID NO:68. In another
embodiment, CDRL1 comprises SEQ ID NO:69, CDRL2 comprises SEQ ID
NO:70 and CDRL3 comprises SEQ ID NO:71. In another embodiment,
CDRL1 comprises SEQ ID NO:69, CDRL2 comprises SEQ ID NO:70 and
CDRL3 comprises SEQ ID NO:72.
[0032] In another aspect, the isolated antigen-binding protein
includes a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a
CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one
embodiment, CDRL1 comprises SEQ ID NO:42, CDRL2 comprises SEQ ID
NO:43, CDRL3 comprises SEQ ID NO:44, CDRH1 comprises SEQ ID NO:73,
CDRH2 comprises SEQ ID NO:74 and CDRH3 comprises SEQ ID NO:75. In
another embodiment, CDRL1 comprises SEQ ID NO:45, CDRL2 comprises
SEQ ID NO:46, CDRL3 comprises SEQ ID NO:47, CDRH1 comprises SEQ ID
NO:76, CDRH2 comprises SEQ ID NO:77 and CDRH3 comprises SEQ ID
NO:78. In another embodiment, CDRL1 comprises SEQ ID NO:48, CDRL2
comprises SEQ ID NO:49, CDRL3 comprises SEQ ID NO:50, CDRH1
comprises SEQ ID NO:79, CDRH2 comprises SEQ ID NO:80 and CDRH3
comprises SEQ ID NO:81. In another embodiment, CDRL1 comprises SEQ
ID NO:51, CDRL2 comprises SEQ ID NO:52, CDRL3 comprises SEQ ID
NO:53, CDRH1 comprises SEQ ID NO:82, CDRH2 comprises SEQ ID NO:83
and CDRH3 comprises SEQ ID NO:84. In another embodiment, CDRL1
comprises SEQ ID NO:54, CDRL2 comprises SEQ ID NO:55, CDRL3
comprises SEQ ID NO:56, CDRH1 comprises SEQ ID NO:85, CDRH2
comprises SEQ ID NO:86 and CDRH3 comprises SEQ ID NO:87. In another
embodiment, CDRL1 comprises SEQ ID NO:57, CDRL2 comprises SEQ ID
NO:58, CDRL3 comprises SEQ ID NO:59, CDRH1 comprises SEQ ID NO:88,
CDRH2 comprises SEQ ID NO:89 and CDRH3 comprises SEQ ID NO:90. In
another embodiment, CDRL1 comprises SEQ ID NO:60, CDRL2 comprises
SEQ ID NO:55, CDRL3 comprises SEQ ID NO:56, CDRH1 comprises SEQ ID
NO:85, CDRH2 comprises SEQ ID NO:86 and CDRH3 comprises SEQ ID
NO:87. In another embodiment, CDRL1 comprises SEQ ID NO:45, CDRL2
comprises SEQ ID NO:61, CDRL3 comprises SEQ ID NO:47, CDRH1
comprises SEQ ID NO:76, CDRH2 comprises SEQ ID NO:91 and CDRH3
comprises SEQ ID NO:78. In another embodiment, CDRL1 comprises SEQ
ID NO:62, CDRL2 comprises SEQ ID NO:63, CDRL3 comprises SEQ ID
NO:64, CDRH1 comprises SEQ ID NO:92, CDRH2 comprises SEQ ID NO:93
and CDRH3 comprises SEQ ID NO:94. In another embodiment, CDRL1
comprises SEQ ID NO:45, CDRL2 comprises SEQ ID NO:61, CDRL3
comprises SEQ ID NO:47, CDRH1 comprises SEQ ID NO:76, CDRH2
comprises SEQ ID NO:95 and CDRH3 comprises SEQ ID NO:78. In another
embodiment, CDRL1 comprises SEQ ID NO:65, CDRL2 comprises SEQ ID
NO:55, CDRL3 comprises SEQ ID NO:56, CDRH1 comprises SEQ ID NO:85,
CDRH2 comprises SEQ ID NO:86 and CDRH3 comprises SEQ ID NO:87. In
another embodiment, CDRL1 comprises SEQ ID NO:42, CDRL2 comprises
SEQ ID NO:43, CDRL3 comprises SEQ ID NO:44, CDRH1 comprises SEQ ID
NO:73, CDRH2 comprises SEQ ID NO:74 and CDRH3 comprises SEQ ID
NO:96. In another embodiment, CDRL1 comprises SEQ ID NO:66, CDRL2
comprises SEQ ID NO:67, CDRL3 comprises SEQ ID NO:68, CDRH1
comprises SEQ ID NO:97, CDRH2 comprises SEQ ID NO:98 and CDRH3
comprises SEQ ID NO:99. In another embodiment, CDRL1 comprises SEQ
ID NO:69, CDRL2 comprises SEQ ID NO:70, CDRL3 comprises SEQ ID
NO:71, CDRH1 comprises SEQ ID NO:100, CDRH2 comprises SEQ ID NO:101
and CDRH3 comprises SEQ ID NO:102. In another embodiment, CDRL1
comprises SEQ ID NO:69, CDRL2 comprises SEQ ID NO:70, CDRL3
comprises SEQ ID NO:72, CDRH1 comprises SEQ ID NO:100, CDRH2
comprises SEQ ID NO:101 and CDRH3 comprises SEQ ID NO:102.
[0033] In any of the above-mentioned sequence-defined embodiments,
the isolated antigen-binding protein may be, for example, a
monoclonal antibody, a polyclonal antibody, a recombinant antibody,
a human (e.g., fully human) antibody, a humanized antibody, a
chimeric antibody, a multi-specific antibody, or an antigen binding
fragment thereof. Further, the antibody fragment of the isolated
antigen-binding proteins may be a Fab fragment, and Fab' fragment,
an F(ab').sub.2 fragment, an Fv fragment, a diabody, or a single
chain antibody molecule. For example, the isolated antigen binding
protein may be a human monoclonal antibody, and may be, e.g., an
IgG1-, IgG2-, IgG3-, or IgG4-type antibody. Further, the isolated
antigen binding proteins may be neutralizing antigen binding
proteins.
[0034] In any of the above-mentioned sequence-defined embodiments,
the isolated antigen-binding protein may specifically bind to both
human CRLR and human RAMP1 and not specifically bind AM1, AM2 or a
human amylin receptor (e.g., AMY1), for example, the isolated
antigen binding protein may specifically bind to human CGRP R with
a K.sub.D.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, or
.ltoreq.5 nM, e.g., as determined using a FACS binding assay and
analyzed, for example, using methods described in Rathanaswami, et
al., Biochemical and Biophysical Research Communications 334(2005)
1004-1013. In any of the above-mentioned sequence-defined
embodiments, the isolated antigen-binding protein may selectively
inhibit human CGRP R, relative to the human the AM1, AM2 or AMY1
receptors, e.g., with a selectivity ratio of 100 or more, 250 or
more, 500 or more, 750 or more, 1,000 or more, 2,500 or more, 5,000
or more or 10,000 or more, where the degree of selective inhibition
may be determined using any suitable method, e.g., using a cAMP
assay as described in the Examples herein. In any of the
above-mentioned sequence-defined embodiments, the isolated.
antigen-binding protein may have a Ki of .ltoreq.100 nM, .ltoreq.10
nM, .ltoreq.1 nM, .ltoreq.0.5 nM or .ltoreq.0.1 nM in a CGRP
binding competition assay, e.g., in a radiolabeled .sup.125I-CGRP
binding competition assay to membranes from cells expressing human
CGRP R, e.g., the assay described in Example 5 herein.
[0035] Another set of embodiment includes isolated antigen-binding
proteins that include one or a combination of CDRs having the
consensus sequences described below, and optionally, bind human
CGRP R. The consensus sequences are derived from phylogenetically
related CDR sequences. In one aspect, the CDRs from the various
groups may be mixed and matched in any particular isolated
antigen-binding protein that binds human CGRP R. In another aspect,
the antigen binding protein comprises heavy and light chain CDRs
that are derived from the same phylogenetically-related group of
antibody clones. Exemplary CDR consensus sequences are as
follows:
[0036] K1 Consensus
[0037] CDR1 RASQGIRX.sub.1DLG (SEQ ID NO:103), wherein X.sub.1 is
selected from the group consisting of N and K.
[0038] CDR2 X.sub.1ASSLQS (SEQ ID NO:104), wherein X.sub.1 is
selected from the group consisting of A and G.
[0039] CDR3 LQYNX.sub.1X.sub.2PWT (SEQ ID NO:105), wherein X.sub.1
is selected from the group consisting of I and S, and X.sub.2 is
selected from the group consisting of Y and F.
[0040] K4 Consensus
[0041] CDR3 QQYGNSLX.sub.1R (SEQ ID NO:106), wherein X.sub.1 is
selected from the group consisting of S and C.
[0042] K1,4 Consensus
[0043] CDR1 RASQX.sub.1X.sub.2X.sub.3X.sub.4GX.sub.5LX.sub.6 (SEQ
ID NO:107), wherein X.sub.1 is selected from the group consisting
of S and G, X.sub.2 is selected from the group consisting of V and
I, X.sub.3is selected from the group consisting of S and R, X.sub.4
is selected from the group consisting of S, N and K, X.sub.5 is
selected from the group consisting of Y and D, and X.sub.6 is
selected from the group consisting of T and G.
[0044] CDR2 X.sub.1ASSX.sub.2X.sub.3X.sub.4 (SEQ ID NO:108),
wherein X1 is selected from the group consisting of G and A,
X.sub.2 is selected from the group consisting of R and L, X.sub.3
is selected from the group consisting of A and Q, and X.sub.4 is
selected from the group consisting of T and S.
[0045] CDR3 X.sub.1QYX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7
(SEQ ID NO:109), wherein X.sub.1 is selected from the group
consisting of Q and L, X.sub.2 is selected from the group
consisting of G and N, X.sub.3 is selected from the group
consisting of N and T, X.sub.4 is selected from the group
consisting of S, Y and F, X.sub.5 is selected from the group
consisting of L and P, X.sub.6 is selected from the group
consisting of C, W and S, and X.sub.7 is selected from the group
consisting of R and T.
[0046] K3 Consensus
[0047] CDR1 X.sub.1KSSQSLLHSX.sub.1GX.sub.2X.sub.3YLY (SEQ ID
NO:110), wherein X.sub.1 is selected from the group consisting of D
and A, X.sub.2 is selected from the group consisting of R and K,
and X.sub.3 is selected from the group consisting of N and T.
[0048] K2,3 Consensus
[0049] CDR1 X.sub.1SSQSLLHSX.sub.2GX.sub.3X.sub.4YLX.sub.5 (SEQ ID
NO:111), wherein X.sub.1 is selected from the group consisting of R
and K, X.sub.2 is selected from the group consisting of F, D and A,
X.sub.3 is selected from the group consisting of Y, R and K,
X.sub.4 is selected from the group consisting of N and T, and
X.sub.5 is selected from the group consisting of D and Y.
[0050] CDR2 X.sub.1X.sub.2SNRX.sup.3S (SEQ ID NO:112), wherein
X.sub.1 is selected, from the group consisting of L and E, X.sub.2
is selected from the group consisting of G and V, and X.sub.3 is
selected from the group consisting of A and F.
[0051] CDR3 MQX.sub.1X.sub.2X.sub.3X.sub.4PX.sub.5T (SEQ ID
NO:113), wherein X.sub.1 is selected from the group consisting of A
and S, X.sub.2 is selected from the group consisting of L and F,
X.sub.3 is selected from the group consisting of Q and P, X.sub.4
is selected from the group consisting of T and L, and X.sub.5 is
selected from the group consisting of F and L.
[0052] Lm3 Consensus
[0053] CDR2 RX.sub.1NQRPS (SEQ ID NO:114), wherein X.sub.1 is
selected from the group consisting of N and S.
[0054] Lm1,2,3 Consensus
[0055] CDR1 SGSSSNIGX.sub.1NX.sub.2VX.sub.3 (SEQ ID NO:115),
wherein X.sub.1 is selected from the group consisting of N and S,
X.sub.2 is selected from the group consisting of Y and T, and
X.sub.3 is selected from the group consisting of S, N and Y.
[0056] CDR2 X.sub.1X.sub.2NX.sub.3RPS (SEQ ID NO:116), wherein
X.sub.1 is selected from the group consisting of D, T and R,
X.sub.2 is selected from the group consisting of N and S, and
X.sub.3 is selected from the group consisting of K and Q.
[0057] CDR3 X.sub.1X.sub.2X.sub.3DX.sub.4X.sub.5LX.sub.6X.sub.7VV
(SEQ ID NO:117), wherein X.sub.1 is selected from the group
consisting of G and A, X.sub.2 is selected from the group
consisting of T and A, X.sub.3 is selected from the group
consisting of W and R, X.sub.4 is selected from the group
consisting of S and D, X.sub.3 is selected from the group
consisting of R and S, X.sub.6 is selected from the group
consisting of S and N, and X.sub.7 is selected from the group
consisting of A and G.
[0058] LmAll Consensus
[0059] CDR1
X.sub.1GX.sub.2X.sub.3SX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10-
X.sub.11 (SEQ ID NO:118), wherein X.sub.1 is selected from the
group consisting of S and Q, X.sub.2 is present or absent, and if
present, is S, X.sub.3 is selected from the group consisting of S
and D, X.sub.4 is present or absent, and if present, is N, X.sub.5
is selected from the group consisting of I and L, X.sub.6 is
selected from the group consisting of G and R, X.sub.7 is selected
from the group consisting of N and S, X.sub.8 is selected from the
group consisting of N and F, X.sub.9 is selected, from the group
consisting of Y and T, X.sub.10 is selected from the group
consisting of V and A, and X.sub.11 is selected from the group
consisting of S, N and Y.
[0060] CDR2 X.sub.1X.sub.2NX.sub.3RPS (SEQ ID NO:119), wherein
X.sub.1 is selected from the group consisting of D, G, T, and R,
X.sub.2 is selected from the group consisting of N, K and S, and
X.sub.3 is selected from the group consisting of K, N and Q.
[0061] CDR3
X.sub.1X.sub.2X.sub.3DX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9V
(SEQ ID NO:120), wherein X.sub.1 is selected from the group
consisting of G, N and A, X.sub.2 is selected from the group
consisting of T, S and A, X.sub.3 is selected from the group
consisting of W and R, X.sub.4 is selected from the group
consisting of S and D, X.sub.5 is selected from the group
consisting of R and S, X.sub.6 is selected from the group
consisting of L and V, X.sub.7 is selected from the group
consisting of S, Y and N, X.sub.8 is selected from the group
consisting of A, H and G, and X.sub.9 is selected from the group
consisting of V and L.
[0062] HC1 Consensus
[0063] CDR1 X.sub.1YYMX.sub.2 (SEQ ID NO:121), wherein X.sub.1 is
selected from the group consisting of G and D, X.sub.2 is selected
from the group consisting of H and Y.
[0064] CDR2 WIX.sub.1PNSGGTNYAQKFQG (SEQ ID NO:122), wherein
X.sub.1 is selected from the group consisting of N and S.
[0065] CDR3
X.sub.1X.sub.2X.sub.3SX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8GX.sub.9X.sub.10-
X.sub.11X.sub.12YYX.sub.13GMDV (SEQ ID NO:123), wherein X.sub.1 is
selected from the group consisting of D and G, X.sub.2 is selected
from the group consisting of Q and, G, X.sub.3 is selected from the
group consisting of M and Y, X.sub.4 is selected from the group
consisting of I and G, X.sub.5 is selected from the group
consisting of I and Y, X.sub.6 is selected from the group
consisting of M and A, X.sub.7 is present or absent, and if
present, is L, X.sub.8 is present or absent, and if present, is R,
X.sub.9 is selected from the group consisting of V and L, X.sub.10
is selected from the group consisting of F and Y, X.sub.11 is
selected from the group consisting of P and S, X.sub.12 is selected
from the group consisting of P and H, and X.sub.13 is present or
absent, and if present, is Y.
[0066] HC2 Consensus
[0067] CDR2 RIKSX.sub.1TDGGTTDYX.sub.2APVKG (SEQ ID NO:124),
wherein X.sub.1 is selected from the group consisting of K and T,
and X.sub.2 is selected from the group consisting of T and A.
[0068] HC3 Consensus
[0069] CDR1 X.sub.1YX.sub.2MX.sub.3 (SEQ ID NO:125), wherein
X.sub.1 is selected from the group consisting of T and S, X.sub.2
is selected from the group consisting of S and A, and X.sub.3 is
selected from the group consisting of N and S.
[0070] CDR2 X.sub.1ISX.sub.2SX.sub.3X.sub.4X.sub.5X.sub.6YYADSVKG
(SEQ ID NO:126), wherein X.sub.1is selected from the group
consisting of S and A, X.sub.2 is selected from the group
consisting of S and G, X.sub.3 is selected from the group
consisting of S and G, X.sub.4 is selected from the group
consisting of S and G, X.sub.5 is selected from the group
consisting of Y and R, and X.sub.6 is selected from the group
consisting of R and T.
[0071] CDR3
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7PYSX.sub.8X.sub.9WYDYYYG-
MDV (SEQ ID NO:127), wherein X.sub.1 is selected from the group
consisting of E and D, X.sub.2 is selected from the group
consisting of G and Q, X.sub.3 is selected from the group
consisting of V and R, X.sub.4 is selected from the group
consisting of S and E, X.sub.5 is selected from the group
consisting of G and V, X.sub.6 is selected from the group
consisting of S and G, X.sub.7 is present or absent, and if
present, is S, X.sub.8 is selected from the group consisting of I
and S, and X.sub.9 is selected from the group consisting of S and
G.
[0072] HC4 Consensus
[0073] CDR1 SX.sub.1GMH (SEQ ID NO:128), wherein X.sub.1 is
selected from the group consisting of F and Y.
[0074] CDR2 VISX.sub.1DGSX.sub.2KYX.sub.3X.sub.4DSVKG (SEQ ID
NO:129), wherein X.sub.1 is selected from the group consisting of F
and Y, X.sub.2 is selected from the group consisting of I and H,
X.sub.3 is selected from the group consisting of S and Y, and
X.sub.4 is selected from the group consisting of V and A.
[0075] CDR3
X.sub.1RX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6SX.sub.7X.sub.8YYX.sub.9X.sub.-
10X.sub.11YYGX.sub.12X.sub.13V (SEQ ID NO:130), wherein X.sub.1 is
selected from the group consisting of D and E, X.sub.2 is selected
from the group consisting of L and K, X.sub.3 is selected from the
group consisting of N and R, X.sub.4 is selected from the group
consisting of Y and V, X.sub.5 is selected from the group
consisting of Y and T, X.sub.6 is selected from the group
consisting of D and M, X.sub.7 is selected from the group
consisting of S and T, X.sub.8 is selected from the group
consisting of G and L, X.sub.9 is selected from the group
consisting of H and Y, X.sub.10 is present or absent, and if
present, is Y, X.sub.11 is selected from the group consisting of K
and F, X.sub.12 is selected from the group consisting of M and L,
and X.sub.13 is selected from the group consisting of A and D.
[0076] HCA Consensus
[0077] CDR1 X.sub.1X.sub.2X.sub.3MX.sub.4 (SEQ ID NO:131), wherein
X.sub.1 is selected from the group consisting of N and S, X.sub.2
is selected from the group consisting of A, Y and F, X.sub.3 is
selected from the group consisting of W, A and G, and X.sub.4 is
selected from the group consisting of S and H.
[0078] CDR2
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6GX.sub.7X.sub.8X.sub.9X.sub.10-
X.sub.11X.sub.12X.sub.13X.sub.14VKG (SEQ ID NO:132), wherein
X.sub.1 is selected from the group consisting of R, A and V,
X.sub.2 is selected from the group consisting of K, S and W,
X.sub.3 is selected from the group consisting of S, G, F and Y,
X.sub.4 is present or absent, and if present, is selected from the
group consisting of K and T, X.sub.5 is present or absent, and if
present, is T, X.sub.6 is selected from the group consisting of D
and S, X.sub.7 is selected from the group consisting of G and S,
X.sub.8 is selected from the group consisting of T, R, I, N and H,
X.sub.9 is selected from the group consisting of T and K, X.sub.10
is selected from the group consisting of D and Y, X.sub.11 is
selected from the group consisting of Y and S, X.sub.12 is selected
from the group consisting of T, V and V, X.sub.13 is selected from
the group consisting of A and D, and X.sub.14 is selected from the
group consisting of P and S.
[0079] CDR3
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17GX.sub.18X.sub.19V
(SEQ ID NO:133), wherein X.sub.1 is selected from the group
consisting of D, A and E, X.sub.2 is selected from the group
consisting of R, Q and G, X.sub.3 selected from the group
consisting of T, R, L, G and K, X.sub.4 is selected from the group
consisting of G, E, N, I and R, X.sub.5 is selected from the group
consisting of Y, V and A, X.sub.6 is selected from the group
consisting of S, G, Y, A and T, X.sub.7 is selected from the group
consisting of I, P, D, A and M, X.sub.8 is present or absent, and
if present, is selected from the group consisting of S and Y,
X.sub.9 is present or absent, and if present, is selected. from the
group consisting of W, S and T, X.sub.10 is selected from the group
consisting of S, G and L, X.sub.11 is selected from the group
consisting of S, G, L, and Y, X.sub.12 is present or absent, and if
present, is selected from the group consisting of W and Y, X.sub.13
is selected from the group consisting of Y and H, X.sub.14 is
present or absent, and if present, is selected from the group
consisting of Y and D, X.sub.15 is selected from the group
consisting of Y, K and F, X.sub.16 is present or absent, and if
present, is Y, X.sub.17 is present or absent, and if present, is Y,
X.sub.18 is selected from the group consisting of M and L, and
X.sub.19 is selected from the group consisting of D and A.
[0080] HCB Consensus
[0081] CDR1 X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO:134),
wherein X.sub.1 is selected from the group consisting of N, G, D, S
and A, X.sub.2 is selected from the group consisting of A, F and Y,
X.sub.3 is selected from the group consisting of W, Y, A and G,
X.sub.4 is selected from the group consisting of M and L, and
X.sub.5 is selected from the group consisting of S and H.
[0082] CDR2
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X-
.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17G (SEQ ID
NO:135), wherein X.sub.1 is selected from the group consisting of
R, W, A, V, S and F, X.sub.2 is selected from the group consisting
of K, N, S, W and R, X.sub.3 is selected from the group consisting
of S, P, G, F and Y, X.sub.4 is present or absent, and if present,
is selected from the group consisting of K, T and R, X.sub.5 is
present or absent, and if present, is selected from the group
consisting of T and A, X.sub.6 is selected. from the group
consisting of D, N, H, S and Y, X.sub.7 is selected from the group
consisting of G and S, X.sub.8 is selected. from the group
consisting of G and S, X.sub.9 is selected from the group
consisting of T, G, R, I, N, H and Y, X.sub.10 is selected from the
group consisting of T, K, R and P, X.sub.11 is selected from the
group consisting of D, N, Y and E, X.sub.12 is selected from the
group consisting of Y and S, X.sub.13 is selected from the group
consisting of T, A and V, X.sub.14 is selected from the group
consisting of A, Q and D, X.sub.15 is selected from the group
consisting of P, K and S, X.sub.16 is selected from the group
consisting of V and F, and X.sub.17 is selected from the group
consisting of K and Q.
[0083] CDR3
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5SX.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X-
.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16GX.sub.17X.sub.18V
(SEQ ID NO:136), wherein X.sub.1 is selected from the group
consisting of D, G, A and E, X.sub.2 is selected from the group
consisting of R, G and Q, X.sub.3 is selected from the group
consisting of T, M, Y, R, L, G and K, X.sub.4 is selected from the
group consisting of G, S, E, N, I and R, X.sub.5 is selected from
the group consisting of Y, I, G, V and A, X.sub.6 is selected from
the group consisting of S, I, Y, G, A and T, X.sub.7 is selected
from the group consisting of I, M, A, P and D, X.sub.8 is present
or absent, and if present, is selected from the group consisting of
S, L and Y, X.sub.9 is present or absent, and if present, is
selected from the group consisting of W, R, S and T, X.sub.10 is
selected from the group consisting of S, G and L, X.sub.11 is
selected from the group consisting of S, V, L, G and Y, X.sub.12 is
present or absent, and if present, is selected from the group
consisting of F, Y and W, X.sub.13 is selected from the group
consisting of Y, P, S and H, X.sub.14 is present or absent, and if
present, is selected from the group consisting of Y, P, D and H,
X.sub.15 is selected from the group consisting of Y, K and F,
X.sub.16 is present or absent, and if present, is Y, X.sub.17 is
present or absent, and if present, is Y and X.sub.18 is selected
from the group consisting of M and L.
[0084] In any of the above-mentioned consensus sequence defined
embodiments, the isolated antigen-binding protein may be, for
example, an AVIMER polypeptide, a monoclonal antibody, a polyclonal
antibody, a recombinant antibody, a human (e.g., fully human)
antibody, a humanized antibody, a chimeric antibody, a
multi-specific antibody, or an antigen binding fragment thereof.
Further, the antibody fragment of the isolated antigen-binding
proteins may be a Fab fragment, and Fab' fragment, an F(ab').sub.2
fragment, an Fv fragment, a diabody, or a single chain antibody
molecule. For example, the isolated antigen binding protein may be
a human monoclonal antibody, and may be, e.g., an IgG1-, IgG2-,
IgG3-, IgG4-type antibody. Further, the isolated antigen binding
proteins may be neutralizing antigen binding proteins.
[0085] In any of the above-mentioned consensus sequence defined
embodiments, the isolated antigen-binding protein may specifically
bind to both human CRLR and human RAMP1 and not specifically bind
to AM1, AM2 or a human amylin receptor (e.g., AMY1), for example,
the isolated antigen binding protein may specifically bind to human
CGRP R with a K.sub.D.ltoreq.1 nM, .ltoreq.100 nM, .ltoreq.10 nM,
or .ltoreq.5 nM, e.g., as determined using a FACS binding assay and
analyzed, for example, using methods described in Rathanaswami, et
al., Biochemical and Biophysical Research Communications 334 (2005)
1004-1013. In any of the above-mentioned consensus sequence defined
embodiments, the isolated antigen-binding protein may selectively
inhibit human CGRP R, relative to the human the AM1, AM2 AMY1
receptors, e.g., with a selectivity ratio of 100 or more, 250 or
more, 500 or more, 750 or more, 1,000 or more, 2,500 or more, 5,000
or more or 10,000 or more, where the degree of selective inhibition
may be determined using any suitable method, e.g., using a cAMP
assay as described in the Examples herein. In any of the
above-mentioned consensus sequence defined embodiments, the
isolated antigen-binding protein may have a Ki of .ltoreq.100 nM,
--10 nM, .ltoreq.1 nM, .ltoreq.0.5 nM or .ltoreq.0.1 nM in a CGRP
binding competition assay, e.g., in a radiolabeled .sup.125I-CGRP
binding competition assay to membranes from cells expressing human
CGRP R, e.g., the assay described in Example 5 herein.
[0086] Some of the isolated antigen-binding proteins described
comprise a heavy chain variable region (V.sub.H) sequence that has
at least 80%, 85%, and 90% or 95% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID
NOs:158-170. Some of the isolated antigen-binding proteins
described comprise a light chain variable region (V.sub.L) sequence
that has at least 80%, 85%, and 90% or 95% sequence identity with
an amino acid sequence selected from the group consisting of SEQ ID
NOs:137-153. Some of the isolated antigen-binding proteins
described comprise a V.sub.H sequence that has at least 80%, 85%,
90% or 95% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NOs:158-170, and a V.sub.L that
has at least 80%, 85%, 90% or 95% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID
NOs:137-153. In some embodiments, the isolated antigen-binding
proteins comprise (A) a heavy chain variable region (V.sub.H)
comprising a sequence (i) selected from the group consisting of SEQ
ID NOs:158-170, (ii) as defined by (i) and containing one or more
(e.g., five, ten, fifteen or twenty) amino acid substitutions
(e.g., conservative amino acid substitutions), deletions or
insertions; (B) a V.sub.L comprising a sequence (iii) selected from
the group consisting of SEQ ID NOs:137-153, or (iv) as defined by
(iii) containing one or more (e.g., five, ten, fifteen or twenty)
amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; or (C) V.sub.L of (A) and
a V.sub.L of (B). In some embodiments, the isolated antigen-binding
proteins comprise a heavy chain variable region (V.sub.H)
comprising a sequence selected from the group consisting of SEQ ID
NOs:158-170 and a V.sub.L comprising a sequence selected from the
group consisting of SEQ ID NOs:137-153.
[0087] In one embodiment, the isolated antigen-binding protein
comprises a heavy chain variable region (V.sub.H) comprising an
amino acid sequence selected from the group consisting of (i) SEQ
ID NO:158, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:159, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:160, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:161, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:162, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:163, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:164, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:165, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:166, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:167, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid. substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:168, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.H comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:169, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.H comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:170, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions.
[0088] In one embodiment, the isolated antigen-binding protein
comprises a light chain variable region (V.sub.L) comprising an
amino acid sequence selected from the group consisting of (i) SEQ
ID NO:137, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:138, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:139, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:140, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:141, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:142, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:143, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:144, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:145, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO: 146, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:147, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:148, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:149, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:150, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:151, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions. In another
embodiment, the isolated antigen-binding protein comprises a
V.sub.L comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:152, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions. In another embodiment, the isolated
antigen-binding protein comprises a V.sub.L comprising an amino
acid sequence selected from the group consisting of (i) SEQ ID
NO:153, (ii) a sequence that is at least 90% or 95% identical to
the sequence defined by (i), and (iii) a sequence as defined by (i)
containing up to ten amino acid substitutions (e.g., conservative
amino acid substitutions), deletions or insertions.
[0089] In any of the above-mentioned V.sub.L and V.sub.H sequence
defined embodiments, the isolated antigen-binding protein may be,
for example, a monoclonal antibody, a polyclonal antibody, a
recombinant antibody, a human (e.g., fully human) antibody, a
humanized antibody, a chimeric antibody, a multi-specific antibody,
or an antigen binding fragment thereof. Further, the antibody
fragment of the isolated antigen-binding proteins may be a Fab
fragment, and Fab' fragment, an F(ab').sup.2 fragment, an Fv
fragment, a diabody, or a single chain antibody molecule. For
example, the isolated. antigen binding protein may be a human
monoclonal antibody, and may be, e.g., an IgG1-, IgG2-, IgG3-, or
IgG4-type antibody. Further, the isolated antigen binding proteins
may be neutralizing antigen binding proteins.
[0090] In any of the above-mentioned V.sub.L and V.sub.H sequence
defined embodiments, the isolated antigen-binding protein may
specifically bind to both human CRLR and human RAMP1 and not
specifically bind to AM1, AM2 or a human amylin receptor (e.g.,
AMY1), for example, the isolated antigen binding protein may
specifically bind to human CGRP R with a K.sub.D23 1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, or .ltoreq.5 nM, e.g., as determined
using a FACS binding assay and analyzed, for example, using methods
described in Rathanaswami, et al., Biochemical and Biophysical
Research Communications 334 (2005) 1004-1013. In any of the
above-mentioned V.sub.L and V.sub.H sequence defined embodiments,
the isolated antigen-binding protein may selectively inhibit human
CGRP R, relative to the human the AM1, AM2 or AMY1 receptors, e.g.,
with a selectivity ratio of 100 or more, 250 or more, 500 or more,
750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000
or more, where the degree of selective inhibition may be determined
using any suitable method, e.g., using a cAMP assay as described in
the Examples herein. In any of the above-mentioned V.sub.L and
V.sub.H sequence-defined embodiments, the isolated antigen-binding
protein may have a Ki of .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1
nM, .ltoreq.0.5 nM or .ltoreq.0.1 nM in a CGRP binding competition
assay, e.g., in a radiolabeled .sup.125I-CGRP binding competition
assay to membranes from cells expressing human CGRP R, e.g., the
assay described in Example 5 herein.
[0091] In one aspect, the isolated antigen-binding proteins
comprise a heavy chain sequence that has at least 80%, 85%, 90% or
95% sequence identity with an amino acid sequence selected from the
group consisting of SEQ ID NOs:29-41. Some of the isolated
antigen-binding proteins described comprise a light chain sequence
that has at least 80%, 85%, 90% or 95% sequence identity with an
amino acid sequence selected from the group consisting of SEQ ID
NOs:12-28. Some of the isolated antigen-binding proteins comprise a
heavy chain sequence that has at least 80%, 85%, 90% or 95%
sequence identity with an amino acid sequence selected from the
group consisting of SEQ ID NOs: 29-41, and a light chain sequence
that has at least 80%, 85%, 90% or 95% sequence identity with an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 12-28. In some embodiments, the isolated antigen-binding
proteins comprise (A) a heavy chain comprising a sequence (i)
selected from the group consisting of SEQ ID NOs: 29-41, or (ii) as
defined by (i) and containing one or more (e.g., five, ten, fifteen
or twenty) amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; (B) a light chain
comprising a sequence (iii) selected from the group consisting of
SEQ ID NOs: 12-28, or (iv) as defined by (iii) containing one or
more (e.g., five, ten, fifteen or twenty) amino acid substitutions
(e.g., conservative amino acid substitutions), deletions or
insertions; or (C) a heavy chain of (A) and a light chain of (B).
In some embodiments, the isolated antigen-binding proteins comprise
a heavy chain comprising a sequence selected from the group
consisting of SEQ ID NOs: 29-41 and a light chain comprising a
sequence selected from the group consisting of SEQ ID NOs:
12-28.
[0092] In one embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:29, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:12, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0093] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:30, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:13, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0094] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:31, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:14, (ii) sequence that is at least 90%
or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions,
[0095] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:32, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid. substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:15, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0096] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:33, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:16, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0097] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:29, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:17, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0098] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:34, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:18, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0099] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:33, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:19, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0100] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:29, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:20, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0101] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:35, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:21, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0102] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:36, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:22, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0103] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:37, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:23, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0104] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:38, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:23, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0105] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:33, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:24, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0106] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:39, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:25, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0107] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:40, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:26, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0108] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:41, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:27, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0109] In another embodiment, the isolated antigen-binding protein
comprises (A) a heavy chain comprising an amino acid sequence
selected from the group consisting of (i) SEQ ID NO:41, (ii) a
sequence that is at least 90% or 95% identical to the sequence
defined by (i), and (iii) a sequence as defined by (i) containing
up to ten amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions; and (B) a light chain
comprising an amino acid sequence selected from the group
consisting of (i) SEQ ID NO:28, (ii) a sequence that is at least
90% or 95% identical to the sequence defined by (i), and (iii) a
sequence as defined by (i) containing up to ten amino acid
substitutions (e.g., conservative amino acid substitutions),
deletions or insertions.
[0110] In any of the above-mentioned light and heavy chain sequence
defined embodiments, the isolated antigen-binding protein may
comprise the specified heavy and/or light chain sequence, but with
a different signal peptide or with no signal peptide. In any of the
above-mentioned light and heavy chain sequence defined embodiments,
the isolated antigen-binding protein may be, for example, a
monoclonal antibody, a polyclonal antibody, a recombinant antibody,
a human (e.g., fully human) antibody, a humanized antibody, a
chimeric antibody, a multi-specific antibody, or an antigen binding
fragment thereof. Further, the antibody fragment of the isolated
antigen-binding proteins may be a Fab fragment, and Fab' fragment,
an F(ab').sub.2 fragment, an Fv fragment, a diabody, or a single
chain antibody molecule. For example, the isolated antigen binding
protein may be a human monoclonal antibody, and may be, e.g., an
IgG1-, IgG2-, IgG3-, or IgG4-type antibody. Further, the isolated
antigen binding proteins may be neutralizing antigen binding
proteins.
[0111] In any of the above-mentioned light and heavy chain sequence
defined embodiments, the isolated antigen-binding protein may
specifically bind to both human CRLR and human RAMP1 and not
specifically bind to AM1, AM2 or a human amylin receptor (e.g.,
AMY1), for example, the isolated antigen binding protein may
specifically bind to human CGRP R with a K.sub.D.ltoreq.1 .mu.M,
.ltoreq.100 nm, .ltoreq.10 nM, or .ltoreq.5 nM, e.g., as determined
using a FACS binding assay and analyzed, for example, using methods
described in Rathanaswami, et al., Biochemical and Biophysical
Research Communications 334 (2005) 1004-1013. In any of the
above-mentioned light and heavy chain sequence defined embodiments,
the isolated antigen-binding protein may selectively inhibit human
CGRP R, relative to the human the AM1, AM2 or AMY1 receptors, e.g.,
with a selectivity ratio of 100 or more, 250 or more, 500 or more,
750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000
or more, where the degree of selective inhibition may be determined
using any suitable method, e.g., using a cAMP assay as described in
the Examples herein. In any of the above-mentioned light and heavy
chain sequence-defined embodiments, the isolated antigen-binding
protein may have a Ki of .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1
nM, .ltoreq.0.5 nM or .ltoreq.0.1 nM in a CGRP binding competition
assay, e.g., in a radiolabeled .sup.125I-CGRP binding competition
assay to membranes from cells expressing human CGRP R, the assay
described in Example 5 herein.
[0112] In a further aspect, also provided are isolated nucleic acid
polynucleotides that encode any of the CGRP R antigen-binding
proteins summarized above. In one embodiment, the isolated
polynucleotide comprises a sequence selected from the group
consisting of SEQ ID NOs:175, 176, 178, 179, 180, 181, 182, 183,
186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209 and 210. In another
embodiment, the isolated polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NOs:224-258. In
another embodiment, the isolated polynucleotide comprises a
sequence capable of hybridizing under stringent hybridization
conditions with a sequence selected from the group consisting of
SEQ ID NOs:224-258. In another embodiment, the isolated
polynucleotide comprises a sequence that is about 80%, 85%, 90% or
95% or more identical to a sequence selected from the group
consisting of SEQ ID NOs:224-258. In some instances, the isolated
nucleic acid molecules are operably-linked to a control sequence.
In related embodiments, the isolated polynucleotides are
incorporated into an expression vector.
[0113] Also included are cell lines transformed with expression
vectors comprising isolated polynucleotides as described above. In
a related aspect, also provided are expression vectors and host
cells transformed or transfected with the expression vectors that
comprise the aforementioned isolated nucleic acid molecules that
encode CGRP R antigen-binding proteins described above
[0114] In another aspect, also provided is a method of preparing
the antigen-binding proteins that includes the step of preparing
the antigen binding protein from a host cell that secretes the
antigen-binding protein. In some embodiments, the antigen binding
protein is generated using an immunogen comprising soluble CGRP
receptor. In some embodiments, such soluble CGRP receptor is
obtained by co-expressing and purifying an N-terminal extracellular
domain (ECD) of human CRLR and an ECD of human RAMP1, e.g., an ECD
of human CRLR comprising SEQ ID NO: 6 and an ECD of RAMP1
comprising SEQ ID NO: 8, for example, as described in Examples 1
and 2 herein.
[0115] In yet another aspect, a pharmaceutical composition is
provided comprising at least one of the antigen-binding proteins
summarized above and a pharmaceutically acceptable excipient. In
one embodiment, the pharmaceutical composition may comprise an
additional active agent that is selected from the group consisting
of a radioisotope, radionuclide, a toxin, or a therapeutic and a
chemotherapeutic group.
[0116] In one aspect, the isolated antigen binding protein is
effective to inhibit vasodialation and/or decrease neurogenic
inflammation when administered to a patient. In one embodiment, the
isolated antigen binding protein is effective to reduce the
frequency and/or severity of headaches, for example, migraine
headaches. For example, the antigen binding protein may be used as
an acute treatment of migraine, and/or as a prophylactic treatment
to prevent or reduce the frequency and/or severity of symptoms,
particularly pain symptoms, associated with a migraine attack.
[0117] Other aspects further provide methods for treating or
preventing a condition associated with CGRP R in a patient,
comprising administering to a patient an effective amount of at
least one isolated antigen-binding protein summarized above. In one
embodiment, the condition is a headache, for example, a migraine
headache or a cluster headache or another type of pain, e.g., a
chronic pain; in another embodiment it is diabetes mellitus (type
II); in another embodiment it is inflammation, particularly
neurogenic inflammation; in another embodiment it is a
cardiovascular disorder; in another embodiment it is a hemodynamic
derangement associated with endotoxemia and sepsis; in another
embodiment it is vasodialation.
[0118] In another aspect, also provided is a method of inhibiting
binding of CGRP to human CGRP R, e.g., the extracellular portion of
CGRP R, in a patient comprising administering an effective amount
of at least one antigen-binding protein provided herein and/or
summarized above.
[0119] These and other aspects will be described in greater detail
herein. Each of the aspects provided can encompass various
embodiments provided herein. It is therefore anticipated that each
of the embodiments involving one element or combinations of
elements can be included in each aspect described, and all such
combinations of the above aspects and embodiments are expressly
considered. Other features, objects, and advantages of the
invention are apparent in the detailed description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0120] FIG. 1 shows an alignment of RAMP-1 sequences from human,
cynomolgus monkey and rat.
[0121] FIG. 2 shows an alignment of CRLR sequences from human,
cynomolgus monkey and rat.
[0122] FIGS. 3A and 3B show phylogenetically-based sequence
alignments of light chain CDRs from the indicated anti-CGRP
receptor antibody clones having kappa light chains, and certain
corresponding consensus sequences.
[0123] FIG. 4 shows phylogenetically-based sequence alignments of
light chain CDRs from the indicated anti-CGRP receptor antibody
clones having lambda light chains, and certain corresponding
consensus sequences.
[0124] FIGS. 5A, 5B, 5C, 5D and 5E show phylogenetically-based
sequence alignments of heavy chain CDRs from the indicated
anti-CGRP receptor antibody clones, and certain corresponding
consensus sequences.
[0125] FIG. 5F shows consensus sequences of exemplary anti-CGRP
receptor antibody heavy chain CDRs disclosed herein.
[0126] FIG. 6 is a plot of data from two experiments showing
percent inhibition of labeled ligand binding to CGRP R by 1092
anti-CGRP R hybridoma supernatants (diamonds) and 68 negative
control supernatants (squares).
[0127] FIGS. 7A-D show exemplary cAMP assay IC50 data from cells
expressing hCGRP receptor (FIG. 7A), hAM1 (FIG. 7B), hAM2 (FIG. 7C)
and human amylin receptors (FIG. 7D) for three indicated anti-CGRP
R mAbs.
[0128] FIG. 8 shows an example of .sup.125I-CGRP binding data such
as may be used to determine the Ki of mAbs to human CGRP
receptor.
[0129] FIGS. 9A-D show Biacore competition data for selected
antibodies disclosed herein.
[0130] FIG. 10 shows a FACS Kd determination of mAb 12G8.
[0131] FIG. 11 shows an alignment of cynomolgus, human, human
chimeras, rat, and rhesus RAMP1 sequences.
[0132] FIGS. 12A-B show an alignment of human, cynomolgus, rhesus,
rat, human chimera and consensus CRLR sequences,
[0133] FIGS. 13A-13C show representative FACS data of different
chimeric CGRP receptors binding to anti-CGRP R antibodies.
[0134] FIG. 14 shows peptide maps derived from AspN digestions of
CGRP R alone (chromatogram A) and from digestion of a control
sample containing CGRP R monoclonal antibody 12G8 (chromatogram
B).
[0135] FIG. 15 shows AspN digestions of CGRP R in the presence of
different concentrations of CGRP R neutralizing antibody.
[0136] FIG. 16 shows AspN digestions of CGRP R in the presence of
different concentration of CGRP R neutralizing antibody, 4E4.
[0137] FIG. 17 shows immunohistochemistry staining intensity of
cells expressing various receptor components with antibody
32H7.
DETAILED DESCRIPTION
[0138] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0139] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Further, unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular.
[0140] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present application are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification unless otherwise
indicated. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols
in Molecular Biology, Greene Publishing Associates (1992), and
Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are
incorporated herein by reference. Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The terminology used in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques can be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery,
and treatment of patients.
[0141] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0142] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages means .+-.1%.
Definitions
[0143] The term "polynueleotide" or "nucleic acid" includes both
single-stranded and double-stranded nucleotide polymers. The
nucleotides comprising the polynueleotide can be ribonucleotides or
deoxyribonucleotides or a modified form of either type of
nucleotide. Said modifications include base modifications such as
bromouridine and inosine derivatives, ribose modifications such as
2',3'-dideoxyribose, and internucleotide linkage modifications such
as phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and
phosphoroamidate.
[0144] The term "oligonucleotide" means a polynucleotide comprising
200 or fewer nucleotides. In some embodiments, oligonucleotides are
10 to 60 bases in length. In other embodiments, oligonucleotides
are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in
length. Oligonucleotides may be single stranded or double stranded,
e.g., for use in the construction of a mutant gene.
Oligonucleotides may be sense or antisense oligonucleotides. An
oligonucleotide can include a label, including a radiolabel, a
fluorescent label, a hapten or an antigenic label for detection
assays. Oligonucleotides may be used, for example. as PCR primers,
cloning primers or hybridization probes.
[0145] An "isolated nucleic acid molecule" means a DNA or RNA of
genomic, mRNA, cDNA, or synthetic origin or some combination
thereof which is not associated with all or a portion of a
polynucleotide in which the isolated polynucleotide is found in
nature, or is linked to a polynucleotide to which it is not linked
in nature. For purposes of this disclosure, it should be understood
that "a nucleic acid molecule comprising" a particular nucleotide
sequence does not encompass intact chromosomes. Isolated nucleic
acid molecules "comprising" specified nucleic acid sequences may
include, in addition to the specified sequences, coding sequences
for up to ten or even up to twenty other proteins or portions
thereof, or may include operably linked regulatory sequences that
control expression of the coding region of the recited nucleic acid
sequences, and/or may include vector sequences.
[0146] Unless specified otherwise, the left-hand end of any
single-stranded polynucleotide sequence discussed herein is the 5'
end; the left-hand direction of double-stranded polynucleotide
sequences is referred to as the 5' direction. The direction of 5'
to 3' addition of nascent RNA transcripts is referred to as the
transcription direction; sequence regions on the DNA strand having
the same sequence as the RNA transcript that are 5' to the 5' end
of the RNA transcript are referred to as "upstream sequences;"
sequence regions on the DNA strand having the same sequence as the
RNA transcript that are 3' to the 3' end of the RNA transcript are
referred to as "downstream sequences."
[0147] The term "control sequence" refers to a polynucleotide
sequence that can affect the expression and processing of coding
sequences to which it is ligated. The nature of such control
sequences may depend upon the host organism. In particular
embodiments, control sequences for prokaryotes may include a
promoter, a ribosomal binding site, and a transcription termination
sequence. For example, control sequences for eukaryotes may include
promoters comprising one or a plurality of recognition sites for
transcription factors, transcription enhancer sequences, and
transcription termination sequence. "Control sequences" can include
leader sequences and/or fusion partner sequences.
[0148] The term "vector" means any molecule or entity (e.g.,
nucleic acid, plasmid, bacteriophage or virus) used to transfer
protein coding information into a host cell.
[0149] The term "expression vector" or "expression construct"
refers to a vector that is suitable for transformation of a host
cell and contains nucleic acid sequences that direct and/or control
(in conjunction with the host cell) expression of one or more
heterologous coding regions operatively linked thereto. An
expression construct may include, but is not limited to, sequences
that affect or control transcription, translation, and, if introns
are present, affect RNA splicing of a coding region operably linked
thereto.
[0150] As used herein, "operably linked" means that the components
to which the term is applied are in a relationship that allows them
to carry out their inherent functions under suitable conditions.
For example, a control sequence in a vector that is "operably
linked" to a protein coding sequence is ligated thereto so that
expression of the protein coding sequence is achieved under
conditions compatible with the transcriptional activity of the
control sequences.
[0151] The term "host cell" means a cell that has been transformed,
or is capable of being transformed, with a nucleic acid sequence
and thereby expresses a gene of interest. The term includes the
progeny of the parent cell, whether or not the progeny is identical
in morphology or in genetic make-up to the original parent cell, so
long as the gene of interest is present.
[0152] The term "transduction" means the transfer of genes from one
bacterium to another, usually by bacteriophage. "Transduction" also
refers to the acquisition and transfer of eukaryotic cellular
sequences by replication defective retroviruses.
[0153] The term "transfection" means the uptake of foreign or
exogenous DNA by a cell, and a cell has been "transfected" when the
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are well known in the art and are
disclosed herein, See, e.g., Graham et al., 1973, Virology 52:456;
Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual,
supra; Davis et al., 1986, Basic Methods in Molecular Biology,
Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be
used to introduce one or more exogenous DNA moieties into suitable
host cells.
[0154] The term "transformation" refers to a change in a cell's
genetic characteristics, and a cell has been transformed when it
has been modified to contain new DNA or RNA. For example, a cell is
transformed where it is genetically modified from its native state
by introducing new genetic material via transfection, transduction,
or other techniques. Following transfection or transduction, the
transforming DNA may recombine with that of the cell by physically
integrating into a chromosome of the cell, or may be maintained
transiently as an episomal element without being replicated, or may
replicate independently as a plasmid. A cell is considered to have
been "stably transformed" when the transforming DNA is replicated
with the division of the cell.
[0155] The terms "polypeptide" or "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms also apply to amino acid polymers in which one
or more amino acid residues is an analog or mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The terms can also
encompass amino acid polymers that have been modified, e.g., by the
addition of carbohydrate residues to form glycoproteins, or
phosphorylated. Polypeptides and proteins can be produced by a
naturally-occurring and non-recombinant cell; or it is produced by
a genetically-engineered or recombinant cell, and comprise
molecules having the amino acid sequence of the native protein, or
molecules having deletions from, additions to, and/or substitutions
of one or more amino acids of the native sequence. The terms
"polypeptide" and "protein" specifically encompass antigen binding
proteins, e.g., CGRP R antigen-binding proteins, CGRP R binding
proteins, antibodies, or sequences that have deletions from,
additions to, and/or substitutions of one or more amino acids of an
antigen-binding protein. The term "polypeptide fragment" refers to
a polypeptide that has an amino-terminal deletion, a
carboxyl-terminal deletion, and/or an internal deletion as compared
with the full-length protein. Such fragments may also contain
modified amino acids as compared with the full-length protein. In
certain embodiments, fragments are about five to 500 amino acids
long. For example, fragments may be at least 5, 6, 8, 10, 14, 20,
50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids
long. Useful polypeptide fragments include immunologically
functional fragments of antibodies, including binding domains. In
the case of a CGRP R-binding antibody, useful fragments include but
are not limited to a CDR region, a variable domain of a heavy or
light chain, a portion of an antibody chain or just its variable
domain including two CDRs, and the like. The"CGRP receptor", or
"CGRP R", is understood to comprise RAMP1 and CRLR.
[0156] The term "isolated protein" (e.g., isolated antigen binding
protein), "isolated polypeptide" or "isolated antibody" means that
a subject protein, polypeptide or antibody (1) is free of at least
some other proteins with which it would normally be found, (2) is
essentially free of other proteins from the same source, e.g., from
the same species, (3) is expressed by a cell from a different
species, (4) has been separated from at least about 50 percent of
polynucleotides, lipids, carbohydrates, or other materials with
which it is associated in nature, (5) is operably associated (by
covalent or noncovalent interaction) with a polypeptide with which
it is not associated in nature, or (6) does not occur in nature.
Typically, an "isolated protein", "isolated polypeptide" or
"isolated antibody" constitutes at least about 5%, at least about
10%, at least about 25%, or at least about 50% of a given sample.
Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any
combination thereof may encode such an isolated protein.
Preferably, the isolated protein polypeptide or antibody is
substantially free from other proteins or other polypeptides or
other contaminants that are found in its natural environment that
would interfere with its therapeutic, diagnostic, prophylactic,
research or other use.
[0157] A "variant" of a polypeptide (e.g., an antigen binding
protein, or an antibody) comprises an amino acid sequence wherein
one or more amino acid residues are inserted into, deleted from
and/or substituted into the amino acid sequence relative to another
polypeptide sequence. Variants include fusion proteins.
[0158] A "derivative" of a polypeptide is a polypeptide (e.g., an
antigen binding protein, or an antibody) that has been chemically
modified in some manner distinct from insertion, deletion, or
substitution variants, e.g., via conjugation to another chemical
moiety.
[0159] The term "naturally occurring" as used throughout the
specification in connection with biological materials such as
polypeptides, nucleic acids, host cells, and the like, refers to
materials which are found in nature.
[0160] An "antigen binding protein" as used herein means a protein
that specifically binds a specified target antigen, such as CGRP R,
particularly primate, e.g., human CGRP R. A CGRP R antigen binding
protein specifically binds the human CGRP receptor.
[0161] An antigen binding protein is said to "specifically bind"
its target when the dissociation constant (K.sub.D) is
.ltoreq.10.sup.-6 M. The antibody specifically binds the target
antigen with "high affinity" when the K.sub.D is
.ltoreq.1.times.10.sup.-8 M. In one embodiment, the antibodies will
bind to CGRP R, or human CGRP R with a K.sub.D5.times.10.sup.-7; in
another embodiment the antibodies will bind with a
K.sub.D.ltoreq.1.times.10.sup.-7; in another embodiment the
antibodies will bind with a K.sub.D.ltoreq.5.times.10.sup.-8; in
another embodiment the antibodies will bind with a
K.sub.D.ltoreq.1.times.10.sup.-8; in another embodiment the
antibodies will bind with a K.sub.D.ltoreq.5.times.10.sup.-9; in
another embodiment the antibodies will bind with
K.sub.D.ltoreq.1.times.10.sup.-9; in another embodiment the
antibodies will bind with a K.sub.D.ltoreq.5.times.10.sup.-10; in
another embodiment the antibodies will bind with a
K.sub.D.ltoreq.1.times.10.sup.-10.
[0162] An antibody, antigen binding fragment thereof or antigen
binding protein "selectively inhibits" a specific receptor relative
to other receptors when the IC50 of the antibody, antigen binding
fragment thereof or antigen binding protein in an inhibition assay
of the specific receptor is at least 50-fold lower than the IC50 in
an inhibition assay of another "reference" receptor. The
"selectivity ratio" is the IC50 of the reference receptor divided
by IC50 of the specific receptor. An antibody, antigen binding
fragment thereof or antigen binding protein selectively inhibits
the human CGRP receptor if the IC50 of the antibody, antigen
binding fragment thereof or antigen binding protein in a cAMP
assay, e.g., the cAMP inhibition assay as described in Example 4
herein, is at least 50-fold lower than the IC50 of that same
antibody, antigen binding fragment thereof or antigen binding
protein in an inhibition assay of the human AM1, AM2 or an amylin
receptor (e.g., AMY1). By way of non-limiting example, if the IC50
of a specific anti-CGRP R antibody in a cAMP assay of hCGRP R is,
e.g., between 0.1 nM and 20 nM. and the IC50 of the same antibody
in a cAMP assay of the hAM1, hAM2 or human AMY1 receptor is 1000 nM
or more, that antibody selectively inhibits the hCGRP receptor. An
antigen binding protein that selectively inhibits a specific
receptor is also understood to be a neutralizing antigen binding
protein with respect to that receptor.
[0163] "Antigen binding region" means a protein, or a portion of a
protein, that specifically binds a specified antigen. For example,
that portion of an antigen binding protein that contains the amino
acid residues that interact with an antigen and confer on the
antigen binding protein its specificity and affinity for the
antigen is referred to as "antigen binding region." An antigen
binding region typically includes one or more "complementary
binding regions" ("CDRs"). Certain antigen binding regions also
include one or more "framework" regions. A "CDR" is an amino acid
sequence that contributes to antigen binding specificity and
affinity. "Framework" regions can aid in maintaining the proper
conformation of the CDRs to promote binding between the antigen
binding region and an antigen.
[0164] certain aspects, recombinant antigen binding proteins that
bind CGRP R protein, or human CGRP R, are provided. In this
context, a "recombinant protein" is a protein made using
recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as described herein. Methods and
techniques for the production of recombinant proteins are well
known in the art.
[0165] The term "antibody" refers to an intact immunoglobulin of
any isotype, or an antigen binding fragment thereof that can
compete with the intact antibody for specific binding to the target
antigen, and includes, for instance, chimeric, humanized, fully
human, and bispecific antibodies. An "antibody" as such is a
species of an antigen binding protein. An intact antibody generally
will comprise at least two full-length heavy chains and two
full-length light chains, but in some instances may include fewer
chains such as antibodies naturally occurring in camelids which may
comprise only heavy chains. Antibodies may be derived solely from a
single source, or may be "chimeric," that is, different portions of
the antibody may be derived from two different antibodies as
described further below. The antigen binding proteins, antibodies,
or binding fragments may be produced in hybridomas, by recombinant
DNA techniques, or by enzymatic or chemical cleavage of intact
antibodies. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and mutations thereof, examples of which are
described below.
[0166] The term "light chain" includes a full-length light chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length light chain includes a
variable region domain, V.sub.L, and a constant region domain,
C.sub.L. The variable region domain of the light chain is at the
amino-terminus of the polypeptide. Light chains include kappa
chains and lambda chains.
[0167] The term "heavy chain" includes a full-length heavy chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length heavy chain includes a
variable region domain, V.sub.H, and three constant region domains,
C.sub.H1, C.sub.H2, and C.sub.H3. The V.sub.H domain is at the
amino-terminus of the polypeptide, and the C.sub.H domains are at
the carboxyl-terminus, with the C.sub.H3 being closest to the
carboxy-terminus of the polypeptide. Heavy chains may be of any
isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4
subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
[0168] The term "signal sequence", "leader sequence" or "signal
peptide" refers to a short (3-60 amino acids long) peptide chain
that directs the transport of a protein. Signal peptides may also
be called targeting signals, signal sequences, transit peptides, or
localization signals. Some signal peptides are cleaved from the
protein by signal peptidase after the proteins are transported,
such that the biologically active form of the protein (e.g., an
antigen binding protein as described herein) is the cleaved,
shorter form. Accordingly, terms such as "antibody comprising a
heavy chain . . . ", "antibody comprising a light chain . . . ",
etc., where the antibody is characterized as having a heavy and/or
light chain with a particular identified sequence, are understood
to include antibodies having the specific identified sequences,
antibodies having the specific identified sequences except that the
signal sequences are replaced by different signal sequences, as
well as antibodies having the identified sequences, minus any
signal sequences.
[0169] The term "antigen binding fragment" (or simply "fragment")
of an antibody or immunoglobulin chain (heavy or light chain), as
used herein, comprises a portion (regardless of how that portion is
obtained or synthesized) of an antibody that lacks at least some of
the amino acids present in a full-length chain but which is capable
of specifically binding to an antigen. Such fragments are
biologically active in that they bind specifically to the target
antigen and can compete with other antigen binding proteins,
including intact antibodies, for specific binding to a given
epitope. In one aspect, such a fragment will retain at least one
CDR present in the full-length light or heavy chain, and in some
embodiments will comprise a single heavy chain and/or light chain
or portion thereof. These biologically active fragments may be
produced by recombinant DNA techniques, or may be produced by
enzymatic or chemical cleavage of antigen binding proteins,
including intact antibodies immunologically functional
immunoglobulin fragments include, but are not limited to, Fab,
Fab', F(ab').sub.2, Fv, domain antibodies and single-chain
antibodies, and may be derived from any mammalian source, including
but not limited to human, mouse, rat, camelid or rabbit. It is
contemplated further that a functional portion of the antigen
binding proteins disclosed herein, for example, one or more CDRs,
could be covalently bound to a second protein or to a small
molecule to create a therapeutic agent directed to a particular
target in the body, possessing bifunctional therapeutic properties,
or having a prolonged serum half-life.
[0170] An "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0171] An "Fc" region contains two heavy chain fragments comprising
the C.sub.H1 and C.sub.H2 domains of an antibody. The two heavy
chain fragments are held together by two or more disulfide bonds
and by hydrophobic interactions of the C.sub.H3 domains.
[0172] An "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form an
F(ab').sub.2 molecule.
[0173] An "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0174] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0175] "Single-chain antibodies" are Fv molecules in which the
heavy and light chain variable regions have been connected by a
flexible linker to form a single polypeptide chain, which forms an
antigen-binding region. Single chain antibodies are discussed in
detail in International Patent Application Publication No. WO
88/01649 and U.S. Pat. No. 4,946,778 and No. 5,260,203, the
disclosures of which are incorporated by reference.
[0176] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more V.sub.H regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two
V.sub.H regions of a bivalent domain antibody may target the same
or different antigens.
[0177] A "bivalent antigen binding protein" or "bivalent antibody"
comprises two antigen binding sites. In some instances, the two
binding sites have the same antigen specificities. Bivalent antigen
binding proteins and bivalent antibodies may be bispecific, see,
infra.
[0178] A "multispecific antigen binding protein" or "multispecific
antibody" is one that targets more than one antigen or epitope.
[0179] A "bispecific," "dual-specific" or "bifunctional" antigen
binding protein or antibody is a hybrid antigen binding protein or
antibody, respectively, having two different antigen binding sites.
Bispecific antigen binding proteins and antibodies are a species of
multispecific antigen binding protein or multispecific antibody and
may be produced by a variety of methods including, but not limited
to, fusion of hybridomas or linking of Fab' fragments. See, e.g.,
Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321;
Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding
sites of a bispecific antigen binding protein or antibody will bind
to two different epitopes, which may reside on the same or
different protein targets.
[0180] The term "neutralizing antigen binding protein" or
"neutralizing antibody" refers to an antigen binding protein or
antibody, respectively, that binds to a ligand, prevents binding of
the ligand to its binding partner and interrupts the biological
response that otherwise would result from the ligand binding to its
binding partner. In assessing the binding and specificity of an
antigen binding protein, e.g., an antibody or immunologically
functional antigen binding fragment thereof, an antibody or
fragment will substantially inhibit binding of a ligand to its
binding partner when an excess of antibody reduces the quantity of
binding partner bound to the ligand by at least about 20%, 30%,
40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as
measured in an in vitro competitive binding assay). In the case of
a CGRP R binding protein, such a neutralizing molecule will
diminish the ability of CGRP R to bind CGRP.
[0181] The term "compete", when used in the context of antigen
binding proteins that may bind the same region on a target antigen,
means competition between antigen binding proteins is determined by
an assay in which the antigen binding protein (e.g., antibody or
immunologically functional antigen binding fragment thereof) under
test prevents or inhibits specific binding of a reference antigen
binding protein (e.g., a ligand, or a reference antibody) to a
common antigen (e.g., CGRP R or an antigen binding fragment
thereof). Any of a number of competitive binding assays can be
used, for example: solid phase direct or indirect radioimmunoassay
(RIA), solid phase direct or indirect enzyme immunoassay (EIA),
sandwich competition assay (see, e.g., Stahli et al., 1983, Methods
in Enzymology 9:242-253); solid phase direct biotin-avidin EIA
(see, e.g., Kirkland. et al., 1986, J. Immunol. 137:3614-3619)
solid phase direct labeled assay, solid phase direct labeled
sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A
Laboratory Manual, Cold Spring Harbor Press); solid phase direct
label RIA using I-125 label (see, e.g., Morel et al., 1988, Molec.
Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g.,
Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA
(Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Such an
assay may involve the use of purified antigen bound to a solid
surface or cells bearing either of these, an unlabelled test
antigen binding protein and a labeled reference antigen binding
protein. Competitive inhibition may measured by determining the
amount of label bound to the solid surface or cells in the presence
of the test antigen binding protein. Antigen binding proteins
identified by competition assay (competing antigen binding
proteins) include antigen binding proteins binding to the same
epitope as the reference antigen binding proteins and antigen
binding proteins binding to an adjacent epitope sufficiently
proximal to the epitope bound by the reference antigen binding
protein for stearic hindrance to occur. Usually, when a competing
antigen binding protein is present in excess, it will inhibit
specific binding of a reference antigen binding protein to a common
antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In
some instance, binding is inhibited by at least 80%, 85%, 90%, 95%,
or 97% or more. Competitive inhibition may also be measured by
immobilizing a reference antigen binding protein to a substrate,
e.g., a "sensor chip", capturing antigen on the substrate via
binding to the reference antibody, and assaying whether a different
antigen binding protein (a competing antigen binding protein) can
additionally bind to the antigen. An example of the latter
competitive binding assay employs a Biacore analysis, and is
described in Example 7 herein.
[0182] The term "antigen" or "immunogen" refers to a molecule or a
portion of a molecule capable of being bound by a selective binding
agent, such as an antigen binding protein (including, e.g., an
antibody or immunological functional antigen binding fragment
thereof), and additionally capable of being used in an animal to
produce antibodies capable of binding to that antigen. An antigen
may possess one or more epitopes that are capable of interacting
with different antigen binding proteins, e.g., antibodies.
[0183] The term "epitope" is the portion of a molecule that is
bound by an antigen binding protein (for example, an antibody). The
term includes any determinant capable of specifically binding to an
antigen binding protein, such as an antibody or to a T-cell
receptor. An epitope can be contiguous or non-contiguous (e.g., (i)
in a single-chain polypeptide, amino acid residues that are not
contiguous to one another in the polypeptide sequence but that
within in context of the molecule are bound by the antigen binding
protein, or (ii) in a multimeric receptor, e.g., CGRP R, comprising
two or more individual components, e.g., RAMP1 and CRLR, amino acid
residues present on two or more of the individual components, but
that within the context of the multimeric receptor are bound by the
antigen binding protein). In certain embodiments, epitopes may be
mimetic in that they comprise a three dimensional structure that is
similar to an epitope used to generate the antigen binding protein,
yet comprise none or only some of the amino acid residues found in
that epitope used to generate the antigen binding protein. Most
often, epitopes reside on proteins, but in some instances may
reside on other kinds of molecules, such as nucleic acids. Epitope
determinants may include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl or
sulfonyl groups, and may have specific three dimensional structural
characteristics, and/or specific charge characteristics. Generally,
antibodies specific for a particular target antigen will
preferentially recognize an epitope on the target antigen in a
complex mixture of proteins and/or macromolecules.
[0184] The term "identity" refers to a relationship between the
sequences of two or more polypeptide molecules or two or more
nucleic acid molecules, as determined by aligning and comparing the
sequences. "Percent identity" means the percent of identical
residues between the amino acids or nucleotides in the compared
molecules and is calculated based on the size of the smallest of
the molecules being compared. For these calculations, gaps in
alignments (if any) must be addressed by a particular mathematical
model or computer program (i.e., an "algorithm"). Methods that can
be used to calculate the identity of the aligned nucleic acids or
polypeptides include those described in Computational Molecular
Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University
Press; Biocomputing Informatics and Genome Projects, (Smith, D. W.,
ed.), 1993, New York: Academic Press; Computer Analysis of Sequence
Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New
Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in
Molecular Biology, New York: Academic Press; Sequence Analysis
Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.
Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.
48:1073.
[0185] In calculating percent identity, the sequences being
compared are aligned in a way that gives the largest match between
the sequences. The computer program used to determine percent
identity is the GCG program package, which includes GAP (Devereux
et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group,
University of Wisconsin, Madison, Wis.). The computer algorithm GAP
is used to align the two polypeptides or polynucleotides for which
the percent sequence identity is to be determined. The sequences
are aligned for optimal matching of their respective amino acid or
nucleotide (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal, wherein the "average diagonal" is the average of the
diagonal of the comparison matrix being used; the "diagonal" is the
score or number assigned to each perfect amino acid match by the
particular comparison matrix) and a gap extension penalty (which is
usually 1/10 times the gap opening penalty), as well as a
comparison matrix such as PAM 250 or BLOSUM 62 are used in
conjunction with the algorithm. In certain embodiments, a standard
comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein
Sequence and Structure 5:345-352 for the PAM 250 comparison matrix;
Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919
for the BLOSUM 62 comparison matrix) is also used by the
algorithm.
[0186] Recommended parameters for determining percent identity for
polypeptides or nucleotide sequences using the GAP program are the
following:
[0187] Algorithm: Needleman et al., 1970, J. Mol. Biol.
48:443-453;
[0188] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992,
supra;
[0189] Gap Penalty: 12 (but with no penalty for end gaps)
[0190] Gap Length Penalty: 4
[0191] Threshold of Similarity: 0
[0192] Certain alignment schemes for aligning two amino acid
sequences may result in matching of only a short region of the two
sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, the selected
alignment method (GAP program) can be adjusted if so desired to
result in an alignment that spans at least 50 contiguous amino
acids of the target polypeptide.
[0193] As used herein, "substantially pure" means that the
described species of molecule is the predominant species present,
that is, on a molar basis it is more abundant than any other
individual species in the same mixture. In certain embodiments, a
substantially pure molecule is a composition wherein the object
species comprises at least 50% (on a molar basis) of all
macromolecular species present. In other embodiments, a
substantially pure composition will comprise at least 80%, 85%,
90%, 95%, or 99% of all macromolecular species present in the
composition. In other embodiments, the object species is purified
to essential homogeneity wherein contaminating species cannot be
detected in the composition by conventional detection methods and
thus the composition consists of a single detectable macromolecular
species.
[0194] The term "treating" refers to any indicia of success in the
treatment or amelioration of an injury, pathology or condition,
including any objective or subjective parameter such as abatement;
remission; diminishing of symptoms or making the injury, pathology
or condition more tolerable to the patient; slowing in the rate of
degeneration or decline; making the final point of degeneration
less debilitating; improving a patient's physical or mental
well-being, The treatment or amelioration of symptoms can be based
on objective or subjective parameters; including the results of a
physical examination, neuropsychiatric exams, and/or a psychiatric
evaluation. For example, certain methods presented herein
successfully treat migraine headaches either prophylactically or as
an acute treatment, decreasing the frequency of migraine headaches,
decreasing the severity of migraine headaches, and/or ameliorating
a symptom associated with migraine headaches.
[0195] An "effective amount" is generally an amount sufficient to
reduce the severity and/or frequency of symptoms, eliminate the
symptoms and/or underlying cause, prevent the occurrence of
symptoms and/or their underlying cause, and/or improve or remediate
the damage that results from or is associated with migraine
headache. In some embodiments, the effective amount is a
therapeutically effective amount or a prophylactically effective
amount. A "therapeutically effective amount" is an amount
sufficient to remedy a disease state (e.g. migraine headache) or
symptoms, particularly astute or symptoms associated with the
disease state, or otherwise prevent, hinder, retard or reverse the
progression of the disease state or any other undesirable symptom
associated with the disease in any way whatsoever. A
"prophylactically effective amount" is an amount of a
pharmaceutical composition that, when administered to a subject,
will have the intended prophylactic effect, e.g., preventing or
delaying the onset (or reoccurrence) of migraine headache, or
reducing the likelihood of the onset (or reoccurrence) of migraine
headache or migraine headache symptoms. The full therapeutic or
prophylactic effect does not necessarily occur by administration of
one dose, and may occur only after administration of a series of
doses. Thus, a therapeutically or prophylactically effective amount
may be administered in one or more administrations.
[0196] "Amino acid" includes its normal meaning in the art. The
twenty naturally-occurring amino acids and their abbreviations
follow conventional usage. See, Immunology-A Synthesis, 2nd
Edition, (E. S. Golub and D. R. Green, eds.), Sinauer Associates:
Sunderland, Mass. (1991), incorporated herein by reference for any
purpose. Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as
.alpha.-,.alpha.-disubstituted amino acids, N-alkyl amino acids,
and other unconventional amino acids may also be suitable
components for polypeptides and are included in the phrase "amino
acid." Examples of unconventional amino acids include:
4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxyl-terminal direction, in accordance with standard usage and
convention.
General Overview
[0197] Antigen-binding proteins that bind CGRP R protein, including
human CGRP R (hCGRP R) protein are provided herein. The antigen
binding proteins provided are polypeptides into which one or more
complementary determining regions (CDRs), as described herein, are
embedded and/or joined. In some antigen binding proteins, the CDRs
are embedded into a "framework" region, which orients the CDR(s)
such that the proper antigen binding properties of the CDR(s) is
achieved. In general, antigen binding proteins that are provided
can interfere with, block, reduce or modulate the interaction
between CGRP and CGRP R.
[0198] Certain antigen binding proteins described herein are
antibodies or are derived from antibodies. In certain embodiments,
the polypeptide structure of the antigen binding proteins is based
on antibodies, including, but not limited to, monoclonal
antibodies, bispecific antibodies, minibodies, domain antibodies,
synthetic antibodies (sometimes referred to herein as "antibody
mimetics"), chimeric antibodies, humanized antibodies, human
antibodies, antibody fusions (sometimes referred to herein as
"antibody conjugates"), and fragments thereof. The various
structures are further described herein below.
[0199] The antigen binding proteins provided herein have been
demonstrated to bind to CGRP R, in particular human CGRP R. As
described further in the examples below, certain antigen binding
proteins were tested and found to bind to epitopes different from
those bound by a number of other antibodies directed against one or
the other of the components of CGRP R. The antigen binding proteins
that are provided compete with CGRP and thereby prevent CGRP from
binding to its receptor. As a consequence, the antigen binding
proteins provided herein are capable of inhibiting CGRP R activity.
In particular, antigen binding proteins binding to these epitopes
can have one or more of the following activities: inhibiting, inter
alia, induction of CGRP R signal transduction pathways, inhibiting
vasodialation, causing vasoconstriction, decreasing inflammation,
e.g., neurogenic inflammation, and other physiological effects
induced by CGRP R upon CGRP binding.
[0200] The antigen binding proteins that are disclosed herein have
a variety of utilities. Some of the antigen binding proteins, for
instance, are useful in specific binding assays, affinity
purification of CGRP R, in particular hCGRP R or its ligands and in
screening assays to identify other antagonists of CGRP R activity.
Some of the antigen-binding proteins are useful for inhibiting
binding of CGRP to CGRP R.
[0201] The antigen-binding proteins can be used in a variety of
treatment applications, as explained herein. For example, certain
CGRP R antigen-binding, proteins are useful for treating conditions
associated with CGRP R mediated signaling, such as reducing,
alleviating, or treating the frequency and/or severity of migraine
headache, reducing, alleviating, or treating cluster headache,
reducing, alleviating, or treating chronic pain, alleviating or
treating diabetes mellitus (type II), reducing, alleviating, or
treating cardiovascular disorders, and reducing, alleviating, or
treating hemodynamic derangements associated with endotoxemia and
sepsis in a patient. Other uses for the antigen binding proteins
include, for example, diagnosis of CGRP R-associated diseases or
conditions and screening assays to determine the presence or
absence of CGRP R. Some of the antigen binding proteins described
herein are useful in treating consequences, symptoms, and/or the
pathology associated with CGRP R activity. These include, but are
not limited to, various types of migraine headaches.
CGRP Receptor
[0202] The antigen binding proteins disclosed herein bind to CGRP
R, in particular human CGRP R. CGRP R is a multimer that includes
both CRLR and RAMP1. The nucleotide sequence of human CRLR is
provided herein as SEQ ID NO: 1. The amino acid sequence of human
CRLR is provided herein as SEQ ID NO:2. The nucleotide sequence of
human RAMP1 is provided herein as SEQ ID NO:3. The amino acid
sequence of human RAMP1 is provided herein as SEQ ID NO:4. The
antigen binding proteins described herein bind the extracellular
portion of CGRP R, which comprises the extracellular portions of
CRLR and RAMP1. An exemplary extracellular domain ("ECD") of human
CRLR is encoded by the nucleotide sequence presented as SEQ ID
NO:5, and has the amino acid sequence presented as SEQ ID NO:6.
This sequence includes a signal peptide; an exemplary mature (minus
the signal peptide) CRLR ECD has the amino acid sequence presented
as SEQ ID NO:10. An exemplary ECD of human RAMP1 is encoded by the
nucleotide sequence presented as SEQ ID NO:7, and has the amino
acid sequence presented as SEQ ID NO:8. This sequence includes a
signal peptide; an exemplary mature (minus the signal peptide)
RAMP1 ECD has the amino acid sequence presented as SEQ ID NO:11. As
described below, CGRP R proteins may also include fragments. As
used. herein, the terms are used interchangeably to mean a
receptor, in particular, unless otherwise specified, a human
receptor that binds specifically to CGRP.
[0203] The term CGRP R also includes post-translational
modifications of the CGRP R amino acid sequence, for example,
possible N-linked glycosylation sites. Thus, the antigen binding
proteins may bind to or be generated from proteins glycosylated at
one or more of the positions.
CGRP Receptor Binding Proteins
[0204] A variety of selective binding agents useful for regulating
the activity of CGRP R are provided. These agents include, for
instance, antigen binding proteins that contain an antigen binding
domain (e.g., single chain antibodies, domain antibodies,
immunoadhesions, and polypeptides with an antigen binding region)
and specifically bind to CGRP R, in particular human CGRP R. Some
of the agents, for example, are useful in inhibiting the binding of
CGRP to CGRP R, and can thus be used to inhibit, interfere with or
modulate one or more activities associated with CGRP R
signaling.
[0205] In general, the antigen binding proteins that are provided
typically comprise one or more CDRs as described herein (e.g., 1,
2, 3, 4, 5 or 6). In some instances, the antigen binding protein
comprises (a) a polypeptide structure and (b) one or more CDRs that
are inserted into and/or joined to the polypeptide structure. The
polypeptide structure can take a variety of different forms. For
example, it can be, or comprise, the framework of a naturally
occurring antibody, or fragment or variant thereof, or may be
completely synthetic in nature. Examples of various polypeptide
structures are further described below.
[0206] In certain embodiments, the polypeptide structure of the
antigen binding proteins is an antibody or is derived from an
antibody, including, but not limited to, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies, synthetic
antibodies (sometimes referred to herein as "antibody mimetics"),
chimeric antibodies, humanized antibodies, antibody fusions
(sometimes referred to as "antibody conjugates"), and portions or
fragments of each, respectively. In some instances, the antigen
binding protein is an immunological fragment of an antibody (e.g.,
a Fab, a Fab', a F(ab').sub.2, or a scFv). The various structures
are further described and defined herein.
[0207] Certain of the antigen binding proteins as provided herein
specifically bind to human CGRP R. In a specific embodiment, the
antigen binding protein specifically binds to human CGRP R protein
comprising human CRLR having the amino acid sequence of SEQ ID NO:2
and human RAMP1 having the amino acid sequence of SEQ ID NO:4.
[0208] In embodiments where the antigen binding protein is used for
therapeutic applications, an antigen binding protein can inhibit,
interfere with or modulate one or more biological activities of
CGRP R. In this case, an antigen binding protein binds specifically
and/or substantially inhibits binding of human CGRP R to CGRP when
an excess of antibody reduces the quantity of human CGRP R bound to
CGRP, or vice versa, by at least about 20%, 30%, 40%, 50%, 60%,
70%, 80%, 85%, 90%, 95%, 97%, 99% or more (for example by measuring
binding in an in vitro competitive binding assay).
Naturally Occurring Antibody Structure
[0209] Some of the antigen binding proteins that are provided have
the structure typically associated with naturally occurring
antibodies. The structural units of these antibodies typically
comprise one or more tetramers, each composed of two identical
couplets of polypeptide chains, though some species of mammals also
produce antibodies having only a single heavy chain. In a typical
antibody, each pair or couplet includes one full-length "light"
chain (in certain embodiments, about 25 kDa) and one full-length
"heavy" chain (in certain embodiments, about 50-70 kDa). Each
individual immunoglobulin chain is composed of several
"immunoglobulin domains", each consisting of roughly 90 to 110
amino acids and expressing a characteristic folding pattern. These
domains are the basic units of which antibody polypeptides are
composed. The amino-terminal portion of each chain typically
includes a variable domain that is responsible for antigen
recognition. The carboxy-terminal portion is more conserved
evolutionarily than the other end of the chain and is referred to
as the "constant region" or "C region". Human light chains
generally are classified as kappa and lambda light chains, and each
of these contains one variable domain and one constant domain.
Heavy chains are typically classified as mu, delta, gamma, alpha,
or epsilon chains, and these define the antibody's isotype IgM,
IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes,
including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM
subtypes include IgM, and IgM2. IgA subtypes include IgA1 and IgA2.
In humans, the IgA and IgD isotypes contain four heavy chains and
four light chains; the IgG and IgE isotypes contain two heavy
chains and two light chains; and the IgM isotype contains five
heavy chains and five light chains. The heavy chain C region
typically comprises one or more domains that may be responsible for
effector function. The number of heavy chain constant region
domains will depend on the isotype. IgG heavy chains, for example,
each contain three C region domains known as C.sub.H1, C.sub.H2 and
C.sub.H3. The antibodies that are provided can have any of these
isotypes and subtypes. In certain embodiments, the CGRP R antibody
is of the IgG1, IgG2, or IgG4 subtype.
[0210] In full-length light and heavy chains, the variable and
constant regions are joined by a "J" region of about twelve or more
amino acids, with the heavy chain also including a "D" region of
about ten more amino acids. See, e.g., Fundamental Immunology, 2nd
ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press (hereby
incorporated by reference in its entirety for all purposes). The
variable regions of each light/heavy chain pair typically form the
antigen binding site.
[0211] One example of an IgG2 heavy constant domain of an exemplary
CGRP R monoclonal antibody has the amino acid sequence:
TABLE-US-00002 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK
TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. (the last 326 residues of
the sequence shown as SEQ. ID NO: 29)
[0212] One example of a kappa light Constant domain of an exemplary
CGRP R monoclonal antibody has the amino acid sequence:
TABLE-US-00003 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (the
last 107 residues of the sequence shown as SEQ ID NO: 14)
[0213] Variable regions of immunoglobulin chains generally exhibit
the same overall structure, comprising relatively conserved
framework regions (FR) joined by three hypervariable regions, more
often called "complementarity determining regions" or CDRs. The
CDRs from the two chains of each heavy chain/light chain pair
mentioned above typically are aligned by the framework regions to
form a structure that binds specifically with a specific epitope on
the target protein (e.g., CGRP R). From N-terminal to C-terminal,
naturally-occurring light and heavy chain variable regions both
typically conform with the following order of these elements: FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been
devised for assigning numbers to amino acids that occupy positions
in each of these domains. This numbering system is defined in Kabat
Sequences of Proteins of Immunological Interest (1987 and 1991,
NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J Mol. Biol.
196:901-917; Chothia et al., 1989, Nature 342:878-883.
[0214] The various heavy chain and light chain variable regions
provided herein are depicted in Table 3. Each of these variable
regions may be attached to the above heavy and light chain constant
regions to form a complete antibody heavy and light chain,
respectively. Further, each of the so generated heavy and light
chain sequences may be combined to form a complete antibody
structure. It should be understood that the heavy chain and light
chain variable regions provided herein can also be attached to
other constant domains having different sequences than the
exemplary sequences listed above.
[0215] Specific examples of some of the full length light and heavy
chains of the antibodies that are provided and their corresponding
amino acid sequences are summarized in Tables 2A and 2B. Table 2A
shows exemplary light chain sequences, and Table 2B shows exemplary
heavy chain sequences.
TABLE-US-00004 TABLE 2A Exemplary Antibody Light Chain Amino Acid
Sequences SEQ ID Desig- Contained NO: nation in Clone Sequence 12
L1 01E11 LC MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSVSEAPGQKVTISC
SGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGS
KSGTSATLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 13
L2 01H7 LC MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSASGTPGQRVTISC
SGSSSNIGSNYVYWYQQLPGAAPKLLIFRSNQRPSGVPDRFSGS
KSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 14
L3 02E7 LC MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTIT
CRASQGIRNDLGWFQQKPGKAPKRLIYAASSLQSGVPSRFSGS
GSGTEFTLTISSLQPEDLATYYCLQYNIYPWTFGQGTKVEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 15 L4
03B6 LC MDMRVPAQLLGLLLLWLRGARCSSELTQDPTVSVALGQTVKITC
QGDSLRSFYASWYQQKPGQAPVLVFYGKNNRPSGIPDRFSGSS
SGNTASLTITGAQAEDEADYYCNSRDSSVYHLVLGGGTKLTVLG
QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADG
SPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS 16 L5
0308 LC MDMRVPAQLLGLLLLWLRGARCDIILAQTPLSLSVTPGQPASISC
KSSQSLLHSAGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRF
SGSGSGTDFTLKISRVEAEDVGIYYCMQSFPLPLTFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 17
L6 04E4 LC MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSVSAAPGQKVTISC
SGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGS
KSGTSTTLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL
GQPKANPTVTLFFPSSEELQANKATLVCLISDFYFGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 18
L7 04H6 LC MDMRVPAQLLGLLLLWLRGARCDIVMTQSPLSLPVTPGEPASISC
RSSQSLLHSFGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRF
SGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGPGTKVDI
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC
19 L8 05F5 LC MDMRVPAQLLGLLLLWLRGARCDIILTQTPLSLSVTPGQPASISC
KSSQSLLHSDGKTYLYVVYLQKPGQPPQLLIYEVSNRFSGEPDRF
SGSGSGTDFTLKISRVEAEDVGTYYCMQSFPLPLTFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 20
L9 09D4 LC MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSVSAAPGQKVTISC
SGSSSNIGNNYVSWYQQFPGTAPKLLIYDNNKRPSGIPDRFSGS
KSGTSATLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL
GQFKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 21
L10 09F5 LC MDMRVPAQLLGLLLLVVLRGARCQSVLTQSPSASGTPGQRVTISC
SGSSSNIGSNYVYWYQQLPGAAPKLLILRNNQRPSGVPDRFSGS
KSGTSASLTISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 22
L11 10E4 LC MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSASGTPGQRVTISC
SGSSSNIGSNIVNWYQQLPGTAPKLLIYINNQRPSGVPDRFSGS
KSGTSASLAISGLQSEDEADFYCAARDESLNGVVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 23
L12 11D11 HL MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSASGTPGQRVTISC 11H9 LC
SGSSSNIGSNYVYWYQQLPGAAPKLLIFRNNQRPSGVPDRFSGS
KSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 24
L13 12E8 LC MDMRVPAQLLGLLLLWLRGARCDITLTQTPLSLSVSPGQPASISC
KSSQSLLHSDGRNYLYWYLQKPGQPPQLLIYEVSNRFSGLPDRF
SGSGSGTDFTLKISRVEAEDVGIYYCMQSFPLPLTFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 25
L14 12G8 HL MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSVSAAPGQKVTISC
SGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGS
KSGTSATLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL
GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 26
L15 13H2 LC MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTIT
CRASQGIRKDLGWYQQKPGKAPKRLIYGASSLQSGVPSRFSGS
GSGTEFTLTISSLQPEDFATYYCLQYNSFPWTFGQGTKVEIKRTV
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 27
L16 32H7 LC METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRA
SQSVSSGYLTWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGNSLCRFGQGTKLEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 28 L17
32H7 CS LC METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRA
SQSVSSGYLTWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQYGNSLSRFGQGTKLEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
TABLE-US-00005 TABLE 2B Exemplary Antibody Heavy Chain Amino Acid
Sequences SEQ ID Design- Contained NO: ation in Clone Sequence 29
H1 01E11 HC MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLS 04E4 HC
CAASGFTFSSFGMHWVRQAPGKGLEWVAVISFDGSIKYSVDSVK 09D4 HC
GRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDRLNYYDSSGY
YHYKYYGMAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 30 H2 01H7 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGGSLRLS
CAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSTTDGGTTDYAA
PVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDRTGYSISW
SSYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKE
YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY
SKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 31 H3 02E7 HC
MDMRVPAQLLGLLLLWLRGARCEVQLLESGGGLVQPGESLRLS
CAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGRTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQREVGPYSS
GWYDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLKSPGK 32 H4 03B6 HC
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVS
CKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQK
FQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCARDQMSIIMLRG
VFPPYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTERVVSVLTWHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33 H5 0308 HC
MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLS 05F5 HC
CAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSHESYADSV 12E8 HC
KGRFTISRDISKNTLYLQMNSLRAEDTAVYFCARERKRVTMSTLY
YYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAP
PVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK
CKVSNKGLPAPIEKTSKTKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 34 H6 0416 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGRSLRLS
CTASGFTFGDYAMSWFRQAPGKGLEWIGFIRSRAYGGTPEYAAS
VKGRFTISRDDSKTIAYLQMNSLKTEDTAVYFCARGRGIAARWDY
WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG
TQTYTCNVDHPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA
PIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPMLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK 35 H7 09F5 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGGSLRLS
CAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYTA
PVKGRFTISRDDSKNTLYLQMNSLKAEDTAVYYCTTDRTGYSISW
SSYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36 H8 10E4 HC
MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVS
CKASGYTFTDYYMYWVRQAPGQGLEWMGWISPNSGGTNYAQK
FQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRGGYSGYAGL
YSHYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA
PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 H9 11D11 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGGSLRLS
CAASGFTFGNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYA
APVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYFCTTDRTGYSIS
WSSYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSE
STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP
CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL
YSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 38 H10 11H9 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGGSLRLS
CAASGFTFGNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYA
APVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDRTGYSIS
WSSYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSE
STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPP
CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 39 H11 12G8 HC
MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLS
CAASGFTFSSFGMHWVRQAPGKGLEWVAVISFDGSIKYSVDSVK
GRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDRLNYYDSSGY
YHYKYYGLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA
PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 40 H12 13H2 HC
MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVKPGGSLRLS
CAASGYTFSTYSMNWVRQAPGKGLEWVSSISSSSSYRYYADSV
KGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREGVSGSSPYSI
SWYDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 H13 32H7 HC
MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLS
CAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADS
VKGRFIISRDKSKNTLYLQMNSLRAEDTAVYYCARAGGIAAAGLY
YYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW
TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP
VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0216] The first 22 amino acids of each of the light chain
sequences in Table 2A, except 32H7 and 32H7 CS, is a signal
sequence. In the case of 32H7 and 32H7 CS, the signal sequence is
20 amino acids. Similarly, the first 22 amino acids of each of the
heavy chain sequences in Table 2B is a signal sequence. The signal
peptides may be changed to signal peptides having different
sequences, e.g., for more optimal expression in certain host cells.
It will be therefore be understood that the invention also includes
antibodies having the light and/or heavy chain sequences as
specified in Tables 2A and 2B, but with different signal
sequences.
[0217] Again, each of the exemplary heavy chains (H1, H2, H3 etc.)
listed in Table 2B can be combined with any of the exemplary light
chains shown in Table 2A to form an antibody. Examples of such
combinations include H1 combined with any of L1 through L17; H2
combined with any of L1 through L17; H3 combined with any of L1
through L17, and so on. In some instances, the antibodies include
at least one heavy chain and one light chain from those listed in
Tables 2A and 2B. In some instances, the antibodies comprise two
different heavy chains and two different light chains listed in
Tables 2A and 2B. In other instances, the antibodies contain two
identical light chains and two identical heavy chains. As an
example. an antibody or immunologically functional fragment may
include two H1 heavy chains and two L1 light chains, or two H2
heavy chains and two L2 light chains, or two H3 heavy chains and
two L3 light chains and other similar combinations of pairs of
light chains and pairs of heavy chains as listed in Tables 2A and
2B.
[0218] Other antigen binding proteins that are provided are
variants of antibodies formed by combination of the heavy and light
chains shown in Tables 2A and 2B and comprise light and/or heavy
chains that each have at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or
99% identity to the amino acid sequences of these chains. In some
instances, such antibodies include at least one heavy chain and one
light chain, whereas in other instances the variant forms contain
two identical light chains and two identical heavy chains.
Variable Domains of Antibodies
[0219] Also provided are antigen binding proteins that contain an
antibody heavy chain variable region selected from the group
consisting of V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5,
V.sub.H6, V.sub.H7, V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11,
V.sub.H12, and V.sub.H13, and/or an antibody light chain variable
region selected from the group consisting of V.sub.L1, V.sub.L2,
V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8,
V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14,
V.sub.L15, V.sub.L16, and V.sub.L17, as shown in Table 3 below, and
immunologically functional fragments. derivatives, muteins and
variants of these light chain and heavy chain variable regions.
[0220] Sequence alignments of the various heavy and light chain
variable regions, respectively, are provided in FIGS. 1A and
1B.
[0221] Antigen binding proteins of this type can generally be
designated by the formula "V.sub.Hx/V.sub.Ly," where "x"
corresponds to the number of heavy chain variable regions and "y"
corresponds to the number of the light chain variable regions.
TABLE-US-00006 TABLE 3 Exemplary V.sub.H and V.sub.L, Chain Amino
Acid Sequences SEQ Contained ID in Gone Designation NO. Amino Acid
Sequence 1E11 V.sub.L1 137
QSVLTQPPSVSEAFGQKVTISCSGSSSNIGNNYVSWYQQLP
GTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDE
ADYYCGTWDSRLSAVVFGGGTKLTVL 1H7 V.sub.L2 138
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLP
GAAPKLLIFRSNQRPSGVPDRFSGSKSGTSASLAISGLRSED
EADYYCAAWDDSLSGWVFGGGTKLTVL 2E7 V.sub.L3 139
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPG
KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLA TYYCLQYNIYPWTFGQGTKVEIK
3B6 V.sub.L4 140 SSELTQDPTVSVALGQTVKITCQGDSLRSFYASWYQQKPGQ
APVLVFYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEA
DYYCNSRDSSVYHLVLGGGTKLTVL 3C8 V.sub.L5 141
DIILAQTPLSLSVTPGQPASISCKSSQSLLHSAGKTYLYWYLQ
KFGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
EDVGIYYCMQSFPLPLTFGGGTKVEIK 4E4 V.sub.L6 142
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLP
GTAPKLLIYDNNKRPSGIPDRFSGSKSGTSTTLGITGLQTGDE
ADYYCGTWDSRLSAVVFGGGTKLTVL 4H6 V.sub.L7 143
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSFGYNYLDWL
QKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCMQALQTPFTFGFGTKVDIK 5F5 V.sub.L8 144
DIILTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQ
KPGQPPQLLIYEVSNRFSGEPDRFSGSGSGTDFTLKISRVEA
EDVGTYYCMQSFPLPLTFGGGTKVEIK 9D4 V.sub.L9 145
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQFP
GTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDE
ADYYCGTWDSRLSAVVFGGGTKLTVL 9F5 V.sub.L10 146
QSVLTQSPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLP
GAAPKLLILRNNQRPSGVPDRFSGSKSGTSASLTISGLRSED
EADYYCAAWDDSLSGWVFGGGTKLTVL 10E4 V.sub.L11 147
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLP
GTAFKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLQSED
EADFYCAARDESLNGVVFGGGTKLTVL 11D11 V.sub.L12 148
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLP 11H9
GAAPKLLIFRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED
EADYYCAAWDDSLSGWVFGGGTKLTVL 12E8 V.sub.L13 149
DITLTQTPLSLSVSPGQPASISCKSSQSLLHSDGRNYLYWYL
QKPGQPPQLLIYEVSNRFSGLPDRFSGSGSGTDFTLKISRVE
AEDVGIYYCMQSFPLPLTFGGGTKVEIK 12G8 V.sub.L14 150
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLP
GTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDE
ADYYCGTWDSRLSAVVFGGGTKLTVL 13H2 V.sub.L15 151
DIQMTQSPSSLSASVGDRVTITCRASQGIRKDLGWYQQKPG
KAPKRLIYGASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFA TYYCLQYNSFPWTFGQGTKVEIK
32H7 V.sub.L16 152 EIVLTQSPGTLSLSPGERATLSCRASQSVSSGYLTWYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYGNSLCRFGQGTKLEIK 32H7 CS V.sub.L17 153
EIVLTQSPGTLSLSPGERATLSCRASQSVSSGYLTWYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYGNSLSRFGQGTKLEIK 32H8 V.sub.L18 154
DIVMTQSPDSLAVSLGERATINCKSSQSILDSSNNDNYLAWY
QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSL
QAEDVAVYYCQQYYNTPFTFGPGTKVDIK 33B5 V.sub.L19 155
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG
KAPKRLIYVASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFA TYYCLQYNTYPLTFGGGTKVEIK
33E4 V.sub.L20 156 EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPG
QAPRLLIHDASPRTAGIPARFSGSGSGTEFTLTINSLQSEDFA
VYYCQQYNYWTPITFGQGTRLEIK 34E3 V.sub.L21 157
QSVLTQPPSMSAAPGQKVTISCSGSSSNIGNNYVSWYQQLP
GTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDE
ANYCCGTWDIGLSVWVFGGGTKLTVL 4E4 V.sub.H1 158
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQA 9D4
PGKGLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQ 1E11
MNSLRAEDTAVYYCARDRLNYYDSSGYYHYKYYGMAVWGQ GTTVTVSS 1H7 V.sub.H2 159
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA
PGKGLEWVGRIKSTTDGGTTDYAAPVKGRFTISRDDSKNTLY
LQMNSLKTEDTAVYYCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS 2E7 V.sub.H3 160
EVQLLESGGGLVQPGESLRLSCAASGFTFSSYAMSWVRQA
PGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKDQREVGPYSSGWYDYYYGMDVW GQGTTVTSS 3B6 V.sub.H4 161
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA
PGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAY
MELSRLRSDDTAVYFCARDQMSIIMLRGVFPPYYYGMDVWG QGTTVTVSS 3C8 V.sub.H5
162 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA 12E8
PGKGLEWVAVISYDGSHESYADSVKGRFTISRDISKNTLYLQ 5F5
MNSLRAEDTAVYFCARERKRVTMSTLYYYFYYGMDVWGQG TTVTVSS 4H6 V.sub.H6 163
EVQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQA
PGKGLEWIGFIRSRAYGGTPEYAASVKGRFTISRDDSKTIAYL
QMNSLKTEDTAVYFCARGRGIAARWDYWGQGTLVTVSS 9F5 V.sub.H7 164
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA
PGKGLEWVGRIKSKTDGGTTDYTAPVKGRFTISRDDSKNTLY
WMNSLKAEDTAVYYCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS 10E4 V.sub.H8 165
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMYWVRQA
PGQGLEWMGWISPNSGGTNYAQKFQGRVTMTRDTSISTAY
MELSRLRSDDTAVYYCVRGGYSGYAGLYSHYYGMDVWGQ GTTVTVSS 11D11 V.sub.H9 166
EVQLVESGGGLVKPGGSLRLSCAASGFTFGNAWMSWVRQA
PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLY
LQMNSLKTEDTAVYFCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS 11H9 V.sub.H10
167 EVQLVESGGGLVKPGGSLRLSCAASGFTFGNAWMSWVRQA
PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLY
LQMNSLKTEDTAVYYCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS 12G8 V.sub.H11
168 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQA
PGKGLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQ
MNSLRAEDTAVYYCARDRLNYYDSSGYYHYKYYGLAVWGQ GTTVTVSS 13H2 V.sub.H12
169 EVQLVESGGGLVKPGGSLRLSCAASGYTFSTYSMNWVRQA
PGKGLEWVSSISSSSSYRYYADSVKGRFTISRDNAKNSLYLQ
MSSLRAEDTAVYYCAREGVSGSSPYSISWYDYYYGMDVWG QGTTVTVSS 32H7 V.sub.H13
170 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA
PGKGLEWVAVIWYDGSNKYYADSVKGRFIISRDKSKNTLYLQ
MNSLRAEDTAVYYCARAGGIAAAGLYYYYGMDVWGQGTTV TVSS 32H8 V.sub.H14 171
QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYYLHWVRQA
PGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAY
MELSRLRSDDTAVFYCARGRQWLGFDYWGQGTLVTVSS 33E4 V.sub.H15 172
QVQLQQWGAGLLKPSETLSLSCAVYGGSFGGYYWSWIRQP
PGKGLEWIGEINHSGGTKYNPSLKSRVTISVDTSKNQFSLKL
SSVTAADTAVYFCARGDVVGFFDYWGQGTLVTVSS 3365 V.sub.H16 173
QVQLVQSGAEVKKSGASVKVSCKASGYTFTGYYMHWVRQA
PGQGLEWMGWINPNSGGTNYVQKFQGRVTMTRDSISTAY
MELSRLRSDDTAVYYCARNEYSSAWPLGYWGQGTLVTVSS 34E3 V.sub.H17 174
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVAWIRQPP
GKALEWLALIYWTDDKRYSPSLKSRLTITKDTSKNQVVLRMT
NMDPLDTATYFCAHRPGGWFDPWGQGTLVTVSS
[0222] Each of the heavy chain variable regions listed in Table 3
may be combined with any of the light chain variable regions shown
in Table 3 to form an antigen binding protein. Examples of such
combinations include V.sub.H1 combined with any of V.sub.L1,
V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7,
V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13,
V.sub.L14, V.sub.L15, V.sub.L16, or V.sub.L17; V.sub.H2 combined
with any of V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5,
V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11,
V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, or
V.sub.L17; V.sub.H3 combined with any of V.sub.L1, V.sub.L2,
V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8,
V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14,
V.sub.L15, V.sub.L16, or V.sub.L17; and so on.
[0223] In some instances, the antigen binding protein includes at
least one heavy chain variable region and/or one light chain
variable region from those listed in Table 3. In some instances,
the antigen binding protein includes at least two different heavy
chain variable regions and/or light chain variable regions from
those listed in Table 3. An example of such an antigen binding
protein comprises (a) one V.sub.H1, and (b) one of V.sub.H2,
V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8,
V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, or V.sub.H13. Another
example comprises (a) one V.sub.H2, and (b) one of V.sub.H1,
V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8,
V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, or V.sub.H13. Again
another example comprises (a) one V.sub.H3, and (b) one of
V.sub.H1, V.sub.H2, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7,
V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, or V.sub.H13,
etc. Again another example of such an antigen binding protein
comprises (a) one V.sub.L1, and (b) one of V.sub.L2, V.sub.L3,
V.sup.L4, V.sub.L5, V.sup.L6, V.sub.L7, V.sub.L8, V.sub.L9,
V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15,
V.sub.L16, or V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, or
V.sub.L21. Again another example of such an antigen binding protein
comprises (a) one V.sub.L2, and (b) one of V.sub.L1, V.sub.L3,
V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9,
V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15,
V.sub.L16, V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, or
V.sub.L21. Again another example of such an antigen binding protein
comprises (a) one V.sub.L3, and (b) one of V.sub.L1, V.sub.L2,
V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9,
V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15,
V.sub.L16, V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, or
V.sub.L21, etc.
[0224] The various combinations of heavy chain variable regions may
be combined with any of the various combinations of light chain
variable regions as is apparent to one of skill in the art.
[0225] In other instances, the antigen binding protein contains two
identical light chain variable regions and/or two identical heavy
chain variable regions. As an example, the antigen binding protein
may be an antibody or immunologically functional fragment that
includes two light chain variable regions and two heavy chain
variable regions in combinations of pairs of light chain variable
regions and pairs of heavy chain variable regions as listed in
Table 3.
[0226] Some antigen binding proteins that are provided comprise a
heavy chain variable domain comprising a sequence of amino acids
that differs from the sequence of a heavy chain variable domain
selected from V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5,
V.sub.H6, V.sub.H7, V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11,
V.sub.H12, and V.sub.H13 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14 or 15 amino acid residues, wherein each such sequence
difference is independently either a deletion, insertion or
substitution of one amino acid, with the deletions, insertions
and/or substitutions resulting in no more than 15 amino acid
changes relative to the foregoing variable domain sequences. The
heavy chain variable region in some antigen binding proteins
comprises a sequence of amino acids that has at least 70%, 75%,
80%, 85%, 90%, 95%, 97% or 99% sequence identity to the amino acid
sequences of the heavy chain variable region of V.sub.H1, V.sub.H2,
V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8,
V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, and V.sub.H13.
[0227] Certain antigen binding proteins comprise a light chain
variable domain comprising a sequence of amino acids that differs
from the sequence of a light chain variable domain selected from
V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6,
V.sup.L7, V.sub.L8, V.sup.L9, V.sub.L10, V.sub.L11, V.sub.L12,
V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, or V.sub.L17 at only 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid
residues, wherein each such sequence difference is independently
either a deletion, insertion or substitution of one amino acid,
with the deletions, insertions and/or substitutions resulting in no
more than 15 amino acid changes relative to the foregoing variable
domain sequences. The light chain variable region in some antigen
binding proteins comprises a sequence of amino acids that has at
least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to
the amino acid sequences of the light chain variable region of
V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6,
V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12,
V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, or V.sub.L17.
[0228] In additional instances, antigen binding proteins comprise
the following pairings of light chain and heavy chain variable
domains: VL1 with VH1, VL2 with VH2, VL3 with VH3, VL4 with VH4,
VL5 with VH5, VL6 with VH1, VL7 with VH6, VL8 with VH5, VL9 with
VH1, VL10 with VH7, VL11 with, H8, VL12 with VH9, VL12 with VH10,
VL13 with VH5, VL14 with VH11, VL15 with VH12, VL16 with VH13, and
VL17 with VH13. In some instances, the antigen binding proteins in
the above pairings may comprise amino acid sequences that have 70%,
75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity with the
specified variable domains.
[0229] Still other antigen binding proteins, e.g., antibodies or
immunologically functional fragments, include variant forms of a
variant heavy chain and a variant light chain as just
described.
CDRs
[0230] The antigen binding proteins disclosed herein are
polypeptides into which one or more CDRs are grafted, inserted
and/or joined. An antigen binding protein can have 1, 2, 3, 4, 5 or
6 CDRs. An antigen binding protein thus can have, for example, one
heavy chain CDR1 ("CDRH1"), and/or one heavy chain CDR2 ("CDRH2"),
and/or one heavy chain CDR3 ("CDRH3"), and/or one light chain CDR1
("CDRL1"), and/or one light chain CDR2 ("CDRL2"), and/or one light
chain CDR3 ("CDRL3"). Some antigen binding proteins include both a
CDRH3 and a CDRL3. Specific heavy and light chain CDRs are
identified in Tables 4A and 4B, respectively.
[0231] Complementarity determining regions (CDRs) and framework
regions (FR) of a given antibody may be identified using the system
described by Kabat et al. in Sequences of Proteins of Immunological
Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH,
NIH Publication no. 91-3242, 1991. Certain antibodies that are
disclosed herein comprise one or more amino acid sequences that are
identical or have substantial sequence identity to the amino acid
sequences of one or more of the CDRs presented in Table 4A (CDRHs)
and Table 4B (CDRLs).
TABLE-US-00007 TABLE 4A Exemplary Heavy Chain CDR Amino Acid
Sequences SEQ Contained Alt ID in Num NO: Reference Designation
Sequence 42 73 1E11HCDR1 CDRH 1-1 SFGMH 4E4HCDR1 9D4HCDR1 12G8HCDR1
43 76 1H7HCDR1 CDRH 1-2 NAWMS 9F5HCDR1 11D11HCDR1 11H9HCDR1 44 79
2E7HCDR1 CDRH 1-3 SYAMS 45 82 3B6HCDR1 CDRH 1-4 GYYMH 46 85
3C8HCDR1 CDRH 1-5 SYGMH 5F5HCDR1 12E8HCDR1 47 88 4H6HCDR1 CDRH 1-6
DYAMS 48 92 10E4HCDR1 CDRH 1-7 DYYMY 49 97 13H2HCDR1 CDRH 1-8 TYSMN
50 100 32H7HCDR1 CDRH 1-9 SYGMH 51 74 1E11HCDR2 CDRH 2-1
VISFDGSIKYSVDSVKG 4E4HCDR2 9D4HCDR2 12GBHCDR2 52 77 1H7HCDR2 CDRH
2-2 RIKSTTDGGTTDYAAPVKG 53 80 2E7HCDR2 CDRH 2-3 AISGSGGRTYYADSVKG
54 83 3B6HCDR2 CDRH 2-4 WINPNSGGTNYAQKFQG 55 86 3C8HCDR2 CDRH 2-5
VISYDGSHESYADSVKG 5F5HCDR2 12E8HCDR2 56 89 4H6HCDR2 CDRH 2-6
FIRSRAYGGTPEYAASVKG 57 91 9F5HCDR2 CDRH 2-7 RIKSKTDGGTTDYTAPVKG 58
93 10E4HCDR2 CDRH 2-8 WISPNSGGINYAQKFQG 59 95 11D11HCDR2 CDRH 2-9
RIKSKTDGGTTDYAAPVKG 11H9HCDR2 60 98 13H2HCDR2 CDRH 2-10
SISSSSSYRYYADSVKG 61 101 32H7HCDR2 CDRH 2-11 VIWYDGSNKYYADSVKG 62
75 1E11HCDR3 CDRH 3-1 DRLNYYDSSGYYHYKYYGMAV 4E4HCDR3 9D4HCDR3 63 78
1H7HCDR3 CDRH 3-2 DRTGYSISWSSYYYYYGMDV 9F5HCDR3 11D11HCDR3
11H9HCDR3 64 81 2E7HCDR3 CDRH 3-3 DQREVGPYSSGWYDYYYGMDV 65 84
3B6HCDR3 CDRH 3-4 DQMSIIMLRGVFPPYYYGMDV 66 87 3C8HCDR3 CDRH 3-5
ERKRVTMSTLYYYFYYGMDV 5F5HCDR3 12E8HCDR3 67 90 4H6HCDR3 CDRH 3-6
GRGIAARWDY 68 94 10E4HCDR3 CDRH 3-7 GGYSGYAGLYSHYYGMDV 69 96
12G8HCDR3 CDRH 3-8 DRLNYYDSSGYYHYKYYGLAV 70 99 13H2HCDR3 CDRH 3-9
EGVSGSSPYSISWYDYYYGMDV 71 102 32H7HCDR3 CDRH 3-10
AGGIAAAGLYYYYGMDV
TABLE-US-00008 TABLE 4B Exemplary Light Chain CDR Amino Acid
Sequences SEQ Contained Alt ID in Desig- Num NO: Reference nation
Sequence 72 42 1E11LCD1 CDRL 1-1 SGSSSNIGNNYVS 4E4LCD1 9D4LCD1
12G8LCD1 73 45 1H7LCD1 CDRL 1-2 SGSSSNIGSNYVY 9F5LCD1 11D11LC1
11H9LCD1 74 48 2E7LCD1 CDRL 1-3 RASQGIRNDLG 75 51 3B6LCD1 CDRL 1-4
QGDSLRSFYAS 76 54 3C8LCD1 CDRL 1-5 KSSQSLLHSAGKTYLY 77 57 4H6LCD1
CDRL 1-6 RSSQSLLHSFGYNYLD 78 60 5F5LCD1 CDRL 1-7 KSSQSLLHSDGKTYLY
79 62 10E4LCD1 CDRL 1-8 SGSSSNIGSNTVN 80 65 12E8LCD1 CDRL 1-9
KSSQSLLHSDGRNYLY 81 66 13H2LCD1 CDRL 1-10 RASQGIRKDLG 82 69 32H7
LCD1 CDRL 1-11 RASQSVSSGYLT 32H7m LCD1 83 43 1E11LCD2 CDRL 2-1
DNNKRPS 4E4LCD2 9D4LCD2 12G8LCD2 84 46 1H7LCD2 CDRL 2-2 RSNQRPS 85
49 2E7LCD2 CDRL 2-3 AASSLQS 86 52 3B6LCD2 CDRL 2-4 GKNNRPS 87 55
3C8LCD2 CDRL 2-5 EVSNRFS 5F5LCD2 12E8LCD2 88 58 4H6LCD2 CDRL 2-6
LGSNRAS 89 61 9F5LCD2 CDRL 2-7 RNNQRPS 11D11LC2 11H9LCD2 90 63
10E4LCD2 CDRL 2-8 TNNQRPS 91 67 13H2LCD2 CDRL 2-9 GASSLQS 92 70
32H7 LCD2 CDRL 2-10 GASSRAT 32H7M LCD2 93 44 1E11LCD3 CDRL 3-1
GTWDSRLSAVV 4E4LCD3 9D4LCD3 12G8LCD3 94 47 1H7LCD3 CDRL 3-2
AAWDDSLSGWV 9F5LCD3 11D11LC3 11H9LCD3 95 50 2E7LCD3 CDRL 3-3
LQYNIYPWT 96 53 3B6LCD3 CDRL 3-4 NSRDSSVYHLV 97 56 3C8LCD3 CDRL 3-5
MQSFPLPLT 5F5LCD3 12E8LCD3 98 59 4H6LCD3 CDRL 3-6 MQALQTPFT 99 64
10E4LCD3 CDRL 3-7 AARDESLNGVV 100 68 13H2LCD3 CDRL 3-8 LQYNSFPWT
101 71 32H7 LCD3 CDRL 3-9 QQYGNSLCR 102 72 32H7m LCD3 CDRL 3-10
QQYGNSLSR
[0232] The structure and properties of CDRs within a naturally
occurring antibody has been described, supra. Briefly, in a
traditional antibody, the CDRs are embedded within a framework in
the heavy and light chain variable region where they constitute the
regions responsible for antigen binding and recognition. A variable
region comprises at least three heavy or light chain CDRs, see,
supra (Kabat et al., 1991, Sequences of Proteins of Immunological
Interest, Public Health Service N.I.H., Bethesda, Md.; see also
Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al.,
1989, Nature 342: 877-883), within a framework region (designated
framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al.,
1991, supra; see also Chothia and Lesk, 1987, supra). The CDRs
provided herein, however, may not only be used to define the
antigen binding domain of a traditional antibody structure, but may
be embedded in a variety of other polypeptide structures, as
described herein.
[0233] In one aspect, the CDRs provided are (a) a CDRH selected
from the group consisting of (i) a CDRH1 selected from the group
consisting of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97, and 100;
(ii) a CDRH2 selected from the group consisting of SEQ ID NO: 74,
77, 80, 83, 86, 89, 91, 93, 95, 98, 101, and 129; (iii) a CDRH3
selected from the group consisting of SEQ ID NO:75, 78, 81, 84, 87,
90, 96, 99, 102, and 123; and (iv) a CDRH of (i), (ii) and (iii)
that contains one or more, e.g., one, two, three, four or more
amino acid substitutions (e.g., conservative amino acid
substitutions), deletions or insertions of no more than five, four,
three, two, or one amino acids; (B) a CDRL selected from the group
consisting of (i) a CDRL1 selected from the group consisting of SEQ
ID NO:42, 45, 48, 51, 54, 57, 62, 65, 66, and 69; (ii) a CDRL2
selected from the group consisting of SEQ ID NO:43, 46, 49, 52, 55,
58, 61, 63, 67, and 70; (iii) a CDRL3 selected from the group
consisting of SEQ ID NO:44, 47, 50, 53, 56, 59, 64, 68, 71, and 72;
and (iv) a CDRL of (i), (ii) and (iii) that contains one or more,
e.g., one, two, three, four or more amino acid substitutions (e.g.,
conservative amino acid substitutions), deletions or insertions of
no more than five, four, three, two, or one amino acids amino
acids.
[0234] In another aspect, an antigen binding protein includes 1, 2,
3, 4, 5, or 6 variant forms of the CDRs listed in Tables 4A and 4B,
each having at least 80%, 85%, 90% or 95% sequence identity to a
CDR sequence listed in Tables 4A and 4B. Some antigen binding
proteins include 1, 2, 3, 4, 5, or 6 of the CDRs listed in Tables
4A and 4B, each differing by no more than 1, 2, 3, 4 or 5 amino
acids from the CDRs listed in these tables.
[0235] In yet another aspect, the CDRs disclosed herein include
consensus sequences derived from groups of related monoclonal
antibodies. As described herein, a "consensus sequence" refers to
amino acid sequences having conserved amino acids common among a
number of sequences and variable amino acids that vary within a
given amino acid sequences. The CDR consensus sequences provided
include CDRs corresponding to each of CDRH1, CDRH2, CDRH3, CDRL1,
CDRL2 and CDRL3.
[0236] In still another aspect, an antigen binding protein includes
the following associations of CDRL1, CDRL2 and CDRL3: SEQ ID NOs:
42, 43, and 44; SEQ ID NOs: 45, 46, and 47; SEQ ID NOs: 48, 49, and
50; SEQ ID NOs: 51, 52, and 53; SEQ NOs: 54, 55, and 56; SEQ ID
NOs: 57, 58, and 59: SEQ ID NOs: 60, 55, and 56; SEQ ID NOs: 45,
61, and 47; SEQ ID NOs: 62, 63, and 64; SEQ ID NOs: 65, 55, and 56;
SEQ ID NOs: 66, 67, and 68; SEQ ID NOs: 69, 70, and 71; and SEQ ID
NOs: 69, 70, and 72.
[0237] In an additional aspect, an antigen binding protein includes
the following associations of CDRH1, CDRH2 and CDRH3: SEQ ID NOs:
73, 74, and 75; SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 79, 80, and
81; SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 85, 86, and 87; SEQ ID
NOs: 88, 89, and 90; SEQ ID NOs: 76, 91, and 78; SEQ ID NOs: 92,
93, and, 94; SEQ ID NOs: 76, 95, and 78; SEQ ID NOs: 73, 74, and
96; SEQ ID NOs: 97, 98, and 99; and SEQ ID NOs: 100, 101, and
102.
[0238] In another aspect, an antigen binding protein includes the
following associations of CDRL1, CDRL2 and CDRL3 with CDRH1, CDRH2
and CDRH3: SEQ ID NOs: 42, 43, and 44 with SEQ ID NOs: 73, 74, and
75; SEQ ID NOs: 45, 46, and 47 with SEQ ID NOs: 76, 77, and 78; SEQ
ID NOs: 48, 49, and 50 with SEQ ID NOs: 79, 80, and 81; SEQ ID NOs:
51, 52, and 53 with SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 54, 55,
and 56 with SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 57, 58, and 59
with SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 60, 55, and 56 with
SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 45, 61, and 47 with SEQ ID
NOs: 76, 91, and 78; SEQ ID NOs: 62, 63, and 64 with SEQ ID NOs:
92, 93, and 94; SEQ ID NOs: 45, 61, and 47 with SEQ ID NOs: 76, 95,
and 78; SEQ ID NOs: 65, 55, and 56 with SEQ ID NOs: 85, 86, and 87;
SEQ ID NOs: 42, 43, and 44 with SEQ ID NOs: 73, 74, and 96; SEQ ID
NOs: 66, 67, and 68 with SEQ ID NOs: 97, 98, and 99; SEQ ID NOs:
69, 70, and 71 with SEQ ID NOs: 100, 101, and 102; and SEQ ID NOs:
69, 70, and 72 with SEQ ID NOs: 100, 101, and 102.
[0239] Consensus sequences were determined using standard
phylogenic analyses of the CDRs corresponding to the V.sub.H and
V.sub.L of anti-CGRP R antibodies. The consensus sequences were
determined by keeping the CDRs contiguous within the same sequence
corresponding to a V.sub.H or V.sub.L.
[0240] As illustrated in FIGS. 3A, 3B, 4, 5A, 5B, 5C, 5D and 5E,
lineage analysis of a variety of the antigen binding proteins
provided herein resulted in groups of related sequences, designated
as light chain CDR groups K1, K2, K3, and K4 (FIGS. 3A and 3B),
light chain CDR groups L1, L2, L3, and L4 (FIG. 4), and heavy chain
CDR groups HC1 (FIG. 5A), HC2 (FIG. 5B), HC3 (FIG. 5C), HC4 (FIG.
5C), HC5 (FIG. 5D) and HC6 (FIG. 5E). Some of the above groups were
used to generate additional consensus sequences, as illustrated in
FIGS. 3A, 3B, 4, and 5F, to yield light chain CDR groups K1,4 (FIG.
3A), K2,3 (FIG. 3B), L1,2,3 (FIG. 4), and LA11 (FIG. 4), and heavy
chain CDR groups HCA and HCB (FIG. 5F).
[0241] The consensus sequences of the various CDR region groups are
provided below:
[0242] K1 Consensus
[0243] CDR1 RASQGIRX.sub.1DLG (SEQ ID NO:103), wherein X.sub.1 is
selected from the group consisting of N and K.
[0244] CDR2 X.sub.1ASSLQS (SEQ ID NO:104), wherein X.sub.1 is
selected from the group consisting of A and G.
[0245] CDR3 LQYNX.sub.1X.sub.2PWT (SEQ ID NO:105), wherein X.sub.1
is selected from the group consisting of I and S, and X.sub.2
selected from the group consisting of Y and F.
[0246] K4 Consensus
[0247] CDR3 QQYGNSLX.sub.1R (SEQ ID NO:106), wherein X.sub.1 is
selected from the group consisting of S and C.
[0248] K1,4 Consensus
[0249] CDR1 RASQX.sub.1X.sub.2X.sub.3X.sub.4GX.sub.5LX.sub.6 (SEQ
ID NO:107), wherein X.sub.1 is selected from the group consisting
of S and G, X.sub.2 is selected from the group consisting of V and
I, X.sub.3 is selected from the group consisting of S and R,
X.sub.4 is selected from the group consisting of S, N and K,
X.sub.5 is selected from the group consisting of Y and D, and
X.sub.6 is selected from the group consisting of T and G.
[0250] CDR2 X.sub.1ASSX.sub.2X.sub.3X.sub.4 (SEQ ID NO:108),
wherein X.sub.1 is selected from the group consisting of G and A,
X.sub.2 is selected from the group consisting of R and L, X.sub.3
is selected from the group consisting of A and Q, and X.sub.4 is
selected from the group consisting of T and S.
[0251] CDR3 X.sub.1QYX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7
(SEQ ID NO:109), wherein X.sub.1 is selected from the group
consisting of Q and L, X.sub.2 is selected from the group
consisting of G and N, X.sub.3 is selected from the group
consisting of N and T, X.sub.4 is selected from the group
consisting of S, Y and F, X.sub.5 is selected from the group
consisting of L and P, X.sub.6 is selected from the group
consisting of C, W and S, and X.sub.7 is selected from the group
consisting of R and T.
[0252] K3 Consensus
[0253] CDR1 KSSQSLLHSX.sub.1GX.sub.2X.sub.3YLY (SEQ ID NO:110),
wherein X.sub.1 is selected from the group consisting of D and A,
X.sub.2 is selected from the group consisting of R and K, and
X.sub.3 is selected from the group consisting of N and T.
[0254] K2,3 Consensus
[0255] CDR1 X.sub.1SSQSLLHSX.sub.2GX.sub.3X.sub.4YLX.sub.5 (SEQ ID
NO:111), wherein X.sub.1 is selected from the group consisting of R
and K, X.sub.2 is selected from the group consisting of F, D and A,
X.sub.3 is selected from the group consisting of Y, R and K,
X.sub.4 is selected from the group consisting of N and T, and
X.sub.5 is selected from the group consisting of D and Y.
[0256] CDR2 X.sub.1X.sub.2SNRX.sub.3S (SEQ ID NO:112), wherein
X.sub.1 is selected from the group consisting of L and E, X.sub.2
is selected from the group consisting of G and V, and X.sub.3 is
selected from the group consisting of A and F.
[0257] CDR3 MQX.sub.1X.sub.2X.sub.3X.sub.4PX.sub.5T (SEQ ID
NO:113), wherein X.sub.1 is selected from the group consisting of A
and S, X.sub.2 is selected from the group consisting L and F,
X.sub.3 is selected from the group consisting of Q and P, X.sub.4
is selected from the group consisting of T and L, and X.sub.5 is
selected from the group consisting of F and L.
[0258] Lm3 Consensus
[0259] CDR2 RX.sub.1NQRPS (SEQ ID NO:114), wherein X.sub.1 is
selected from the group consisting of N and S.
[0260] Lm1,2,3 Consensus
[0261] CDR1 SGSSSNIGX.sub.1NX.sub.2VX.sub.3 (SEQ ID NO:115),
wherein X.sub.1 is selected from the group consisting of N and S,
X.sub.2 is selected from the group consisting of Y and T, and
X.sub.3 is selected from the group consisting of S, N and Y.
[0262] CDR2 X.sub.1X.sub.2NX.sub.3RPS (SEQ ID NO:116), wherein
X.sub.1 is selected from the group consisting of D, T and R,
X.sub.7 is selected from the group consisting of N and S, and
X.sub.3 is selected from the group consisting of K and Q.
[0263] CDR3 X.sub.1X.sub.2X.sub.3DX.sub.4X.sub.5LX.sub.6X.sub.7VV
(SEQ ID NO:117), wherein X.sub.1 is selected from the group
consisting of G and A, X.sub.2 selected from the group consisting
of T and A, X.sub.3 is selected from the group consisting of W and
R, X.sub.4 is selected from the group consisting of S and D,
X.sub.5 is selected from the group consisting of R and S, X.sub.6
is selected from the group consisting of S and N, and X.sub.7 is
selected from the group consisting of A and G.
[0264] LA11 Consensus
[0265] CDR1
X.sub.1GX.sub.2X.sub.3SX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10-
X.sub.11(SEQ ID NO:118), wherein X.sub.1 is selected from the group
consisting of S and Q, X.sub.2 is present or absent, and if
present, is S, X.sub.3 is selected. from the group consisting of S
and D, X.sub.4 is present or absent, and if present, is N, X.sub.5
is selected from the group consisting of I and L, X.sub.6 is
selected from the group consisting of G and R, X.sub.7 is selected
from the group consisting of N and S, X.sub.8 is selected from the
group consisting of N and F, X.sub.9 is selected from the group
consisting of Y and T, X.sub.10 is selected from the group
consisting of V and A, and X.sub.11 is selected from the group
consisting of S, N and Y.
[0266] CDR2 X.sub.1X.sub.2NX.sub.3RPS (SEQ ID NO:119), wherein
X.sub.1 is selected from the group consisting of D, G, T, and R, V
is selected from the group consisting of N, K and S, and X.sub.3 is
selected from the group consisting of K, N and Q,
[0267] CDR3
X.sub.1X.sub.2X.sub.3DX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9V
(SEQ ID NO:120), wherein X.sub.1 is selected from the group
consisting of G, N and A, X.sub.2 is selected from the group
consisting of T, S and A, X.sub.3 is selected from the group
consisting of W and R, X.sub.4 is selected from the group
consisting of S and. D, X.sub.5 is selected from the group
consisting of R and S, X.sub.6 is selected from the group
consisting of L and V, X.sub.7 is selected from the group
consisting of S, Y and N, X.sub.8 is selected from the group
consisting of A, H and G, and X.sub.9 is selected from the group
consisting of V and L.
[0268] HC1 Consensus
[0269] CDR1 X.sub.1YYMX.sub.2 (SEQ ID NO:121), wherein X.sub.1 is
selected from the group consisting of G and D, X.sub.2 is selected
from the group consisting of H and Y.
[0270] CDR2 WIX.sub.1PNSGGTNYAQKFQG (SEQ ID NO:122), wherein
X.sub.1 is selected from the group consisting of N and S.
[0271] CDR3
X.sub.1X.sub.2X.sub.3SX.sub.4X.sub.5X.sub.6X.sub.7X.sub.8GX.sub.9X.sub.10-
X.sub.11X.sub.12YYX.sub.13GMDV (SEQ ID NO:123), wherein X.sub.1 is
selected from the group consisting of D and G, X.sub.2 is selected
from the group consisting of Q and G, X.sub.3 is selected from the
group consisting of M and Y, X.sub.4 is selected from the group
consisting of I and G, X.sub.5 is selected from the group
consisting of I and Y, X.sub.6 is selected from the group
consisting of M and A, X.sub.7 is present or absent, and if
present, is L, X.sub.8 is present or absent, and if present, is R,
X.sub.9 is selected from the group consisting of V and L, X.sub.10
is selected from the group consisting of F and Y, X.sub.11 is
selected from the group consisting of P and S, X.sub.12 is selected
from the group consisting of P and H, and X.sub.13 is present or
absent, and if present, is Y.
[0272] HC2 Consensus
[0273] CDR2 RIKSX.sub.1TDGGTTDYX.sub.2APVKG (SEQ ID NO:124),
wherein X.sub.1 is selected from the group consisting of K and T,
and X.sub.2 is selected from the group consisting of T and A.
[0274] HC3 Consensus
[0275] CDR1 X.sub.1YX.sub.2MX.sub.3 (SEQ ID NO:125), wherein
X.sub.1 is selected from the group consisting of T and S, X.sub.2
is selected from the group consisting of S and A, and X.sub.3 is
selected from the group consisting of N and S.
[0276] CDR2 X.sub.1ISX.sub.2SX.sub.3X.sub.4X.sub.5X.sub.6YYADSVKG
(SEQ ID NO:126), wherein X.sub.1is selected. from the group
consisting of S and A, X.sub.2 is selected from the group
consisting of S and G, X.sub.3 is selected from the group
consisting of S and G, X.sub.4 is selected from the group
consisting of S and G, X.sub.5 is selected from the group
consisting of Y and R, and X.sub.6 is selected from the group
consisting of R and T.
[0277] CDR3
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7PYSX.sub.8X.sub.9WYDYYYG-
MDV (SEQ ID NO:127), wherein X.sub.1 is selected from the group
consisting of E and D, X.sub.2 is selected from the group
consisting of G and Q, X.sub.3 is selected from the group
consisting of V and R, X.sub.4 is selected from the group
consisting of S and E, X.sub.5 is selected from the group
consisting of G and V, X.sub.6 is selected from the group
consisting of S and G, X.sub.7 is present or absent, and if
present, is S, X.sub.8 is selected from the group consisting of I
and S, and X.sub.9 is selected from the group consisting of S and
G.
[0278] HC4 Consensus
[0279] CDR1 SX.sub.IGMH (SEQ ID NO:128), wherein X.sub.1 is
selected from the group consisting of F and Y.
[0280] CDR2 VISX.sub.1DGSX.sub.2KYX.sub.3X.sub.4DSVKG (SEQ ID
NO:129), wherein X.sub.1 is selected from the group consisting of F
and Y, X.sub.2 is selected from the group consisting of I and H,
X.sub.3 is selected from the group consisting of S and Y, and
X.sub.4 is selected from the group consisting of V and A.
[0281] CDR3
X.sub.1RX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6SX.sub.7X.sub.8YYX.sub.9X.sub.-
10X.sub.11YYGX.sub.12X.sub.13V (SEQ ID NO:130), wherein X.sub.1 is
selected from the group consisting of D and E, X.sub.2 is selected
from the group consisting of L and K, X.sub.3 is selected from the
group consisting of N and R, X.sub.4 is selected from the group
consisting of Y and V, X.sub.5 is selected from the group
consisting of Y and T, X.sub.6 is selected from the group
consisting of D and M, X.sub.7 is selected from the group
consisting of S and T, X.sub.8 is selected from the group
consisting of G and L, X.sub.9 is selected from the group
consisting of H and Y, X.sub.10 is present or absent, and if
present, is Y, X.sub.11 is selected from the group consisting of K
and F, X.sub.12 is selected from the group consisting of M and L,
and X.sub.13 is selected from the group consisting of A and D.
[0282] HCA Consensus
[0283] CDR1 X.sub.1X.sub.2X.sub.3MX.sub.4 (SEQ ID NO:131), wherein
X.sub.1 is selected from the group consisting of N and S, X.sup.2
is selected from the group consisting of A, Y and F, X.sub.3 is
selected from the group consisting of W, A and G, and X.sub.4 is
selected from the group consisting of S and H.
[0284] CDR2
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6GX.sub.7X.sub.8X.sub.9X.sub.10-
X.sub.11X.sub.12X.sub.13X.sub.14VKG (SEQ ID NO:132), wherein
X.sub.1 is selected from the group consisting of R, A and V,
X.sup.2 is selected from the group consisting of K, S and W,
X.sub.3 is selected from the group consisting of S, G, F and Y,
X.sub.4 is present or absent, and if present, is selected from the
group consisting of K and T, X.sub.5 is present or absent, and if
present, is T, X.sub.6 is selected from the group consisting of D
and S, X.sub.7 is selected from the group consisting of G and S,
X.sub.8 is selected from the group consisting of T, R, I, N and H,
X.sub.9 is selected from the group consisting of T and K, X.sub.10
is selected from the group consisting of D and Y, X.sub.11 is
selected from the group consisting of Y and S, X.sub.12 is selected
from the group consisting of T, A and V, X.sub.13 is selected from
the group consisting of A and D, and X.sub.14 is selected from the
group consisting of P and S.
[0285] CDR3 X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7
X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.s-
ub.17GX.sub.18X.sub.19V (SEQ ID NO:133), wherein X.sub.1 is
selected from the group consisting of D, A and E, X.sub.2 is
selected from the group consisting of R, Q and G, X.sub.3 is
selected from the group consisting of T, R, L, G and K, X.sub.4 is
selected from the group consisting of G, E, N, I and R, X.sub.5 is
selected from the group consisting of Y, V and A, X.sub.6 is
selected from the group consisting of S, G, Y, A and T, X.sub.7 is
selected from the group consisting of I, P, D, A and M, X.sub.8 is
present or absent, and if present, is selected from the group
consisting of S and Y, X.sub.9 is present or absent, and if
present, is selected from the group consisting of W, S and T,
X.sub.10 is selected front the group consisting of S, G and L,
X.sub.11 is selected from the group consisting of S, G, L and Y,
X.sub.12 is present or absent, and if present, is selected from the
group consisting of W and Y, X.sub.13 is selected from the group
consisting of Y and H, X.sub.14 is present or absent, and if
present, is selected from the group consisting of Y and D, X.sub.15
is selected from the group consisting of Y, K and F, X.sub.16 is
present or absent, and if present, is Y, X.sub.17 is present or
absent, and if present, is Y, X.sub.18 is selected from the group
consisting of M and L, and X.sub.19 is selected from the group
consisting of D and A.
[0286] HCB Consensus
[0287] CDR1 X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO:134),
wherein X.sub.1 is selected from the group consisting of N, G, D, S
and A, X.sup.2 is selected from the group consisting of A, F and Y,
X.sub.3 is selected from the group consisting of W, Y, A and G,
X.sub.4 is selected from the group consisting of M and L, and
X.sub.5 is selected from the group consisting of S and H.
[0288] CDR2
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X-
.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17G (SEQ ID
NO:135), wherein X.sub.1 is selected from the group consisting of
R, W, A, V, S and F, X.sub.2 is selected from the group consisting
of K, N, S, W and R, X.sub.3 is selected from the group consisting
of S, P, G, F and Y, X.sub.4 is present or absent, and if present,
is selected from the group consisting of K, T and R, X.sub.5 is
present or absent, and if present, is selected from the group
consisting of T and A, X.sub.6 is selected from the group
consisting of D, N, H, S and Y, X.sub.7 is selected from the group
consisting of G and S, X.sub.8 is selected from the group
consisting of G and S, X.sub.9 is selected from the group
consisting of T, G, R, I, N, H and Y, X.sub.10 is selected from the
group consisting of T, K, R and P, X.sub.11 is selected from the
group consisting of D, N, Y and E, X.sub.12 is selected from the
group consisting of Y and S, X.sub.13 is selected from the group
consisting of T, A and V, X.sub.14 is selected from the group
consisting of A, Q and D, X.sub.15 is selected from the group
consisting of P, K and S, X.sub.16 is selected from the group
consisting of V and F, and X.sub.17 is selected from the group
consisting of K and Q.
[0289] CDR3
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5SX.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X-
.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16GX.sub.17X.sub.18V
(SEQ ID NO:136), wherein X.sub.1 is selected from the group
consisting of D, G, A and E, X.sub.2 is selected from the group
consisting of R, G and Q, X.sub.3 is selected from the group
consisting of T, M, Y, R, L, G and K, X.sub.4 is selected from the
group consisting of G, S, E, N, I and R, X.sub.5 is selected from
the group consisting of Y, I, G, V and A, X.sub.6 is selected from
the group consisting of S, I, Y, G, A and T, X.sub.7 is selected
from the group consisting of I, M, A, P and D, X.sub.8 is present
or absent, and if present, is selected from the group consisting of
S, L and Y, X.sub.9 is present or absent, and if present, is
selected from the group consisting of W, R, S and T, X.sub.10 is
selected from the group consisting of S, G and L, X.sub.11 is
selected from the group consisting of S, V, L, G and Y, X.sub.12 is
present or absent, and if present, is selected from the group
consisting of F, Y and W, X.sub.13 is selected from the group
consisting of Y, P, S and H, X.sub.14 is present or absent, and. if
present, is selected from the group consisting of Y, P, D and H,
X.sub.15 is selected from the group consisting of Y, K and F,
X.sub.16 is present or absent, and if present, is Y, X.sub.17 is
present or absent, and if present, is Y, and X.sub.18 is selected
from the group consisting of M and L.
[0290] In some cases the antigen binding protein comprises at least
one heavy chain CDR1, CDR2, or CDR3 having one of the above
consensus sequences. In some cases, the antigen binding protein
comprises at least one light chain CDR1, CDR2, or CDR3 having one
of the above consensus sequences. In other cases, the antigen
binding protein comprises at least two heavy chain CDRs according
to the above consensus sequences, and/or at least two light chain
CDRs according to the above consensus sequences. In still other
cases, the antigen binding protein comprises at least three heavy
chain CDRs according to the above consensus sequences, and/or at
least three light chain CDRs according to the above consensus
sequences.
Exemplary Antigen Binding Proteins
[0291] According to one aspect, provided is an isolated
antigen-binding protein that binds CGRP R comprising (A) one or
more heavy chain complementary determining regions (CDRHs) selected
from the group consisting of: (i) a CDRH1 selected from the group
consisting of SEQ ID NO:73, 76, 79, 82, 85, 88, 92, 97, and 100;
(ii) CDRH2 selected from the group consisting of SEQ ID NO:74, 77,
80, 83, 86, 89, 91, 93, 95, 98, 101, and 129; (iii) a CDRH3
selected from the group consisting of SEQ ID NO:75, 78, 81, 84, 87,
90, 96, 99, 102, and 123; and (iv) a CDRH of (i), (ii) and (iii)
that contains one or more, e.g., one, two, three, four or more
amino acid substitutions, deletions or insertions of no more than
five, four, three, four, two or one amino acids; (B) one or more
light chain complementary determining regions (CDRLs) selected from
the group consisting of: (i) a CDRL1 selected from the group
consisting of SEQ ID NO:42, 45, 48, 51, 54, 57, 62, 65, 66, and 69;
(ii) a CDRL2 selected from the group consisting of SEQ ID NO:43,
46, 49, 52, 55, 58, 61, 63, 67, and 70; (iii) a CDRL3 selected from
the group consisting of SEQ ID NO:44, 47, 50, 53, 56, 59, 64, 68,
71, and 72; and (iv) a CDRL (i), (ii) and (iii) that contains one
or more, e.g., one, two, three, four or more amino acid
substitutions, deletions or insertions of no more than five, four,
three, four, two or one amino acids; or (C) one or more heavy chain
CDRHs of (A) and one or more light chain CDRLs of (B).
[0292] In yet another embodiment, the isolated antigen-binding
protein may comprise (A) a CDRH selected from the group consisting:
of (i) a CDRH1 selected from the group consisting of SEQ ID NO:73,
76, 79, 82, 85, 88, 92, 97, and 100; (ii) a CDRH2 selected from the
group consisting of SEQ ID NO:74, 77, 80, 83, 86, 89, 91, 93, 95,
98, 101, and 129; and (iii) a CDRH3 selected from the group
consisting of SEQ ID NO:75, 78, 81, 84, 87, 90, 96, 99, 102, and
123; (B) a CDRL selected from the group consisting of (i) a CDRL1
selected from the group consisting of SEQ ID NO:42, 45, 48, 51, 54,
57, 62, 65, 66, and 69; (ii) CDRL2 selected from the group
consisting of SEQ ID NO:43, 46, 49, 52, 55, 58, 61, 63, 67, and 70;
and (iii) a CDRL3 selected from the group consisting of SEQ ID
NO:44, 47, 50, 53, 56, 59, 64, 68, 71, and 72; or (C) one or more
heavy chain CDRHs of (A) and one or more light chain CDRLs of (B).
In one embodiment, the isolated antigen-binding protein may include
(A) a CDRH1 of SEQ ID NO:73, 76, 79, 82, 85, 88, 92, 97, and 100, a
CDRH2 of SEQ ID NO:74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101, and
129, and a CDRH3 of SEQ ID NO:75, 78, 81, 84, 87, 90, 96, 99, 102,
and 123, and (B) a CDRL1 of SEQ ID NO:42, 45, 48, 51, 54, 57, 62,
65, 66, and 69, a CDRL2 of SEQ ID NO:43, 46, 49, 52, 55, 58, 61,
63, 67, and 70, and a CDRL3 of SEQ ID NO:44, 47, 50, 53, 56, 59,
64, 68, 71, and 72.
[0293] In another embodiment, the heavy chain variable region
(V.sub.H) has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99%
sequence identity with an amino acid sequence selected from the
group consisting of SEQ ID NO:158-170, and/or the V.sub.L has at
least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity
with an amino acid sequence selected from the group consisting of
SEQ ID NO:137-153. In a further embodiment, the V.sub.H is selected
from the group consisting of SEQ ID NO: 158-170, and/or the V.sub.L
is selected from the group consisting of SEQ ID NO: 137-153.
[0294] In another aspect, also provided is an isolated antigen
binding protein that specifically binds to an epitope formed of
amino acid residues from both the CRLR and RAMP1 components of the
CGRP R.
[0295] In yet another embodiment, the isolated antigen binding
protein described hereinabove comprises a first amino acid sequence
comprising at least one of the CDRH consensus sequences disclosed
herein, and a second amino acid sequence comprising at least one of
the CDRL consensus sequences disclosed herein. In one aspect, the
first amino acid sequence comprises at least two of the CDRH
consensus sequences, and/or the second amino acid sequence
comprises at least two of the CDRL consensus sequences.
[0296] In certain embodiments, the first and the second amino acid
sequence are covalently bonded to each other.
[0297] In a further embodiment, the first amino acid sequence of
the isolated antigen-binding protein includes the CDRH3 of SEQ ID
NO:75, 78, 81, 84, 87, 90, 96, 99, 102, and 123, CDRH2 of SEQ ID
NO:74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101, and 129, and CDRH1
of SEQ ID NO:73, 76, 79, 82, 85, 88, 92, 97, and 100, and/or the
second amino acid sequence of the isolated antigen binding protein
comprises the CDRL3 of SEQ ID NO:44, 47, 50, 53, 56, 59, 64, 68,
71, and 72. CDRL2 of SEQ ID NO:43, 46, 49, 52, 55, 58, 61, 63, 67,
and 70, and CDRL1 of SEQ ID NO:42, 45, 48, 51, 54, 57, 62, 65, 66,
and 69.
[0298] In a further embodiment, the antigen binding protein
comprises at least two CDRH sequences of heavy chain sequences H1,
H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, or H13, as shown in
Table 5A. In again a further embodiment, the antigen binding
protein comprises at least two CDRL sequences of light chain
sequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13,
L14, L15, L16, or L17, as shown in Table 5B. In again a further
embodiment, the antigen binding protein comprises at least two CDRH
sequences of heavy chain sequences H1, H2, H3, H4, H5, H6, H7, H8,
H9, H10, H11, H12, or H13, as shown in Table 5A , and at least two
CDRLs of light chain sequences L1, L2, L3, L4, L5, L6, L7, L8, L9,
L10, L11, L12, L13, L14, L15, L16, or L17, as shown in Table
5B.
[0299] In again another embodiment, the antigen binding protein
comprises the CDRH1, CDRH2, and CDRH3 sequences of heavy chain
sequences H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, or
H13, as shown in Table 5A. In yet another embodiment, the antigen
binding protein comprises the CDRL1, CDRL2, and CDRL3 sequences of
light chain sequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11,
L12, L13, L14, L15, L16, or L17, as shown in Table 5B.
[0300] In yet another embodiment, the antigen binding protein
comprises all six CDRs of L1 and H1, or L2 and H2, or L3 and H3, or
L4 and H4, or L5 and H5, or L6 and H1, or L7 and H6, or L8 and H5,
or L9 and H1, or L10 and H7, or L11 and H8, or L12 and H9, or L12
and H10, or L13 and H5, or L14 and H11, or L15 and H12, or L16 and
H13, or L17 and H13, as shown in Tables 5A and 5B.
TABLE-US-00009 TABLE 5A Exemplary Heavy Chain Amino Acid Sequence
Regions Full Heavy Full Heavy Heavy Chain Heavy Chain Chain Chain
Variable Variable CDRH1 CDRH2 CDRH3 Reference Group SEQ ID NO
Region Group Region SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO 1E11 H1
29 V.sub.H1 158 73 74 75 1H7 H2 30 V.sub.H2 159 76 77 78 2E7 H3 31
V.sub.H3 160 79 80 81 3B6 H4 32 V.sub.H4 161 82 83 84 3C8 H5 33
V.sub.H5 162 85 86 87 4E4 H1 29 V.sub.H1 158 73 74 75 4H6 H6 34
V.sub.H6 163 88 89 90 5F5 H5 33 V.sub.H5 162 85 86 87 9D4 H1 29
V.sub.H1 158 73 74 75 9F5 H7 35 V.sub.H7 164 76 91 78 10E4 H8 36
V.sub.H8 165 92 93 94 11D11 H9 37 V.sub.H9 166 76 95 78 11H9 H10 38
V.sub.H10 167 76 95 78 12E8 H5 33 V.sub.H5 162 85 86 87 12G8 H11 39
V.sub.H11 168 73 74 96 13H2 H12 40 V.sub.H12 169 97 98 99 32H7 H13
41 V.sub.H13 170 100 101 102 32H7 CS H13 41 V.sub.H13 170 100 101
102 32H8 V.sub.H14 171 33B5 V.sub.H15 172 33E4 V.sub.H16 173 34E3
V.sub.H17 174
TABLE-US-00010 TABLE 5B Exemplary Light Chain Amino Acid Sequence
Regions Full Light Full Light Light Chain Light Chain Chain Chain
Variable Region Variable Region CDRL1 CDRL2 CDRL3 Reference Group
SEQ ID NO Group SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO 1E11 L1 12
V.sub.L1 137 42 43 44 1H7 L2 13 V.sub.L2 138 45 46 47 2E7 L3 14
V.sub.L3 139 48 49 50 3B6 L4 15 V.sub.L4 140 51 52 53 3C8 L5 16
V.sub.L5 141 54 55 56 4E4 L6 17 V.sub.L6 142 42 43 44 4H6 L7 18
V.sub.L7 143 57 58 59 5F5 L8 19 V.sub.L8 144 60 55 56 9D4 L9 20
V.sub.L9 145 42 43 44 9F5 L10 21 V.sub.L10 146 45 61 47 10E4 L11 22
V.sub.L11 147 62 63 64 11D11 L12 23 V.sub.L12 148 45 61 47 11H9 L12
23 V.sub.L12 148 45 61 47 12E8 L13 24 V.sub.L13 149 65 55 56 12G8
L14 25 V.sub.L14 150 42 43 44 13H2 L15 26 V.sub.L15 151 66 67 68
32H7 L16 27 V.sub.L16 152 69 70 71 32H7 CS L17 28 V.sub.L17 153 69
70 72 32H8 V.sub.L18 154 33B5 V.sub.L19 155 33E4 V.sub.L20 156 34E3
V.sub.L21 157
[0301] In one aspect, the isolated antigen-binding proteins
provided, herein can be a monoclonal antibody, a polyclonal
antibody, a recombinant antibody, a human antibody, a humanized
antibody, a chimeric antibody, a multispecific antibody, or an
antibody antigen binding fragment thereof.
[0302] In another embodiment, the antibody fragment of the isolated
antigen-binding proteins provided herein can be a Fab fragment, a
Fab' fragment, an F(ab').sub.2 fragment, an Fv fragment, a diabody,
or a single chain antibody molecule.
[0303] In a further embodiment, the isolated antigen binding
protein provided herein is a human antibody and can be of the
IgG1-, IgG2- IgG3- or IgG4-type.
[0304] In another embodiment, the antigen binding protein consists
of a just a light or a heavy chain polypeptide as set forth in
Tables 5A-5B. In some embodiments, the antigen binding protein
consists just of a light chain variable or heavy chain variable
domain such as those listed in Tables 5A-5B. Such antigen binding
proteins can be pegylated with one or more PEG molecules.
[0305] In yet another aspect, the isolated antigen-binding protein
provided herein can be coupled to a labeling group and can compete
for binding to the extracellular portion of human CGRP R with an
antigen binding protein of one of the isolated antigen-binding
proteins provided herein. In one embodiment, the isolated antigen
binding protein provided herein can reduce monocyte chemotaxis,
monocyte migration into tumors or inhibit accumulation and function
of tumor associated macrophage in a tumor when administered to a
patient.
[0306] As will be appreciated by those in the art, for any antigen
binding protein with more than one CDR from the depicted sequences,
any combination of CDRs independently selected from the depicted
sequences is useful. Thus, antigen binding proteins with one, two,
three, four, five or six of independently selected CDRs can be
generated. However, as will be appreciated by those in the art,
specific embodiments generally utilize combinations of CDRs that
are non-repetitive, e.g., antigen binding proteins are generally
not made with two CDRH2 regions, etc.
[0307] Some of the antigen binding proteins provided are discussed
in more detail below,
Antigen Binding Proteins and Binding Epitopes and Binding
Domains
[0308] When an antigen binding protein is said to bind an epitope,
such as one or both components of CGRP R, or the extracellular
domain of CGRP R, for example, what is meant is that the antigen
binding protein specifically binds to a specified portion of CGRP
R, which may be on CRLR, RAMP1, or span portions of both CRLR and
RAMP1. In cases where the antigen binding protein binds only CRLR
(and not RAMP1), the antigen binding protein would not be expected
to selectively bind CGRP R because CRLR is shared, inter alia, with
AM1 and AM1 receptors. Similarly, in cases where the antigen
binding protein binds only RAMP1 (and not CRLR), the antigen
binding protein would not be expected to selectively bind CGRP R
because RAMP1 is shared, inter alia, with AMY1 receptor. In cases
where the antigen binding protein interacts with both CRLR and
RAMP1, the antigen binding protein is expected to bind residues or
sequences of residues, or regions in both CRLR and RAMP1. In none
of the foregoing embodiments is an antigen binding protein expected
to contact every residue within CRLR or RAMP1. Similarly, not every
amino acid substitution or deletion within CRLR, RAMP1 or the
extracellular domains thereof is expected to significantly affect
binding affinity.
[0309] Methods detailed, e.g., in Example 10, may be used to assess
what regions of multimeric receptors, such as CGRP R, may be
involved in binding to selected antigen binding proteins.
Competing Antigen Binding Proteins
[0310] In another aspect, antigen binding proteins are provided
that compete with one of the exemplified, or "reference" antibodies
or functional fragments binding to the epitope described above for
specific binding to CGRP R. Such antigen binding proteins may also
bind to the same epitope as one of the herein exemplified antigen
binding proteins, or an overlapping epitope. Antigen binding
proteins and fragments that compete with or bind to the same
epitope as the exemplified or reference antigen binding proteins
are expected to show similar functional properties. The exemplified
antigen binding proteins and fragments include those with the heavy
and light chains, variable region domains V.sub.L1-V.sub.L17 and
V.sub.H1-V.sub.H13, and CDRs included in Tables 2A, 2B, 3, 4A, 4B,
5A and 5B, Thus, as a specific example, the antigen binding
proteins that are provided include those that compete with an
antibody having: (a) all 6 of the CDRs listed for an antibody
listed in Tables 5A and 5B; (b) a V.sub.H and a V.sub.L selected
from V.sub.L1-V.sub.L17 and V.sub.H1-V.sub.H13 and listed for an
antibody listed in Tables 5A and 5B; or (c) two light chains and
two heavy chains as specified for an antibody listed in Tables 5A
and 5B. Other examples of suitable reference antibodies include
those that have a heavy chain variable region having a sequence
corresponding to any of the sequences identified as SEQ ID
NO:158-170 and a light chain variable region having a sequence
corresponding to any of the sequences identified as SEQ ID
NO:137-153.
[0311] Binding competition may be assessed, for example, using a
binning assays, such as the Biacore assay described in Example 7,
below. In that example, 19 antibodies described herein were tested
against each of six "reference" antibodies--five neutralizing
antibodies (11D11, 3B6, 4H6, 12G8, and 9F5) and one
non-neutralizing antibody (34E3). The assay results, shown in Table
13, indicate that all of the tested neutralizing antibodies (1E11,
1H7, 2E7, 3B6, 3C8, 4E4, 4H6, 5F5, 9D4, 9F5, 10E4, 11D11, 11H9,
12E8, 12G8, 13H2 and 32H7) bind to essentially the same region of
CGRP R, which is distinct from the region of CGRP R that is bound
by the non-neutralizing antibodies tested (32H8, 33B5, 33E4 and
34E3). Based on these data, any of the neutralizing antibodies
would make exemplary reference antigen binding proteins in a
competition assay, particularly any of the neutralizing antibodies
that were immobilized in the assay described in Example 7--11D11,
3B6, 4H6, 12G8, and 9F5.
Monoclonal Antibodies
[0312] The antigen binding proteins that are provided include
monoclonal antibodies that bind to CGRP R. Monoclonal antibodies
may be produced using any technique known in the art, e.g., by
immortalizing spleen cells harvested from the transgenic animal
after completion of the immunization schedule. The spleen cells can
be immortalized using any technique known in the art, e.g., by
fusing them with myeloma cells to produce hybridomas. Myeloma cells
for use in hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas). Examples of suitable cell lines for use in
mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1,Ag 4
1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO
Bul; examples of cell lines used in rat fusions include R210.RCY3,
Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines useful for cell
fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6. An
exemplary method of preparing monoclonal antibodies is described in
Example 2, below,
[0313] In some instances, a hybridoma cell line is produced by
immunizing an animal (e.g., a transgenic animal having human
immunoglobulin sequences) with a CGRP R immunogen; harvesting
spleen cells from the immunized animal; fusing the harvested spleen
cells to a myeloma cell line, thereby generating hybridoma cells;
establishing hybridoma cell lines from the hybridoma cells, and
identifying a hybridoma cell line that produces an antibody that
binds CGRP R (e.g., as described in Examples 1-3, below). Such
hybridoma cell lines, and anti-CGRP R monoclonal antibodies
produced by them, are aspects of the present application.
[0314] Monoclonal antibodies secreted by a hybridoma cell line can
be purified using any technique known in the art. Hybridomas or
mAbs may be further screened to identify mAbs with particular
properties, such as the ability to bind cells expressing CGRP,
ability to block or interfere the binding of the CGRP ligand or
CGRP.sub.8-37 peptide, or the ability to functionally block the
receptor, e.g., using a cAMP assay, e.g., as described below.
Chimeric and Humanized Antibodies
[0315] Chimeric and humanized antibodies based upon the foregoing
sequences are also provided. Monoclonal antibodies for use as
therapeutic agents may be modified in various ways prior to use.
One example is a chimeric antibody, which is an antibody composed
of protein segments from different antibodies that are covalently
joined to produce functional immunoglobulin light or heavy chains
or immunologically functional portions thereof. Generally, a
portion of the heavy chain and/or light chain is identical with or
homologous to a corresponding sequence in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. For methods relating to chimeric antibodies, see, for
example, U.S. Pat. No. 4,816,567; and Morrison et al., 1985, Proc.
Natl. Acad. Sci. USA 81:6851-6855, which are hereby incorporated by
reference. CDR grafting is described, for example, in U.S. Pat. No.
6,180,370, No. 5,693,762, No. 5,693,761, No. 5,585,089, and No.
5,530,101.
[0316] Generally, the goal of making a chimeric antibody is to
create a chimera in which the number of amino acids from the
intended patient species is maximized. One example is the
"CDR-grafted" antibody, in which the antibody comprises one or more
complementarity determining regions (CDRs) from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the antibody chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. For use in humans, the variable region or selected CDRs
from a rodent antibody often are grafted into a human antibody,
replacing the naturally-occurring variable regions or CDRs of the
human antibody.
[0317] One useful type of chimeric antibody is a "humanized"
antibody. Generally, a humanized antibody is produced from a
monoclonal antibody raised initially in a non-human animal. Certain
amino acid residues in this monoclonal antibody, typically from
non-antigen recognizing portions of the antibody, are modified to
be homologous to corresponding residues in a human antibody of
corresponding isotype. Humanization can be performed, for example,
using various methods by substituting at least a portion of a
rodent variable region for the corresponding regions of a human
antibody (see, e.g., U.S. Pat. No. 5,585,089, and No. 5,693,762;
Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988,
Nature 332:323-27; Verhoeyen et al., 1988, Science
239:1534-1536),
[0318] In one aspect, the CDRs of the light and heavy chain
variable regions of the antibodies provided herein (see, Table 4)
are grafted to framework regions (FRs) from antibodies from the
same, or a different, phylogenetic species. For example, the CDRs
of the heavy and light chain variable regions V.sub.H1, V.sub.H2,
V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8,
V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, and V.sub.H13, and/or
V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6,
V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12,
V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, and V.sub.L17 can be
grafted to consensus human FRs. To create consensus human FRs, FRs
from several human heavy chain or light chain amino acid sequences
may be aligned to identify a consensus amino acid sequence. In
other embodiments, the FRs of a heavy chain or light chain
disclosed herein are replaced with the FRs from a different heavy
chain or light chain. In one aspect, rare amino acids in the FRs of
the heavy and light chains of anti-CGRP R antibody are not
replaced, while the rest of the FR amino acids are replaced. A
"rare amino acid" is a specific amino acid that is in a position in
which this particular amino acid is not usually found in an FR.
Alternatively, the grafted variable regions from the one heavy or
light chain may be used with a constant region that is different
from the constant region of that particular heavy or light chain as
disclosed herein. In other embodiments, the grafted variable
regions are part of a single chain Fv antibody.
[0319] In certain embodiments, constant regions from species other
than human can be used along with the human variable region(s) to
produce hybrid antibodies.
Fully Human Antibodies
[0320] Fully human antibodies are also provided. Methods are
available for making fully human antibodies specific for a given
antigen without exposing human beings to the antigen ("fully human
antibodies"). One specific means provided for implementing the
production of fully human antibodies is the "humanization" of the
mouse humoral immune system. Introduction of human immunoglobulin
(Ig) loci into mice in which the endogenous Ig genes have been
inactivated is one means of producing fully human monoclonal
antibodies (mAbs) in mouse, an animal that can be immunized with
any desirable antigen. Using fully human antibodies can minimize
the immunogenic and allergic responses that can sometimes be caused
by administering mouse or mouse-derived mAbs to humans as
therapeutic agents.
[0321] Fully human antibodies can be produced by immunizing
transgenic animals (usually mice) that are capable of producing a
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. Antigens for this purpose typically have
six or more contiguous amino acids, and optionally are conjugated
to a carrier, such as a hapten. See, e.g., Jakobovits et al., 1993,
Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993,
Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol.
7:33. In one example of such a method, transgenic animals are
produced by incapacitating the endogenous mouse immunoglobulin loci
encoding the mouse heavy and light immunoglobulin chains therein,
and inserting into the mouse genome large fragments of human genome
DNA containing loci that encode human heavy and light chain
proteins. Partially modified animals, which have less than the full
complement of human immunoglobulin loci, are then cross-bred to
obtain an animal having all of the desired immune system
modifications. When administered an immunogen, these transgenic
animals produce antibodies that are immunospecific for the
immunogen but have human rather than murine amino acid sequences,
including the variable regions. For further details of such
methods, see, for example, WO96/33735 and WO94/02602. Additional
methods relating to transgenic mice for making human antibodies are
described in U.S. Pat. No. 5,545,807; No. 6,713,610; No. 6,673,986;
No. 6,162,963; No. 5,545,807; No. 6,300,129; No, 6,255,458; No.
5,877,397; No. 5,874,299 and No. 5,545,806; in PCT publications
WO91/10741, WO90/04036, and. in EP 546073B1 and EP 546073A1.
[0322] The transgenic mice described above, referred to herein as
"HuMab" mice, contain a human immunoglobulin gene minilocus that
encodes unrearranged human heavy [mu] and [gamma]) and [kappa]
light chain immunoglobulin sequences, together with targeted
mutations that inactivate the endogenous [mu] and [kappa] chain
loci (Lonberg et al., 1994, Nature 368:856-859). Accordingly, the
mice exhibit reduced expression of mouse IgM or [kappa] and in
response to immunization, and the introduced human heavy and light
chain transgenes undergo class switching and somatic mutation to
generate high affinity human IgG [kappa] monoclonal antibodies
(Lonberg et al, supra.; Lonberg and Huszar, 1995, Intern. Rev.
Immunol. 13: 65-93; Harding and Lonberg, 1995. Ann. N.Y. Acad. Sci.
764:536-546). The preparation of HuMab mice is described in detail
in Taylor et al, 1992, Nucleic Acids Research 20:6287-6295; Chen et
al., 1993, International Immunology 5:647-656; Tuaillon et al.,
1994, Immunol. 152:2912-2920; Lonberg et al., 1994, Nature
368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology
113:49-101; Taylor et al., 1994, International Immunology
6:579-591; Lonberg and Huszar, 1995, Intern. Rev. Immunol.
13:65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.
764:536-546; Fishwild et al., 1996, Nature Biotechnology
14:845-851; the foregoing references are hereby incorporated by
reference in their entirety for all purposes. See, further U.S.
Pat. No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425;
No. 5,789,650; No. 5,877,397; No. 5,661,016; No. 5,814,318; No.
5,874,299; and No. 5,770,429; as well as U.S. Pat. No. 5,545,807;
International Publication Nos. WO 93/1227; WO 92/22646; and WO
92/03918, the disclosures of all of which are hereby incorporated
by reference in their entirety for all purposes. Technologies
utilized for producing human antibodies in these transgenic mice
are disclosed also in WO 98/24893, and Mendez et al., 1997, Nature
Genetics 15:146-156, which are hereby incorporated by reference.
For example, the HCo7 and. HCo12 transgenic mice strains can be
used to generate anti-CGRP R antibodies. Further details regarding
the production of human antibodies using transgenic mice are
provided in the examples below.
[0323] Using hybridoma technology, antigen-specific human mAbs with
the desired specificity can be produced and selected from the
transgenic mice such as those described above. Such antibodies may
be cloned and expressed using a suitable vector and host cell, or
the antibodies can be harvested from cultured hybridoma cells.
[0324] Fully human antibodies can also be derived from
phage-display libraries (as disclosed in Hoogenboom et al., 1991,
J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol.
222:581). Phage display techniques mimic immune selection through
the display of antibody repertoires on the surface of filamentous
bacteriophage, and subsequent selection of phage by their binding
to an antigen of choice. One such technique is described in PCT
Publication No. WO 99/10494 (hereby incorporated, by reference),
which describes the isolation of high affinity and functional
agonistic antibodies for MPL- and msk-receptors using such an
approach.
Bispecific or Bifunctional Antigen Binding Proteins
[0325] The antigen binding proteins that are provided also include
bispecific and bifunctional antibodies that include one or more
CDRs one or more variable regions as described above. A bispecific
or bifunctional antibody in some instances is an artificial hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Bispecific antibodies may be produced by a
variety of methods including, but not limited to, fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and
Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al.,
1992, J. Immunol. 148:1547-1553.
Various Other Forms
[0326] Some of the antigen binding proteins that are provided are
variant forms of the antigen binding proteins disclosed above
(e.g., those having the sequences listed in Tables 2-5). For
instance, some of the antigen binding proteins have one or more
conservative amino acid substitutions in one or more of the heavy
or light chains, variable regions or CDRs listed in Tables 2-5.
[0327] Naturally-occurring amino acids may be divided into classes
based on common side chain properties:
[0328] 1) hydrophobic: norleucine, Met, Ala, Leu, Ile;
[0329] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0330] 3) acidic: Asp, Glu;
[0331] 4) basic: His, Lys, Arg;
[0332] 5) residues that influence chain orientation: Gly, Pro;
and
[0333] 6) aromatic: Trp, Tyr, Phe.
[0334] Conservative amino acid substitutions may involve exchange
of a member of one of these classes with another member of the same
class. Conservative amino acid substitutions may encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties.
[0335] Non-conservative substitutions may involve the exchange of a
member of one of the above classes for a member from another class.
Such substituted residues may be introduced into regions of the
antibody that are homologous with human antibodies, or into the
non-homologous regions of the molecule.
[0336] In making such changes, according to certain embodiments,
the hydropathic index of amino acids may be considered. The
hydropathic profile of a protein is calculated by assigning each
amino acid a numerical value ("hydropathy index") and then
repetitively averaging these values along the peptide chain. Each
amino acid has been assigned a hydropathic index on the basis of
its hydrophobicity and charge characteristics. They are: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(.revreaction.2.8); cysteine/cystine (+2.5); methionine (+1.9);
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine
(-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0337] The importance of the hydropathic profile in conferring
interactive biological function on a protein is understood in the
art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It
is known that certain amino acids may be substituted for other
amino acids having a similar hydropathic index or score and still
retain a similar biological activity. In making changes based upon
the hydropathic index, in certain embodiments, the substitution of
amino acids whose hydropathic indices are within .+-.2 is included.
In some aspects, those which are within .+-.1 are included, and in
other aspects, those within .+-.0.5 are included.
[0338] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as in the present case. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigen-binding or
immunogenicity, that is, with a biological property of the
protein.
[0339] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in other embodiments, those which are within .+-.1 are
included, and in still other embodiments, those within .+-.0.5 are
included. In some instances, one may also identify epitopes from
primary amino acid sequences on the basis of hydrophilicity. These
regions are also referred to as "epitopic core regions."
[0340] Exemplary conservative amino acid substitutions are set
forth in Table 6.
TABLE-US-00011 TABLE 6 Conservative Amino Acid Substitutions
Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln,
His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,
Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr
Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0341] A skilled artisan will be able to determine suitable
variants of polypeptides as set forth herein using well-known
techniques. One skilled in the art may identify suitable areas of
the molecule that may be changed without destroying activity by
targeting regions not believed to be important for activity. The
skilled artisan also will be able to identify residues and portions
of the molecules that are conserved among similar polypeptides. In
further embodiments, even areas that may be important for
biological activity or for structure may be subject to conservative
amino acid substitutions without destroying the biological activity
or without adversely affecting the polypeptide structure.
[0342] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a protein that correspond to amino acid residues
important for activity or structure in similar proteins. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues.
[0343] One skilled in the art can also analyze the 3-dimensional
structure and amino acid sequence in relation to that structure in
similar polypeptides. In view of such information, one skilled in
the art may predict the alignment of amino acid residues of an
antibody with respect to its three dimensional structure. One
skilled in the art may choose not to make radical changes to amino
acid residues predicted to be on the surface of the protein, since
such residues may be involved in important interactions with other
molecules. Moreover, one skilled in the art may generate test
variants containing a single amino acid substitution at each
desired amino acid residue. These variants can then be screened
using assays for CGRP R neutralizing activity, (see examples below)
thus yielding information regarding which amino acids can be
changed and which must not be changed. In other words, based on
information gathered from such routine experiments, one skilled in
the art can readily determine the amino acid positions where
further substitutions should be avoided either alone or in
combination with other mutations.
[0344] A number of scientific publications have been devoted to the
prediction of secondary structure. See, Moult, 1996, Curr. Op. in
Biotech. 7:422-427; Chou et al., 1974, Biochem. 13:222-245; Chou et
al., 1974, Biochemistry 113:211-222; Chou et al., 1978, Adv.
Enzymol. Relat. Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann.
Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J.
26:367-384. Moreover, computer programs are currently available to
assist with predicting secondary structure. One method of
predicting secondary structure is based upon homology modeling. For
example, two polypeptides or proteins that have a sequence identity
of greater than 30%, or similarity greater than 40% can have
similar structural topologies. The recent growth of the protein
structural database (PDB) has provided enhanced predictability of
secondary structure, including the potential number of folds within
a polypeptide's or protein's structure. See, Holm et al., 1999,
Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner et al.,
1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will become dramatically more accurate.
[0345] Additional methods of predicting secondary structure include
"threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-387;
Sippl et al., 1996, Structure 4:15-19), "profile analysis" (Bowie
et al., 1991, Science 253:164-170; Gribskov et al., 1990, Meth.
Enzym. 183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci.
84:4355-4358), and "evolutionary linkage" (See, Holm, 1999, supra;
and Brenner, 1997, supra),
[0346] In some embodiments, amino acid substitutions are made that:
(1) reduce susceptibility to proteolysis, (2) reduce susceptibility
to oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter ligand or antigen binding affinities, and/or
(4) confer or modify other physicochemical or functional properties
on such polypeptides. For example, single or multiple amino acid
substitutions (in certain embodiments, conservative amino acid
substitutions) may be made in the naturally-occurring sequence.
Substitutions can be made in that portion of the antibody that lies
outside the domain(s) forming intermolecular contacts). In such
embodiments, conservative amino acid substitutions can be used that
do not substantially change the structural characteristics of the
parent sequence (e.g., one or more replacement amino acids that do
not disrupt the secondary structure that characterizes the parent
or native antigen binding protein). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed.),
1984, W. H, New York: Freeman and Company; Introduction to Protein
Structure (Branden and Tooze, eds.), 1991, New York: Garland.
Publishing; and Thornton et al., 1991, Nature 354:105, which are
each incorporated herein by reference.
[0347] Additional preferred antibody variants include cysteine
variants wherein one or more cysteine residues in the parent or
native amino acid sequence are deleted from or substituted with
another amino acid (e.g., serine). Cysteine variants are useful,
inter alia when antibodies must be refolded into a biologically
active conformation. Cysteine variants may have fewer cysteine
residues than the native antibody, and typically have an even
number to minimize interactions resulting from unpaired
cysteines.
[0348] The heavy and light chains, variable regions domains and
CDRs that are disclosed can be used to prepare polypeptides that
contain an antigen binding region that can specifically bind to
CGRP R. For example, one or more of the CDRs listed in Tables 4 and
5 can be incorporated into a molecule (e.g., a polypeptide)
covalently or noncovalently to make an immunoadhesion. An
immunoadhesion may incorporate the CDR(s) as part of a larger
polypeptide chain, may covalently link the CDR(s) to another
polypeptide chain, or may incorporate the CDR(s) noncovalently. The
CDR(s) enable the immunoadhesion to bind specifically to a
particular antigen of interest (e.g., CGRP R or epitope
thereof).
[0349] Mimetics (e.g., "peptide mimetics" or "peptidomimetics")
based upon the variable region domains and CDRs that are described
herein are also provided. These analogs can be peptides,
non-peptides or combinations of peptide and non-peptide regions.
Fauchere, 1986, Adv. Drug Res. 15:29; Veber and Freidinger, 1985,
TINS p, 392; and Evans et al., 1987, J. Med. Chem. 30:1229, which
are incorporated herein by reference for any purpose. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce a similar therapeutic or
prophylactic effect. Such compounds are often developed with the
aid of computerized molecular modeling. Generally, peptidomimetics
are proteins that are structurally similar to an antibody
displaying a desired biological activity, such as here the ability
to specifically bind CGRP R, but have one or more peptide linkages
optionally replaced by a linkage selected from: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH--, --CH--CH-(cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used in certain
embodiments to generate more stable proteins. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo and Gierasch, 1992,
Ann. Rev. Biochem. 61:387), incorporated herein by reference), for
example, by adding internal cysteine residues capable of forming
intramolecular disulfide bridges which cyclize the peptide.
[0350] Derivatives of the antigen binding proteins that are
described herein are also provided. The derivatized antigen binding
proteins can comprise any molecule or substance that imparts a
desired property to the antibody or fragment, such as increased
half-life in a particular use. The derivatized antigen binding
protein can comprise, for example, a detectable (or labeling)
moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic
molecule, a detectable bead (such as a magnetic or electrodense
(e.g., gold) bead), or a molecule that binds to another molecule
(e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety
(e.g., a radioactive, cytotoxic, or pharmaceutically active
moiety), or a molecule that increases the suitability of the
antigen binding protein for a particular use (e.g., administration
to a subject, such as a human subject, or other in vivo or in vitro
uses). Examples of molecules that can be used to derivatize an
antigen binding protein include albumin (e.g., human serum albumin)
and polyethylene glycol (PEG). Albumin-linked and PEGylated
derivatives of antigen binding proteins can be prepared using
techniques well known in the art. Certain antigen binding proteins
include a pegylated single chain polypeptide as described herein.
In one embodiment, the antigen binding protein is conjugated or
otherwise linked to transthyretin (TTR) or a TTR variant. The TTR
or TTR variant can be chemically modified with, for example, a
chemical selected from the group consisting of dextran,
poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
[0351] Other derivatives include covalent or aggregative conjugates
of CGRP R binding proteins with other proteins or polypeptides,
such as by expression of recombinant fusion proteins comprising
heterologous polypeptides fused to the N-terminus or C-terminus of
a CGRP R binding protein. For example, the conjugated peptide may
be a heterologous signal (or leader) polypeptide, e.g., the yeast
alpha-factor leader, or a peptide such as an epitope tag. CGRP
antigen binding protein-containing fusion proteins can comprise
peptides added to facilitate purification or identification of the
CGRP R binding protein (e.g., poly-His). A CGRP R binding protein
also can be linked to the FLAG peptide as described in Hopp el al.,
1988, Bio/Technology 6:1204; and U.S. Pat. No. 5,011,912. The FLAG
peptide is highly antigenic and provides an epitope reversibly
bound by a specific monoclonal antibody (mAb), enabling rapid assay
and facile purification of expressed recombinant protein. Reagents
useful for preparing fusion proteins in which the FLAG peptide is
fused to a given polypeptide are commercially available (Sigma, St,
Louis, Mo,).
[0352] Oligomers that contain one or more CGRP R binding proteins
may be employed as CGRP R antagonists. Oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers. Oligomers comprising two or more CGRP R binding
proteins are contemplated for use, with one example being a
homodimer. Other oligomers include heterodimers, homotrimers,
heterotrimers, homotetramers, heterotetramers, etc.
[0353] One embodiment is directed to oligomers comprising multiple
CGRP R-binding polypeptides joined via covalent or non-covalent
interactions between peptide moieties fused to the CGRP R binding
proteins. Such peptides may be peptide linkers (spacers), or
peptides that have the property of promoting oligomerization.
Leucine zippers and certain polypeptides derived from antibodies
are among the peptides that can promote oligomerization CGRP R
binding proteins attached thereto, as described, in more detail
below.
[0354] In particular embodiments, the oligomers comprise from two
to four CGRP R binding proteins. The CGRP R binding protein
moieties of the oligomer may be in any of the forms described,
above, e.g., variants or fragments. Preferably, the oligomers
comprise CGRP R binding proteins that have CGRP R binding
activity.
[0355] In one embodiment, an oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation of fusion
proteins comprising certain heterologous polypeptides fused to
various portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al., 1991, Proc.
Natl. Acad. Sci. USA 88:10535; Byrn el al., 1990, Nature 344:677;
and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion
Proteins", in Current Protocols in Immunology, Suppl. 4, pages
10.19.1-10.19.11.
[0356] One embodiment is directed to a dimer comprising two fusion
proteins created by fusing a CGRP R binding protein to the Fc
region of an antibody. The dimer can be made by, for example,
inserting a gene fusion encoding the fusion protein into an
appropriate expression vector, expressing the gene fusion in host
cells transformed with the recombinant expression vector, and
allowing the expressed fusion protein to assemble much like
antibody molecules, whereupon interchain disulfide bonds form
between the Fc moieties to yield the dimer.
[0357] The term "Fc polypeptide" as used herein includes native
and, mutein forms of polypeptides derived from the Fc region of an
antibody. Truncated forms of such polypeptides containing the hinge
region that promotes dimerization also are included. Fusion
proteins comprising Fc moieties (and oligomers formed therefrom)
offer the advantage of facile purification by affinity
chromatography over Protein A or Protein G columns.
[0358] One suitable Fc polypeptide, described in PCT application WO
93/10151 and U.S. Pat. No. 5,426,048 and No. 5,262,522, is a single
chain polypeptide extending from the N-terminal hinge region to the
native C-terminus of the Fc region of a human IgG1 antibody.
Another useful FC polypeptide is the Fc mutein described in U.S.
Pat. No. 5,457,035, and in Baum et al., 1994, EMBO J. 13:3992-4001.
The amino acid sequence of this mutein is identical to that of the
native Fc sequence presented in WO 93/10151, except that amino acid
19 has been changed from Leu to Ala, amino acid 20 has been changed
from Leu to Glu, and amino acid 22 has been changed from Gly to
Ala. The mutein exhibits reduced affinity for Fc receptors.
[0359] In other embodiments, the variable portion of the heavy
and/or light chains of a CGRP R binding protein such as disclosed
herein may be substituted for the variable portion of an antibody
heavy and/or light chain.
[0360] Alternatively, the oligomer is a fusion protein comprising
multiple CGRP R binding proteins, with or without peptide linkers
(spacer peptides). Among the suitable peptide linkers are those
described in U.S. Pat. No, 4,751,180 and No. 4,935,233.
[0361] Another method for preparing oligomeric CGRP R binding
protein derivatives involves use of a leucine zipper. Leucine
zipper domains are peptides that promote oligomerization of the
proteins in which they are found. Leucine zippers were originally
identified in several DNA-binding proteins (Landschulz et al.,
1988, Science 240:1759), and have since been found in a variety of
different proteins. Among the known leucine zippers are naturally
occurring peptides and derivatives thereof that dimerize or
trimerize. Examples of leucine zipper domains suitable for
producing soluble oligomeric proteins are described in PCT
application WO 94/10308, and the leucine zipper derived from lung
surfactant protein D (SPD) described in Hoppe et al., 1994, FEBS
Letters 344:191, hereby incorporated by reference. The use of a
modified leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described Fanslow et al.,
1994, Semin. Immunol. 6:267-278. In one approach, recombinant
fusion proteins comprising a CGRP R binding protein fragment or
derivative fused to a leucine zipper peptide are expressed in
suitable host cells, and the soluble oligomeric CGRP R binding
protein fragments or derivatives that form are recovered from the
culture supernatant.
[0362] in certain embodiments, the antigen binding protein has a
K.sub.D (equilibrium binding affinity) of less than 1 pM, 10 pM,
100 pM, 1 nM, 2 nM, 5 nM, 10 nM, 25 nM or 50 nM.
[0363] Another aspect provides an antigen-binding protein having a
half-life of at least one day in vitro or in vivo (e.g., when
administered to a human subject). In one embodiment, the antigen
binding protein has a half-life of at least three days. In another
embodiment, the antibody or portion thereof has a half-life of four
days or longer. In another embodiment, the antibody or portion
thereof has a half-life of eight days or longer. In another
embodiment, the antibody or antigen-binding portion thereof is
derivatized or modified such that it has a longer half-life as
compared to the underivatized or unmodified antibody. In another
embodiment, the antigen binding protein contains point mutations to
increase serum half life, such as described in WO 00/09560,
published Feb. 24, 2000, incorporated by reference.
Glycosylation
[0364] The antigen-binding protein may have a glycosylation pattern
that is different or altered from that found in the native species.
As is known in the art, glycosylation patterns can depend on both
the sequence of the protein (e.g., the presence or absence of
particular glycosylation amino acid residues, discussed below), or
the host cell or organism in which the protein is produced.
Particular expression systems are discussed below.
[0365] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tri-peptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked. glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0366] Addition of glycosylation sites to the antigen binding
protein is conveniently accomplished by altering the amino acid
sequence such that it contains one or more of the above-described
tri-peptide sequences (for N-linked glycosylation sites). The
alteration may also be made by the addition of, or substitution by,
one or more serine or threonine residues to the starting sequence
(for O-linked glycosylation sites). For ease, the antigen binding
protein amino acid sequence may be altered through changes at the
DNA level, particularly by mutating the DNA encoding the target
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0367] Another means of increasing the number of carbohydrate
moieties on the antigen binding protein is by chemical or enzymatic
coupling of glycosides to the protein. These procedures are
advantageous in that they do not require production of the protein
in a host cell that has glycosylation capabilities for N- and
O-linked glycosylation. Depending on the coupling mode used, the
sugar(s) may be attached to (a) arginine and histidine, (b) free
carboxyl groups, (c) free sulfhydryl groups such as those of
cysteine, (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline, (e) aromatic residues such as those
of phenylalanine, tyrosine, or tryptophan, or (f) the amide group
of glutamine. These methods are described in WO 87/05330 published
Sep. 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev.
Biochem., pp. 259-306.
[0368] Removal of carbohydrate moieties present on the starting
antigen binding protein may be accomplished chemically or
enzymatically. Chemical deglycosylation requires exposure of the
protein to the compound trifluoromethanesulfonic acid, or an
equivalent compound. This treatment results in the cleavage of most
or all sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the polypeptide intact.
Chemical deglycosylation is described by Hakimuddin et al., 1987,
Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal.
Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on
polypeptides can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., 1987, Meth.
Enzymol. 138:350. Glycosylation at potential glycosylation sites
may be prevented by the use of the compound tunicamycin as
described by Duskin et al., 1982, J. Biol. Chem. 257:3105.
Tunicamycin blocks the formation of protein-N-glycoside
linkages.
[0369] Hence, aspects include glycosylation variants of the antigen
binding proteins wherein the number and/or type of glycosylation
site(s) has been altered compared to the amino acid sequences of
the parent polypeptide. In certain embodiments, antibody protein
variants comprise a greater or a lesser number of N-linked
glycosylation sites than the native antibody. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or,
Asn-X-Thr, wherein the amino acid residue designated as X may be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions that eliminate or alter this sequence will prevent
addition of an N-linked carbohydrate chain present in the native
polypeptide. For example, the glycosylation can be reduced by the
deletion of an Asn or by substituting the Asn with a different
amino acid. In other embodiments, one or more new N-linked sites
are created. Antibodies typically have a N-linked glycosylation
site in the Fc region.
Labels and Effector Groups
[0370] In some embodiments, the antigen-binding comprises one or
more labels. The term "labeling group" or "label" means any
detectable label. Examples of suitable labeling groups include, but
are not limited, to, the following: radioisotopes or radionuclides
(e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc,
.sup.111In, .sup.125I, .sup.131I), fluorescent groups (e.g., FITC,
rhodamine, lanthanide phosphors), enzymatic groups (e.g.,
horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase), chemiluminescent groups, biotinyl groups, or
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In some
embodiments, the labeling group is coupled to the antigen binding
protein via spacer arms of various lengths to reduce potential
steric hindrance. Various methods for labeling proteins are known
in the art and may be used as is seen fit.
[0371] The term "effector group" means any group coupled to an
antigen binding protein that acts as a cytotoxic agent. Examples
for suitable effector groups are radioisotopes or radionuclides
(e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc,
.sup.111In, .sup.125I, .sup.131I). Other suitable groups include
toxins, therapeutic groups, or chemotherapeutic groups. Examples of
suitable groups include calicheamicin, auristatins, geldanamycin
and maytansine. In some embodiments, the effector group is coupled
to the antigen binding protein via spacer arms of various lengths
to reduce potential steric hindrance.
[0372] In general, labels fall into a variety of classes, depending
on the assay in which they are to be detected: a) isotopic labels,
which may be radioactive or heavy isotopes; b) magnetic labels
(e.g., magnetic particles); c) redox active moieties; d) optical
dyes; enzymatic groups (e.g. horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase); e)
biotinylated groups; and f) predetermined polypeptide epitopes
recognized by a secondary reporter (e.g., leucine zipper pair
sequences, binding sites for secondary antibodies, metal binding
domains, epitope tags, etc.). In some embodiments, the labeling
group is coupled to the antigen binding protein via spacer arms of
various lengths to reduce potential steric hindrance. Various
methods for labeling proteins are known in the art.
[0373] Specific labels include optical dyes, including, but not
limited to, chromophores, phosphors and fluorophores, with the
latter being specific in many instances. Fluorophores can be either
"small molecule" fluores, or proteinaceous fluores.
[0374] By "fluorescent label" is meant any molecule that may be
detected via its inherent fluorescent properties. Suitable
fluorescent labels include, but are not limited to, fluorescein,
rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy
5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (Alexa
Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa
Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa
Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and
R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC,
Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7
(Amersham Life Science, Pittsburgh, Pa.). Suitable optical dyes,
including fluorophons, are described in MOLECULAR PROBES HANDBOOK
by Richard P. Haugland, hereby expressly incorporated by
reference.
[0375] Suitable proteinaceous fluorescent labels also include, but
are not limited to, green fluorescent protein, including a Renilla,
Ptilosarcus, or Aequorea species of GPP (Chalfie et al., 1994,
Science 263:802-805), EGFP (Clontech Labs., Inc., Genbank Accession
Number U55762), blue fluorescent protein (BFP, Quantum
Biotechnologies, Inc., Quebec, Canada; Stauber, 1998, Biotechniques
24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced
yellow fluorescent protein (EYFP, Clontech Labs., Inc.), luciferase
(Ichiki et al., 1993, J. Immunol. 150:5408-5417), .beta.
galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605,
WO98/26277, WO99/49019, U.S. Pat. No. 5,292,658, No. 5,418,155, No.
5,683,888, No. 5,741,668, No. 5,777,079, No. 5,804,387, No.
5,874,304, No. 5,876,995, No. 5,925,558).
Nucleic Acid Sequences Encoding CGRP Antigen Binding Proteins
[0376] Nucleic acids that encode for the antigen binding proteins
described herein, or portions thereof, are also provided, including
nucleic acids encoding one or both chains of an antibody, or a
fragment, derivative, mutein, or variant thereof, polynucleotides
encoding heavy chain variable regions or only CDRs, polynucleotides
sufficient for use as hybridization probes, PCR primers or
sequencing primers for identifying, analyzing, mutating or
amplifying a polynucleotide encoding a polypeptide, anti-sense
nucleic acids for inhibiting expression of a polynucleotide, and
complementary sequences of the foregoing. The nucleic acids can be
any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450,
500, 750, 1,000, 1,500 or more nucleotides in length, and/or can
comprise one or more additional sequences, for example, regulatory
sequences, and/or be part of a larger nucleic acid, for example, a
vector. The nucleic acids can be single-stranded or double-stranded
and can comprise RNA and/or DNA nucleotides, and artificial
variants thereof (e.g., peptide nucleic acids).
[0377] Table 7 shows exemplary nucleic acid sequences encoding an
IgG2 heavy chain constant region, a kappa light chain constant
region and a lambda hCL-1 light chain constant region. Any variable
region provided herein may be attached to these constant regions to
form complete heavy and light chain sequences, However. it should
he understood that these constant regions sequences are provided as
specific examples only--one of skill in the art may employ other
constant regions, including IgG1 heavy chain constant region, IgG3
or IgG4 heavy chain constant regions, any of the seven lambda light
chain constant regions, including hCL-1, hCL-2, hCL-3 and hCL-7;
constant regions that have been modified for improved stability,
expression, manufacturability or other desired characteristics, and
the like. In some embodiments, the variable region sequences are
joined to other constant region sequences that are known in the
art. Exemplary nucleic acid sequences encoding heavy and light
chain variable regions are provided in Table 8.
TABLE-US-00012 TABLE 7 Exemplary Heavy And Light Chain Constant
Region Nucleic Acid Sequences Type Nucleic Acid Sequence/SEQ ID NO.
IgG2
gctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcg-
gcc heavy
ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgac-
cagcgg chain
cgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgc-
cctccagc
aacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagt
tgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttc-
ccc
ccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccac
gaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacg
ggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggc
aaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaa
agggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtc
agcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccg
gagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccg-
t
ggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactac
acgcagaagagcctctccctgtctccgggtaaatga [SEQ ID NO: 259] IgG2
cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcc-
tctgttgtgt kappa
gcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg-
ggtaa light
ctcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgc-
tg chain
agcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcc-
cgt cacaaagagcttcaacaggggagagtgttag [SEQ ID NO: 260] IgG2
ggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctgaggagctccaagccaacaa-
ggcca lambda
cactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcctggaaggcagatggcag-
ccccgtc hCL-1
aaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacct-
g light
agcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcac-
cgt chain ggagaagacagtggcccctacagaatgttcatag [SEQ ID NO: 261]
[0378] Table 8 shows exemplary nucleic acid sequences encoding
heavy chain and light chain variable regions, in which the various
CDRL1, CDRL2 and CDRL3, or CDRLH1, CDRH2 and CDRH3, sequences are
embedded.
TABLE-US-00013 TABLE 8 Exemplary Light and Heavy Chain Variable
Region Nucleic Acid Sequences SEQ ID Reference NO. Nucleic Acid
Sequence 2E7 V.sub.L 175
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcactt
gccgggcaagtcagggcattagaaatgatttaggctggtttcagcagaaaccagggaaagccc
ctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtg
gatctgggacagaattcactctcacaatcagcagcctgcagcctgaagatttagcaacttattactg
tctacagtataatatttacccgtggacgttcggccaagggaccaaggtggaaatcaaa 13H2
V.sub.L 176
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcactt
gccgggcaagtcagggcattagaaaggatttaggctggtatcagcagaaaccagggaaagcc
cctaagcgcctgatctatggagcatccagtttgcaaagtggggtcccatcaaggttcagcggcagt
ggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattact
gtctacagtataatagtttcccgtggacgttcggccaagggaccaaggtggaaatcaaa 33B5
V.sub.L 177
aggtgcagctggtgcagtctggggctgaggtgaagaagtctggggcctcagtgaaggtctcctgc
aaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagg
gcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgtacagaagtttcagg
gcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctg
agatctgacgacacggccgtgtattactgtgcgagaaatgagtatagcagtgcctggcccttggg
gtattggggccagggaaccctggtcaccgtctctagt 4H6 V.sub.L 178
gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctg
caggtctagtcagagcctcctgcatagttttgggtacaactatttggattggtacctgcagaagccag
ggcagtctccacagctcctgatctatttgggttctaatcgggcctccggggtccctgacaggttcagt
ggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggatgttgggg
tttattactgcatgcaagctctacaaactccattcactttcggccctgggaccaaagtggatatcaaa
3C8 V.sub.L 179
gatattatactggcccagactccactttctctgtccgtcacccctggacagccggcctccatctcctg
caagtctagtcagagcctcctgcacagtgctggaaagacctatttgtattggtacctgcagaagcc
aggccagcctccacagctcctgatctatgaagtttccaaccggttctctggagtgccagataggttc
agtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttg
ggatttattactgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatc
aaa 5F5 V.sub.L 180
gatattattctgacccagactccactttctctgtccgtcacccctggacagccggcctccatctcctgc
aagtctagtcagagcctcctgcacagtgatggaaagacctatttgtattggtacctgcagaagccc
ggccagcctccacagctcctgatctatgaagtttccaaccggttctctggagagccagataggttca
gtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttgg
gacttattattgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatca
aa 12E8 V.sub.L 181
gatattacactgacccagactccactttctctgtccgtctcccctggacagccggcctccatctcctg
caagtctagtcagagcctcctgcacagtgatggaaggaactatctgtattggtacctgcagaagcc
aggccagcctccacagctcctgatctatgaagtgtccaaccggttctctggactgccagataggttc
agtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttg
ggatttattactgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatc
aaa 32H7 V.sub.L 182
gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcct
gcagggccagtcagagtgttagcagcggctacttaacctggtaccagcagaaacctggccagg
ctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggc
agtgggtctgggacagacttcactctcaccatcgcagactggagcctgaagattttgcagtgtatt
actgtcagcagtatggtaactcactgtgcaggtttggccaggggaccaagctggagatcaaa 32H7
CS 183
gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcct
V.sub.L
gcagggccagtcagagtgttagcagcggctacttaacctggtaccagcagaacctggccagg
ctcccagactcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggc
agtggtctgggacggacttcactctcaccatcagcagactggagcctgaagattttgcagtgtatt
actgtcagcagtatggtaactcactgagcaggtttggccaggggaccaagctggagatcaaa 33E4
V.sub.L 184
gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctc
ctgtagggccagtcagagtgttcgcagcaatttagcctggtaccagcagaaacctggccaggctc
ccaggctcctcattcatgatgcatcccccaggaccgctggtatcccagccaggttcagtggcagtg
gatctgggacagaattcactctcaccatcaacagcctgcagtctgaagattttgcagtttattactgtc
agcagtataattactggactccgatcaccttcggccaagggacacgactggagattaaa 32H8
V.sub.L 185
gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaa
ctgcaagtccagccagagtattttagacagctccaacaatgataactacttagcttggtaccagca
gaaaccaggacagcctcctaaactgctcatttactgggcatctacccgggaatccggggtccctg
accgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaa
gatgtggcagtttattactgtcagcaatattataatactccattcactttcggccctgggaccaaagtg
gatatcaaa 1E11 V.sub.L 186
cagtctgtgttgacgcagccgccctcagtgtctgaggccccaggacagaaggtcaccatctcctg
ctctggaagcagctccaacattgggaataattatgtatcctggtaccagcagctcccaggaacag
cccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctc
caagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattat
tactgcggaacatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgt
ccta 4E4 V.sub.L 187
cagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaagtcaccatctcctg
ctctggaagcagctccaacattgggataattatgtatcctggataccagcagctcccaggaacag
cccccaaactcctcatttatgacaataataagcgaccctcgggattcctgaccgattctctggctc
caagtctggcacgtcaaccaccctgggcatcaccggactccagactggggacgaggccgattat
tactgcggaacatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgt
ccta 9D4 V.sub.L 188
cagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctg
ctctggaagcagctccaacattgggaataattatgtatcctggtaccagcagttcccaggaacagc
ccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctcc
aagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattatt
actgcggaacatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgtc
cta 12G8 V.sub.L 189
cagtctgtgttgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctg
ctctggaagcagctccaacattgggaataattatgtatcctggtaccagcagctcccaggaacag
cccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctc
caagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattat
tactgcggaacatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgt
ccta 34E3 V.sub.L 190
cagtctgtgttgacgcagccgccctcaatgtctgcggccccaggacagaaggtcaccatctcctg
ctctggaagcagctccaacattgggaataattatgtatcctggtaccagcagctcccaggaacag
cccccaaactcctcatttatgacaataataagcgaccctcagggattcctgaccgattctctggctc
caagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccaatta
ctgctgcggaacatgggatatcggcctgagtgtttgggtgttcggcggagggaccaaactgaccg
tccta 10E4 V.sub.L 191
cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt
tctggaagcagttccaatatcggaagtaatactgtgaactggtaccagcagctcccaggaacggc
ccccaaactcctcatctatactaataatcagcggccctcaggggtccctgaccgattctctggctcc
aagtctggcacctcagcctccctggccatcagtggactccagtcgaggatgaggctgatttttact
gtgcagcgcgggatgagagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcct a
11D11 V.sub.L 192
cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagagtcaccatctcttgt
11H9 VL
tctggaagcagctccaacatcggcagtaattatgtatactggtaccagcagctcccaggagcggc
ccccaaactcctcatctttaggaataatcagcggccctcaggggtccctgaccgcttctctggctcc
aagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattact
gtgcagcatgggatgacagcctgagtggttgggtgttcggcggagggaccaagctgaccgtcct a
1H7 V.sub.L 193
cagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagagtcaccatctcttgt
tctggaagcagctccaacatcggcagtaattatgtatactggtaccagcagctcccaggagcggc
ccccaaactcctcatctttaggagtaatcagcggccctcaggggtccctgaccgattctctggctcc
aagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattact
gtgcagcatgggatgacagcctgagtggttgggtgttcggcggagggaccaagctgaccgtcct a
9F5 V.sub.L 194
cagtctgtgctgactcagtcaccctcagcgtctgggacccccgggcagagagtcaccatctcttgtt
ctggaagcagctccaacatcggcagtaattatgtatactggtaccagcagctcccaggagcggc
ccccaaactcctcatccttaggaataatcagcggccctcaggggtccctgaccgattctctggctcc
aagtctggcacctcagcctccctgaccatcagtgggctccggtccgaggatgaggctgactattatt
gtgcagcatgggatgacagcctgagtggttgggtgttcggcggagggaccaagctgaccgtcct a
3B6 V.sub.L 195
tcttctgagctgactcaggaccctactgtgtctgtggccttgggacagacagtcaaaatcacatgcc
aaggagacagcctcagaagtttttatgcaagctggtaccagcagaagccaggacaggcccctgt
acttgtcttctatggtaaaaacaaccggccctcagggatcccagaccgattctctggctccagctca
ggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattattgtaat
tcccgggacagcagtgtttaccatctggtactcggcggagggaccaagctgaccgtccta 3B6
V.sub.H 196
caggtgcagttggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctg
caaggcttctggatacaccttcaccggctactatagcactgggtgcgacaggcccctggacaag
ggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcag
ggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggct
gagatctgacgacacggccgtgtatttctgtgcgagagatcaaatgagtattattatgcttcgggga
gtttttcccccttactattacggtatggacgtctggggccaagggaccacggtcaccgtctctagt
10E4 V.sub.H 197
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcct
gcaaggcttctggatacaccttcaccgactactatatgtactgggtgcgacaggcccctggacaa
gggcttgagtggatgggatggatcagccctaatagtggtggcacaaactatgcccagaagtttca
gggcagggtcaccatgaccagggacacgtctatcagcacagcctacatggagctgagtaggct
gagatctgacgacacggccgtgtattactgtgtgagaggaggatatagtggctacgctgggctcta
ctcccactactacggtatggacgtctggggccaagggaccacggtcaccgtctctagt 32H8
V.sub.H 198
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcct
gcaaggcttctggatacaccttcaccgcctactatttacactgggtgcgacaggcccctggacaag
ggcttgagtggatgggatggatcaaccctcacagtggtggcacaaactatgcacagaagtttcag
ggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggct
gagatctgacgacacggccgtgttctactgtgcgagaggaaggcagtggctgggctttgactact
ggggccagggaaccctggtcaccgtctctagt 33B5 V.sub.H 199
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccattacttg
ccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaagcccc
taagcgcctgatctatgttgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtgga
tctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtct
acagtataacacttacccgctcactttcggcggagggaccaaggtggagatcaag 11D11
V.sub.H 200
gaggtacagctggtggagtctgggggaggcttggtaaagcctggggggtccctcagactctcctg
tgcagcctctggattcactttcggtaacgcctggatgagctgggtccgccaggctccagggaagg
ggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacc
cgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaac
agcctgaaaaccgaggacacagccgtgtatttctgtaccacagatcggaccgggtatagcatca
gctggtctagttactactactactacggtatggacgtctggggccaagggaccacggtcaccgtct
ctagt 9F5 V.sub.H 201
gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgt
gcagcctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggg
gctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacactgcaccc
gtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaatag
cctgaaagccgaggacacagccgtgtattactgtaccacagatcggaccgggtatagcatcagc
tggtctagttactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcta
gt 11H9 V.sub.H 202
gaggtacagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgt
gcagcctctggattcactttcggtaacgcctggatgagctgggtccgccaggctccagggaaggg
gctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcaccc
gtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaaca
gcctgaaaaccgaggacacagccgtgtattactgtaccacagatcggaccgggtatagcatcag
ctggtctagttactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctct
agt 1H7 V.sub.H 203
gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgt
gcagcctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggg
gctggagtgggttggccgtattaaaagcacaactgatggtgggacaacagactacgctgcaccc
gtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaaca
gcctgaaaaccgaggacacagccgtgtattactgtaccacagatcggaccggatatagcatcag
ctggtctagttactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctct
agt 13H2 V.sub.H 204
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctg
tgcagcctctggatacaccttcagtacctatagcatgaactgggtccgccaggctccagggaagg
ggctggagtgggtctcatccattagtagtagtagtagttacagatattacgcagactcagtgaaggg
ccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgagtagcctgaga
gccgaggacacggctgtgtattactgtgcgagagaaggggtgtctggcagttcgccgtatagcat
cagctggtacgactactattacggtatggacgtctggggccaagggaccacggtcaccgtctcta
gt 2E7 V.sub.H 205
gaggtgcagctattggagtctgggggaggcttggtacagcctggggagtccctgagactctcctgt
gcagcctctgggttcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggg
gctggagtgggtctcagctattagtggtagtggtggtcgcacatactacgcagactccgtgaaggg
ccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaatagcctgagag
ccgaggacacggccgtatattactgtgcgaaagatcaaagggaggtagggccgtatagcagtg
gctggtacgactactactacggtatggacgtctggggccaagggaccacggtcaccgtctctagt
3C8 V.sub.H 206
caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctg
12E8 V.sub.H
tgcagcctctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggg
5F5 V.sub.H
gctggagtgggtggcagttatttcatatgatggaagtcatgaatcctatgcagactccgtgaagggc
cgattcaccatctccagagacatttccaagaacacgctgtatctgcaaatgaacagcctgagagc
tgaggacacggctgtgtatttctgtgcgagagagaggaaacgggttacgatgtctaccttatattact
acttctactacggtatggacgtctggggccaagggaccacggtcaccgtctctagt 4E4
V.sub.H 207
caggtgcagctggtggaatctgggggaggcgtggtccagcctgggaggtccctgagactctcctg
9D4 V.sub.H
tgcagcctctggattcaccttcagtagctttggcatgcactgggtccgccaggctccaggcaaggg
1E11 V.sub.H
gctggagtgggtggcagttatatcatttgatggaagtattaagtattctgtagactccgtgaagggcc
gattcaccatctccagagacaattcaaagaacacgctgtttctgcaaatgaacagcctgcgagcc
gaggacacggctgtgtattactgtgcgagagatcggctcaattactatgatagtagtggttattatca
catcaaatactacggtatggccgtctggggccaagggaccacggtcaccgtctctagt 12G8
V.sub.H 208
caggtgcagctggtggaatctgggggaggcgtggtccagcctgggaggtccctgagactctcctg
tgcagcctctggattcaccttcagtagctttggcatgcattgggtccgccaggctccaggcaaggg
gctggagtgggtggcagttatatcatttgatggaagtattaagtactctgtagactccgtgaagggcc
gattcacctctccagagacaattcaaagaacacgctgtttctgcaaatgaacagcctgcgagcc
gaggacacggctgtgtattactgtgcgagagatcggctcaattactatgatagtagtggttattatca
ctacaaatactacggtctggccgtctggggccaagggaccacggtcaccgtctctagt 4H6
V.sub.H 209
gaggtgcagctggtggagtctgggggaggcttggtaaagccagggcggtccctgagactctcct
gtacagcttctggattcacctttggtgattatgctatgagctggttccgccaggctccagggaagggg
ctggagtggataggtttcattagaagcagagcttatggtgggacaccagaatacgccgcgtctgtg
aaaggcagattcaccatctcaagagatgattccaaaaccatcgcctatctgcaaatgaacagcct
gaaaaccgaggacacagccgtgtatttctgtgctagaggacggggtattgcagctcgttgggact
actggggccagggaaccctggtcaccgtctctagt 32H7 V.sub.H 210
caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctg
tgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaagg
ggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagg
gccgattcatcatctccagagataaatccaagaacacgctgtatctgcaaatgaacagcctgaga
gccgaggacacggctgtgtattactgtgcgagagcggggggtatagcagcagctggcctctacta
ctactacggtatggacgtctggggccaagggaccacggtcaccgtctctagt 33E4 V.sub.H
211
caggtgcagttacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcagctg
cgctgtctatggtgggtccttcggtggttactactggagctggatccgccagcccccagggaaggg
gctggagtggattggggaaatcaatcatagtggaggcaccaagtacaacccgtccctcaagagt
cgagtcaccatatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgc
cgcggacacggctgtgtatttctgtgcgagaggcgatgtagtaggtttctttgactattggggccagg
gaaccctggtcaccgtctctagt
[0379] Table 9 shows the SEQ ID NOs of exemplary nucleic acid
sequences encoding complete heavy and light chains, as well as
heavy and light chain variable regions, of exemplary isolated
antigen-binding proteins, specifically, hCGRP R binding proteins,
disclosed herein.
TABLE-US-00014 TABLE 9 Exemplary HC, LC, V.sub.H and V.sub.L
Nucleic Acid Sequence SEQ ID NOs Variable Light Variable Heavy Full
Light Full Heavy Ref SEQ ID NO. SEQ ID NO. SEQ ID NO. SEQ ID NO 2E7
175 205 226 244 13H2 176 204 239 257 4H6 178 209 230 248 3C8 179
206 228 246 5F5 180 206 231 249 12E8 181 206 237 255 1E11 186 207
224 242 4E4 187 207 229 247 9D4 188 207 232 250 12G8 189 208 238
256 10E4 191 197 234 252 11D11 192 200 235 253 11H9 192 202 236 254
1H7 193 203 225 243 9F5 194 201 233 251 3B6 195 196 227 245 32H7
182 210 240 258 32H7 CS 183 210 241 258 32H8 185 198 33B5 177 199
33E4 184 211 34E3 190 212
[0380] Nucleic acids encoding certain antigen binding proteins, or
portions thereof (e.g., full length antibody, heavy or light chain,
variable domain, or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3)
may be isolated from B-cells of mice that have been immunized with
CGRP R or immunogenic components thereof, e.g., by immunizing with
full-length CGRP R (comprising both CRLR and RAMP1), with the
extracellular domain of CGRP R (comprising extracellular domains of
CRLR and RAMP1), with whole cells expressing CGRP R, with membranes
prepared from cells expressing CGRP R, with fusion proteins, e.g.,
Fc fusions, comprising CRLR, RAMP1 (or extracellular domains
thereof) fused to Fc, and other methods known in the art, for
example, as described in the Examples 1-3 herein. The nucleic acid
may be isolated by conventional procedures such as polymerase chain
reaction (PCR). Phage display is another example of a known
technique whereby derivatives of antibodies and other antigen
binding proteins may be prepared. In one approach, polypeptides
that are components of an antigen binding protein of interest are
expressed in any suitable recombinant expression system, and the
expressed polypeptides are allowed to assemble to form antigen
binding protein molecules.
[0381] The nucleic acids provided in Tables 7-9 are exemplary only.
Due to the degeneracy of the genetic code, each of the polypeptide
sequences listed. in Tables 2-5 or otherwise depicted herein are
also encoded by a large number of other nucleic acid sequences
besides those provided. One of ordinary skill in the art will
appreciate that the present application thus provides adequate
written description and enablement for each degenerate nucleotide
sequence encoding each antigen binding protein.
[0382] An aspect further provides nucleic acids that hybridize to
other nucleic acids (e.g., nucleic acids comprising a nucleotide
sequence listed in Table 7, Table 8, Table 9 and/or SEQ ID
NOs:224-258) under particular hybridization conditions. Methods for
hybridizing nucleic acids are well-known in the art. See, e.g.,
Current Protocols in Molecular Biology, John Wiley & Sons. N.Y.
(1989), 6.3.1-6.3.6. As defined herein, a moderately stringent
hybridization condition uses a prewashing solution containing
5.times. sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM
EDTA (pH 8.0), hybridization buffer of about 50% formamide,
6.times. SSC, and a hybridization temperature of 55.degree. C. (or
other similar hybridization solutions, such as one containing about
50% formamide, with a hybridization temperature of 42.degree. C.),
and washing conditions of 60.degree. C., in 0.5.times. SSC, 0.1%
SDS. A stringent hybridization condition hybridizes in 6.times. SSC
at 45.degree. C., followed by one or more washes in 0.1.times. SSC,
0.2% SDS at 68.degree. C. Furthermore, one of skill in the art can
manipulate the hybridization and/or washing conditions to increase
or decrease the stringency of hybridization such that nucleic acids
comprising nucleotide sequences that are at least 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98% or 99% identical to each other typically
remain hybridized to each other.
[0383] The basic parameters affecting the choice of hybridization
conditions and guidance for devising suitable conditions are set
forth by, for example, Sambrook, Fritsch, and Maniatis (2001,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., supra; and Current
Protocols in Molecular Biology, 1995, Ausubel et al., eds., John
Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be
readily determined by those having ordinary skill in the art based
on, e.g., the length and/or base composition of the nucleic
acid.
[0384] Changes can be introduced by mutation into a nucleic acid,
thereby leading to changes in the amino acid sequence of a
polypeptide (e.g., an antibody or antibody derivative) that it
encodes. Mutations can be introduced using an technique known in
the art. In one embodiment, one or more particular amino acid
residues are changed using, for example, a site-directed
mutagenesis protocol. In another embodiment, one or more randomly
selected residues is changed using, for example, a random
mutagenesis protocol. However it is made, a mutant polypeptide can
be expressed and screened for a desired property.
[0385] Mutations can be introduced into a nucleic acid without
significantly altering the biological activity of a polypeptide
that it encodes. For example, one can make nucleotide substitutions
leading to amino acid substitutions at non-essential amino acid
residues. Alternatively, one or more mutations can be introduced
into a nucleic acid that selectively changes the biological
activity of a polypeptide that it encodes. For example, the
mutation can quantitatively, or qualitatively change the biological
activity. Examples of quantitative changes include increasing,
reducing or eliminating the activity. Examples of qualitative
changes include changing the antigen specificity of an antibody. In
one embodiment, a nucleic acid encoding any antigen binding protein
described herein can be mutated to alter the amino acid sequence
using molecular biology techniques that are well-established in the
art.
[0386] Another aspect provides nucleic acid molecules that are
suitable for use as primers or hybridization probes for the
detection of nucleic acid sequences. A nucleic acid molecule can
comprise only a portion of a nucleic acid sequence encoding a
full-length polypeptide, for example, a fragment that can be used
as a probe or primer or a fragment encoding an active portion
(e.g., a CGRP R binding portion) of a polypeptide.
[0387] Probes based on the sequence of a nucleic acid can be used
to detect the nucleic acid or similar nucleic acids, for example,
transcripts encoding a polypeptide. The probe can comprise a label
group, e.g., a radioisotope, a fluorescent compound, an enzyme, or
an enzyme co-factor. Such probes can be used to identify a cell
that expresses the polypeptide.
[0388] Another aspect provides vectors comprising a nucleic acid
encoding a polypeptide or a portion thereof (e.g., a fragment
containing one or more CDRs or one or more variable region
domains). Examples of vectors include, but are not limited to,
plasmids, viral vectors, non-episomal mammalian vectors and
expression vectors, for example, recombinant expression vectors.
The recombinant expression vectors can comprise a nucleic acid in a
form suitable for expression of the nucleic acid in a host cell.
The recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operably linked to the nucleic acid sequence
to be expressed. Regulatory sequences include those that direct
constitutive expression of a nucleotide sequence in many types of
host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus
promoter and cytomegalovirus promoter), those that direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences, see, Voss et al.,
1986, Trends Biochem. Sci. 11: 287, Maniatis et al., 1987, Science
236:1237, incorporated by reference herein in their entireties),
and those that direct inducible expression of a nucleotide sequence
in response to particular treatment or condition (e.g., the
metallothionin promoter in mammalian cells and the tet-responsive
and/or streptomycin responsive promoter in both prokaryotic and
eukaryotic systems (see, id.). It will be appreciated by those
skilled in the art that the design of the expression vector can
depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors can be introduced into host cells to thereby
produce proteins or peptides, including fusion proteins or
peptides, encoded by nucleic acids as described herein.
[0389] Another aspect provides host cells into which a recombinant
expression vector has been introduced. A host cell can be any
prokaryotic cell (for example, E. coli) or eukaryotic cell (for
example, yeast, insect, or mammalian cells (e.g., CHO cells)).
Vector DNA can be introduced into prokaryotic or eukaryotic cells
via conventional transformation or transfection techniques. For
stable transfection of mammalian cells, it is known that, depending
upon the expression vector and transfection technique used, only a
small fraction of cells may integrate the foreign DNA into their
genome. In order to identify and select these integrants, a gene
that encodes a selectable marker (e.g., for resistance to
antibiotics) is generally introduced into the host cells along with
the gene of interest. Preferred selectable markers include those
which confer resistance to drugs, such as G418, hygromycin and
methotrexate. Cells stably transfected with the introduced nucleic
acid can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die), among other methods.
Preparing of Antigen Binding Proteins
[0390] Non-human antibodies that are provided can be, for example,
derived from any antibody-producing animal, such as mouse, rat,
rabbit, goat, donkey, or non-human primate (such as monkey (e.g.,
cynomolgus or rhesus monkey) or ape (e.g., chimpanzee)). Non-human
antibodies can be used, for instance, in in vitro cell culture and
cell-culture based applications, or any other application where an
immune response to the antibody does not occur or is insignificant,
can be prevented, is not a concern, or is desired. In certain
embodiments, the antibodies may be produced by immunizing animals
using methods known in the art, as described above and/or in
Examples 1-3 below. The examples describe the generation of anti
CGRP R antibodies using three different immunogen preparations (i)
whole cells expressing full-length versions of two major components
of CGRP R-RAMP1 and CRLR; (ii) membrane extracts from such cells;
and (iii) soluble CGRP R obtained by co-expressing and purifying
the N-terminal extracellular domains of CRLR and RAMP1. The
antibodies may be polyclonal, monoclonal, or may be synthesized in
host cells by expressing recombinant DNA. Fully human antibodies
may be prepared as described above by immunizing transgenic animals
containing human immunoglobulin loci or by selecting a phage
display library that is expressing a repertoire of human
antibodies.
[0391] The monoclonal antibodies (mAbs) can be produced by a
variety of techniques, including conventional monoclonal antibody
methodology, e.g., the standard somatic cell hybridization
technique of Kohler and Milstein, 1975, Nature 256:495.
Alternatively, other techniques for producing monoclonal antibodies
can be employed, for example, the viral or oncogenic transformation
of B-lymphocytes. One suitable animal system for preparing
hybridomas is the murine system, which is a very well established
procedure. Immunization protocols and techniques for isolation of
immunized splenocytes for fusion are known in the art and.
illustrative approaches are described in the Examples, below. For
such procedures, B cells from immunized mice are typically fused
with a suitable immortalized fusion partner, such as a murine
myeloma cell line. If desired, rats or other mammals besides can be
immunized instead of mice and B cells from such animals can be
fused with the murine myeloma cell line to form hybridomas.
Alternatively, a myeloma cell line from a source other than mouse
may be used. Fusion procedures for making hybridomas also are well
known.
[0392] The single chain antibodies that are provided may be formed
by linking heavy and light chain variable domain (Fv region)
fragments via an amino acid bridge (short peptide linker),
resulting in a single polypeptide chain. Such single-chain Fvs
(scFvs) may be prepared by fusing DNA encoding a peptide linker
between DNAs encoding the two variable domain polypeptides (V.sub.L
and V.sub.H). The resulting polypeptides can fold back on
themselves to form antigen-binding monomers, or they can form
multimers (e.g., dimers, trimers, or tetramers), depending on the
length of a flexible linker between the two variable domains (Kortt
et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng.
18:95-108). By combining different V.sub.L and V.sub.H-comprising
polypeptides, one can form multimeric scFvs that bind to different
epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40).
Techniques developed for the production of single chain antibodies
include those described in U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002,
Methods Mol Biol. 178:379-387. Single chain antibodies derived from
antibodies provided herein include, but are not limited to scFvs
comprising the variable domain combinations of the heavy and light
chain variable regions depicted in Table 3, or combinations of
light and heavy chain variable domains which include CDRs depicted
in Tables 4A and 4B.
[0393] Antibodies provided herein that are of one subclass can be
changed to antibodies from a different subclass using subclass
switching methods. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures, e.g., DNA
encoding the constant domain of an antibody of the desired isotype.
See, e.g., Lantto et al, 2002, Methods Mol. Biol. 178:301-316.
[0394] Accordingly, the antibodies that are provided include those
comprising, for example, the variable domain combinations
described, supra., having a desired isotype (for example, IgA,
IgG1, IgG2, IgG3, IgG4, IgE, and IgD) as well as Fab or
F(ab').sub.2 fragments thereof. Moreover, if an IgG4 is desired, it
may also be desired to introduce a point mutation (CPSCP->CPPCP)
in the hinge region as described in Bloom et al., 1997, Protein
Science 6:407, incorporated by reference herein) to alleviate a
tendency to form intra-H chain disulfide bonds that can lead to
heterogeneity in the IgG4 antibodies.
[0395] Moreover, techniques for deriving antibodies having
different properties (i.e., varying affinities for the antigen to
which they bind) are also known. One such technique, referred to as
chain shuffling, involves displaying immunoglobulin variable domain
gene repertoires on the surface of filamentous bacteriophage, often
referred to as phage display. Chain shuffling has been used to
prepare high affinity antibodies to the hapten
2-phenyloxazol-5-one, as described by Marks et al., 1992,
BioTechnology 10:779.
[0396] Conservative modifications may be made to the heavy and
light chain variable regions described in Table 3, or the CDRs
described in Tables 4A and 4B (and corresponding modifications to
the encoding nucleic acids) to produce a CGRP R binding protein
having certain desirable functional and biochemical
characteristics. Methods for achieving such modifications are
described above.
[0397] CGRP antigen binding proteins may be further modified in
various ways. For example, if they are to be used for therapeutic
purposes, they may be conjugated with polyethylene glycol
(pegylated) to prolong the serum half-life or to enhance protein
delivery. Alternatively. the V region of the subject antibodies or
fragments thereof may be fused with the Fc region of a different
antibody molecule. The Fc region used for this purpose may be
modified so that it does not bind complement, thus reducing the
likelihood of inducing cell lysis in the patient when the fusion
protein is used as a therapeutic agent. In addition, the subject
antibodies or functional fragments thereof may be conjugated with
human serum albumin to enhance the serum half-life of the antibody
or antigen binding fragment thereof. Another useful fusion partner
for the antigen binding proteins or fragments thereof is
transthyretin (TTR). TTR has the capacity to form a tetramer, thus
an antibody-TTR fusion protein can form a mutivalent antibody which
may increase its binding avidity.
[0398] Alternatively, substantial modifications in the functional
and/or biochemical characteristics of the antigen binding proteins
described herein may be achieved by creating substitutions in the
amino acid sequence of the heavy and light chains that differ
significantly in their effect on maintaining (a) the structure of
the molecular backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the
bulkiness of the side chain. A "conservative amino acid
substitution" may involve a substitution of a native amino acid
residue with a nonnative residue that has little or no effect on
the polarity or charge of the amino acid residue at that position.
See, Table 4, supra. Furthermore, any native residue in the
polypeptide may also be substituted with alanine, as has been
previously described for alanine scanning mutagenesis.
[0399] Amino acid substitutions (whether conservative or
non-conservative) of the subject antibodies can be implemented by
those skilled in the art by applying routine techniques. Amino acid
substitutions can be used to identify important residues of the
antibodies provided herein, or to increase or decrease the affinity
of these antibodies for human CGRP R or for modifying the binding
affinity of other antigen-binding proteins described herein.
Methods of Expressing Antigen Binding Proteins
[0400] Expression systems and constructs in the form of plasmids,
expression vectors, transcription or expression cassettes that
comprise at least one polynucleotide as described above are also
provided herein, as well host cells comprising such expression
systems or constructs.
[0401] The antigen binding proteins provided herein may be prepared
by any of a number of conventional techniques. For example, CGRP R
antigen binding proteins may be produced by recombinant expression
systems, using any technique known in the art. See, e.g.,
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Kennet et al. (eds.) Plenum Press, New York (1980); and
Antibodies; A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988).
[0402] Antigen binding proteins can be expressed in hybridoma cell
lines (e.g., in particular antibodies may be expressed in
hybridomas) or in cell lines other than hybridomas. Expression
constructs encoding the antibodies can be used to transform a
mammalian, insect or microbial host cell. Transformation can be
performed using any known method for introducing polynucleotides
into a host cell, including, for example packaging the
polynucleotide in a virus or bacteriophage and transducing a host
cell with the construct by transfection procedures known in the
art, as exemplified by U.S. Pat. No. 4,399,216; No. 4,912,040; No.
4,740,461; No. 4,959,455. The optimal transformation procedure used
will depend upon which type of host cell is being transformed.
Methods for introduction of heterologous polynucleotides into
mammalian cells are well known in the art and include, but are not
limited to, dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, mixing nucleic acid with positively-charged lipids, and
direct microinjection of the DNA into nuclei.
[0403] Recombinant expression constructs typically comprise a
nucleic acid molecule encoding a polypeptide comprising one or more
of the following: one or more CDRs provided herein; a light chain
constant region; a light chain variable region; a heavy chain
constant region (e.g., C.sub.H1, C.sub.H2 and/or C.sub.H3); and/or
another scaffold portion of a CGRP R antigen binding protein. These
nucleic acid sequences are inserted into an appropriate expression
vector using standard ligation techniques. In one embodiment, the
heavy or light chain constant region is appended to the C-terminus
of the anti-CGRP R-specific heavy or light chain variable region
and is ligated into an expression vector. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery,
permitting amplification and/or expression of the gene can occur).
In some embodiments, vectors are used that employ protein-fragment
complementation assays using protein reporters, such as
dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964,
which is hereby incorporated by reference). Suitable expression
vectors can be purchased, for example, from Invitrogen Life
Technologies or BD Biosciences (formerly "Clontech"). Other useful
vectors for cloning and expressing the antibodies and fragments
include those described in Bianchi and McGrew, 2003, Biotech.
Biotechnol. Bioeng. 84:439-44, which is hereby incorporated by
reference. Additional suitable expression vectors are discussed,
for example, in Methods Enzymol., vol. 185 (D. V. Goeddel, ed.),
1990, New York: Academic Press.
[0404] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0405] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the CGRP R binding protein coding sequence; the
oligonucleotide sequence encodes polyHis (such as hexaHis), or
another "tag" such as FLAG.RTM., HA (hemaglutinin influenza virus),
or myc, for which commercially available antibodies exist. This tag
is typically fused to the polypeptide upon expression of the
polypeptide, and can serve as a means for affinity purification or
detection of the CGRP R binding protein from the host cell.
Affinity purification can be accomplished, for example, by column
chromatography using antibodies against the tag as an affinity
matrix. Optionally, the tag can subsequently be removed from the
purified CGRP R binding protein by various means such as using
certain peptidases for cleavage.
[0406] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source),
synthetic or native. As such, the source of a flanking sequence may
be any prokaryotic or eukaryotic organism, any vertebrate or
invertebrate organism, or any plant, provided that the flanking
sequence is functional in, and can be activated by, the host cell
machinery.
[0407] Flanking sequences useful in the vectors may be obtained by
any of several methods well known in the art. Typically, flanking
sequences useful herein will have been previously identified by
mapping, and/or by restriction endonuelease digestion and can thus
be isolated from the proper tissue source using the appropriate
restriction endonucleases. In some cases, the full nucleotide
sequence of a flanking sequence may be known. Here, the flanking
sequence may be synthesized using the methods described herein for
nucleic acid synthesis or cloning.
[0408] Whether all or only a portion of the flanking sequence is
known, it may be obtained using polymerase chain reaction (PCR)
and/or by screening a genomic library with a suitable probe such as
an oligonucleotide and/or flanking sequence fragment from the same
or another species. Where the flanking sequence is not known, a
fragment of DNA containing a flanking sequence may be isolated from
a larger piece of DNA that may contain, for example, a coding
sequence or even another gene or genes. Isolation may be
accomplished by restriction endonuclease digestion to produce the
proper DNA fragment followed by isolation using agarose
purification, Qiagen.RTM. column chromatography (Chatsworth,
Calif.), or other methods known to the skilled artisan. The
selection of suitable enzymes to accomplish this purpose will be
readily apparent to one of ordinary skill in the art.
[0409] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell. If
the vector of choice does not contain an origin of replication
site, one may be chemically synthesized based on a known sequence,
and ligated into the vector. For example, the origin of replication
from the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is
suitable for most gram-negative bacteria, and various viral origins
(e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV),
or papillomaviruses such as HPV or BPV) are useful for cloning
vectors in mammalian cells. Generally, the origin of replication
component is not needed for mammalian expression vectors (for
example, the SV40 origin is often used only because it also
contains the virus early promoter).
[0410] A transcription termination sequence is typically located 3'
to the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly-T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0411] A selectable marker gene encodes a protein necessary for the
survival and growth of a host cell grown in a selective culture
medium. Typical selection marker genes encode proteins that (a)
confer resistance to antibiotics or other toxins, e.g., ampicillin,
tetracycline, or kanamycin for prokaryotic host cells; (b)
complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex or defined media.
Specific selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene.
Advantageously, a neomycin resistance gene may also be used for
selection in both prokaryotic and. eukaryotic host cells.
[0412] Other selectable genes may be used to amplify the gene that
will be expressed. Amplification is the process wherein genes that
are required for production of a protein critical for growth or
cell survival are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and promoterless thymidine kinase genes. Mammalian
cell transformants are placed under selection pressure wherein only
the transformants are uniquely adapted to survive by virtue of the
selectable gene present in the vector. Selection pressure is
imposed by culturing the transformed cells under conditions in
which the concentration of selection agent in the medium is
successively increased, thereby leading to the amplification of
both the selectable gene and the DNA that encodes another gene,
such as an antigen binding protein that binds to CGRP R. As a
result, increased quantities of a polypeptide such as an antigen
binding protein are synthesized from the amplified DNA.
[0413] A ribosome-binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of the polypeptide to be expressed.
[0414] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various pre- or pro-sequences to improve glycosylation or yield.
For example, one may alter the peptidase cleavage site of a
particular signal peptide, or add prosequences, which also may
affect glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein),
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the amino-terminus.
Alternatively, use of some enzyme cleavage sites may result in a
slightly truncated form of the desired polypeptide, if the enzyme
cuts at such area within the mature polypeptide.
[0415] Expression and cloning will typically contain a promoter
that is recognized by the host organism and operably linked to the
molecule encoding a CGRP R binding protein. Promoters are
untranscribed sequences located upstream (i.e., 5') to the start
codon of a structural gene (generally within about 100 to 1000 bp)
that control transcription of the structural gene. Promoters are
conventionally grouped into one of two classes: inducible promoters
and constitutive promoters. Inducible promoters initiate increased
levels of transcription from DNA under their control in response to
some change in culture conditions, such as the presence or absence
of a nutrient or a change in temperature. Constitutive promoters,
on the other hand, uniformly transcribe a gene to which they are
operably linked, that is, with little or no control over gene
expression. A large number of promoters, recognized by a variety of
potential host cells, are well known. A suitable promoter is
operably linked to the DNA encoding heavy chain or light chain
comprising a CGRP R binding protein by removing the promoter from
the source DNA by restriction enzyme digestion and inserting the
desired promoter sequence into the vector.
[0416] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B
virus, and Simian Virus 40 (SV40). Other suitable mammalian
promoters include heterologous mammalian promoters, for example,
heat-shock promoters and the actin promoter.
[0417] Additional promoters which may be of interest include, but
are not limited to: SV40 early promoter (Benoist and Chambon, 1981,
Nature 290:304-310); CMV promoter (Thornsen et al., 1984, Proc.
Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3'
long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980,
Cell 22:787-797); herpes thymidine kinase promoter (Wagner et al,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-1445); promoter and
regulatory sequences from the metallothionine gene (Prinster et
al., 1982, Nature 296:39-42); and prokaryotic promoters such as the
beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.
Acad. Sci. U.S.A. 75:3727-3731); or the tac promoter (DeBoer et
al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Also of
interest are the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: the elastase I gene control region that is
active in pancreatic acinar cells (Swift et al., 1984, Cell
38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.
Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the
insulin gene control region that is active in pancreatic beta cells
(Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene
control region that is active in lymphoid cells (Grosschedl et al.,
1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;
Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); the mouse
mammary tumor virus control region that is active in testicular,
breast, lymphoid and mast cells (Leder et al., 1986, Cell
45:485-495); the albumin gene control region that is active in
liver (Pinkert et 1987, Genes and Devel. 1 :268-276); the
alpha-feto-protein gene control region that is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 253:53-58); the alpha 1-antitrypsin gene control
region that is active in liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171); the beta-globin gene control region that is
active in myeloid cells (Mogram et al., 1985, Nature 315:338-340;
Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene
control region that is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2
gene control region that is active in skeletal muscle (Sani, 1985,
Nature 314:283-286); and the gonadotropic releasing hormone gene
control region that is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378).
[0418] An enhancer sequence may be inserted into the vector to
increase transcription of DNA encoding light chain or heavy chain
comprising a human CGRP R binding protein by higher eukaryotes.
Enhancers are cis-acting elements of DNA, usually about 10-300 bp
in length, that act on the promoter to increase transcription.
Enhancers are relatively orientation and position independent,
having been found at positions both 5' and 3' to the transcription
unit. Several enhancer sequences available from mammalian genes are
known (e.g., globin, elastase, albumin, alpha-feto-protein and
insulin). Typically, however, an enhancer from a virus is used. The
SV40 enhancer, the cytomegalovirus early promoter enhancer, the
polyoma enhancer, and adenovirus enhancers known in the art are
exemplary enhancing elements for the activation of eukaryotic
promoters. While an enhancer may be positioned in the vector either
5' or 3' to a coding sequence, it is typically located at a site 5'
from the promoter. A sequence encoding an appropriate native or
heterologous signal sequence (leader sequence or signal peptide)
can be incorporated into an expression vector, to promote
extracellular secretion of the antibody. The choice of signal
peptide or leader depends on the type of host cells in which the
antibody is to be produced, and a heterologous signal sequence can
replace the native signal sequence. Examples of signal peptides
that are functional in mammalian host cells include the following:
the signal sequence for interleukin-7 (IL-7) described in U.S. Pat.
No. 4,965,195; the signal sequence for interleukin-2 receptor
described in Cosman et al., 1984, Nature 312:768; the interleukin-4
receptor signal peptide described in EP Patent No. 0367 566; the
type I interleukin-1 receptor signal peptide described in U.S. Pat.
No. 4,968,607; the type II interleukin-1 receptor signal peptide
described in EP Patent No. 0 460 846.
[0419] The expression vectors that are provided may be constructed
from a starting vector such as a commercially available vector.
Such vectors may or may not contain all of the desired flanking
sequences. Where one or more of the flanking sequences described
herein are not already present in the vector, they may be
individually obtained and ligated into the vector. Methods used for
obtaining each of the flanking sequences are well known to One
skilled in the art.
[0420] After the vector has been constructed and a nucleic acid
molecule encoding light chain, a heavy chain, or a light chain and
a heavy chain comprising a CGRP R antigen binding sequence has been
inserted into the proper site of the vector, the completed vector
may be inserted into a suitable host cell for amplification and/or
polypeptide expression. The transformation of an expression vector
for an antigen-binding protein into a selected host cell may be
accomplished by well known methods including transfection,
infection, calcium phosphate co-precipitation, electroporation,
microinjection, lipofection, DEAE-dextran mediated transfection, or
other known techniques. The method selected will in part be a
function of the type of host cell to be used. These methods and
other suitable methods are well known to the skilled artisan, and
are set forth, for example, in Sambrook et al., 2001, supra.
[0421] A host cell, when cultured under appropriate conditions,
synthesizes an antigen binding protein that can subsequently be
collected from the culture medium (if the host cell secretes it
into the medium) or directly from the host cell producing it (if it
is not secreted). The selection of an appropriate host cell will
depend upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
[0422] Mammalian cell lines available as hosts for expression are
well known in the art and include, but are not limited to,
immortalized cell lines available from the American Type Culture
Collection (ATCC), including but not limited to Chinese hamster
ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), and a number of other cell lines. In certain
embodiments, cell lines may be selected through determining which
cell lines have high expression levels and constitutively produce
antigen binding proteins with CGRP R binding properties. In another
embodiment, a cell line from the B cell lineage that does not make
its own antibody but has a capacity to make and secrete a
heterologous antibody can be selected.
Use of Human CGRP Antigen Binding Proteins for Diagnostic and
Therapeutic Purposes
[0423] Antigen binding proteins are useful for detecting CGRP R in
biological samples and identification of cells or tissues that
produce CGRP R. For instance, the CGRP R antigen binding proteins
can be used in diagnostic assays, e.g., binding assays to detect
and/or quantify CGRP R expressed in a tissue or cell. Antigen
binding proteins that specifically bind to CGRP R can also be used
in treatment of diseases related to CGRP R in a patient in need
thereof. In addition, CGRP R antigen binding proteins can be used
to inhibit CGRP R from forming a complex with its ligand CGRP,
thereby modulating the biological activity of CGRP R in a cell or
tissue. Examples of activities that can be modulated include, but
are not limited to, inhibiting vasodialation and/or decrease
neurogenic inflammation. Antigen binding proteins that bind to CGRP
R thus can modulate and/or block interaction with other binding
compounds and as such may have therapeutic use in ameliorating
diseases related to CGRP R.
Indications
[0424] A disease or condition associated with human CGRP R includes
any disease or condition whose onset in a patient is caused by, at
least in part, the interaction of CGRP R with its ligand, CGRP. The
severity of the disease or condition can also be increased or
decreased by the interaction of CGRP R with CGRP. Examples of
diseases and conditions that can be treated with the antigen
binding proteins described herein include headaches, such as
cluster headaches, migraine, including migraine headaches, chronic
pain, type II diabetes mellitus, inflammation, e.g., neurogenic
inflammation, cardiovascular disorders, and hemodynamic derangement
associated with endotoxemia and sepsis.
[0425] In particular, antigen binding proteins described herein can
be used to treat migraine, either as an acute treatment commencing
after a migraine attack has commenced, and/or as a prophylactic
treatment administered, e.g., daily, weekly, biweekly, monthly,
bimonthly, biannually, etc.) to prevent or reduce the frequency
and/or severity of symptoms, e.g., pain symptoms, associated with
migraine attacks.
Diagnostic Methods
[0426] The antigen binding proteins described herein can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or conditions associated with CGRP R. Also provided are methods
for the detection of the presence of CGRP R in a sample using
classical immunohistological methods known to those of skill in the
art (e.g., Tijssen, 1993, Practice and Theory of Enzyme
Immunoassays, Vol 15 (Eds R. H. Burdon and P. H. van Knippenberg,
Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual
of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al.,
1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell
Biol. 105:3087-3096). The detection of CGRP R can be performed in
vivo or in vitro.
[0427] Diagnostic applications provided herein include use of the
antigen binding proteins to detect expression of CGRP R and binding
of the ligands to CGRP R. Examples of methods useful in the
detection of the presence of CGRP R include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA).
[0428] For diagnostic applications, the antigen binding protein
typically will be labeled with a detectable labeling group.
Suitable labeling groups include, but are not limited to, the
following: radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C,
.sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I,
.sup.131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide
phosphors), enzymatic groups (e.g., horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase),
chemiluminescent groups, biotinyl groups, or predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags). In some
embodiments, the labeling group is coupled to the antigen binding
protein via spacer arms of various lengths to reduce potential
steric hindrance. Various methods for labeling proteins are known
in the art and may be used.
[0429] In another aspect, an antigen binding protein can be used to
identify a cell or cells that express CGRP R. In a specific
embodiment, the antigen binding protein is labeled with a labeling
group and the binding of the labeled antigen binding protein to
CGRP R is detected. In a further specific embodiment, the binding
of the antigen binding protein to CGRP R detected in vivo. In a
further specific embodiment, the CGRP R antigen binding protein is
isolated and measured using techniques known in the art. See, for
example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor (ed. 1991 and periodic supplements);
John E. Coligan, ed., 1993, Current Protocols in Immunology New
York: John Wiley & Sons.
[0430] Another aspect provides for detecting the presence of a test
molecule that competes for binding to CGRP R with the antigen
binding proteins provided. An example of one such assay would
involve detecting the amount of free antigen binding protein in a
solution containing an amount of CGRP R in the presence or absence
of the test molecule. An increase in the amount of free antigen
binding protein (i.e., the antigen binding protein not bound to
CGRP R) would indicate that the test molecule is capable of
competing for CGRP R binding with the antigen binding protein. In
one embodiment, the antigen binding protein is labeled with a
labeling group. Alternatively, the test molecule is labeled and the
amount of free test molecule is monitored in the presence and
absence of an antigen binding protein.
Methods of Treatment: Pharmaceutical Formulations, Routes of
Administration
[0431] Methods of using the antigen binding proteins are also
provided. In some methods, an antigen binding protein is provided
to patient. The antigen binding protein inhibits binding of CGRP to
human CGRP R.
[0432] Pharmaceutical compositions that comprise a therapeutically
effective amount of one or a plurality of the antigen binding
proteins and a pharmaceutically acceptable diluent, carrier,
solubilizer, emulsifier, preservative, and/or adjuvant are also
provided. In addition, methods of treating a patient, e.g., for
migraine, by administering such pharmaceutical composition are
included. The term "patient" includes human patients.
[0433] Acceptable formulation materials are nontoxic to recipients
at the dosages and concentrations employed. In specific
embodiments, pharmaceutical compositions comprising a
therapeutically effective amount of human CGRP R antigen binding
proteins are provided.
[0434] In certain embodiments, acceptable formulation materials
preferably are nontoxic to recipients at the dosages and
concentrations employed. In certain embodiments, the pharmaceutical
composition may contain formulation materials for modifying,
maintaining or preserving, for example, the pH, osmolarity,
viscosity, clarity, color, isotonicity, odor, sterility, stability,
rate of dissolution or release, adsorption or penetration of the
composition. In such embodiments, suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine or lysine); antimicrobials;
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates or other organic acids); bulking agents (such
as mannitol or glycine); chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES,
18'' Edition, (A. R., Genrmo, ed.), 1990, Mack Publishing
Company.
[0435] In certain embodiments, the optimal pharmaceutical
composition will be determined by one skilled in the art depending
upon, for example, the intended route of administration, delivery
format and desired dosage. See, for example, REMINGTON'S
PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such
compositions may influence the physical state, stability, rate of
in vivo release and rate of in vivo clearance of the antigen
binding proteins disclosed. In certain embodiments, the primary
vehicle or carrier in a pharmaceutical composition may be either
aqueous or non-aqueous in nature. For example, a suitable vehicle
or carrier may be water for injection, physiological saline
solution or artificial cerebrospinal fluid, possibly supplemented
with other materials common in compositions for parenteral
administration. Neutral buffered saline or saline mixed with serum
albumin are further exemplary vehicles. In specific embodiments,
pharmaceutical compositions comprise Tris buffer of about pH
7.0-8.5, or acetate buffer of about pH 4.0-5,5, and may further
include sorbitol or a suitable substitute. In certain embodiments,
human CGRP R antigen binding protein compositions may be prepared
for storage by mixing the selected composition having the desired
degree of purity with optional formulation agents (REMINGTON'S
PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake
or an aqueous solution. Further, in certain embodiments, the human
CGRP R antigen binding protein may be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0436] The pharmaceutical compositions can be selected for
parenteral delivery. Alternatively, the compositions may be
selected for inhalation or for delivery through the digestive
tract, such as orally. Preparation of such pharmaceutically
acceptable compositions is within the skill of the art.
[0437] The formulation components are present preferably in
concentrations that are acceptable to the site of administration.
In certain embodiments, buffers are used to maintain the
composition at physiological pH or at a slightly lower pH,
typically within a pH range of from about 5 to about 8.
[0438] When parenteral administration is contemplated, the
therapeutic compositions may be provided in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising
the desired human CGRP R binding protein in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which the human CGRP R
antigen binding protein is formulated as a sterile, isotonic
solution, properly preserved. In certain embodiments, the
preparation can involve the formulation of the desired molecule
with an agent, such as injectable microspheres, bio-erodible
particles, polymeric compounds (such as polylactic acid or
polyglycolic acid), beads or liposomes, that may provide controlled
or sustained release of the product which can be delivered via
depot injection. In certain embodiments, hyaluronic acid may also
be used, having the effect of promoting sustained duration in the
circulation. In certain embodiments, implantable drug delivery
devices may be used to introduce the desired antigen binding
protein.
[0439] Certain pharmaceutical compositions are formulated for
inhalation. In some embodiments, human CGRP R antigen binding
proteins are formulated as a dry, inhalable powder. In specific
embodiments, human CGRP R antigen binding protein inhalation
solutions may also be formulated with a propellant for aerosol
delivery. In certain embodiments, solutions may be nebulized.
Pulmonary administration and formulation methods therefore are
further described in International Patent Application No.
PCT/US94/001875, which is incorporated by reference and describes
pulmonary delivery of chemically modified proteins. Some
formulations can be administered orally. Human CGRP R antigen
binding proteins that are administered in this fashion can be
formulated with or without carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. In
certain embodiments, a capsule may be designed to release the
active portion of the formulation at the point in the
gastrointestinal tract when bioavailability is maximized and
pre-systemic degradation is minimized. Additional agents can be
included to facilitate absorption of the human CGRP R antigen
binding protein. Diluents, flavorings, low melting point waxes,
vegetable oils, lubricants, suspending agents, tablet
disintegrating agents, and binders may also be employed.
[0440] Some pharmaceutical compositions comprise an effective
quantity of one or a plurality of human CGRP R antigen binding
proteins in a mixture with non-toxic excipients that are suitable
for the manufacture of tablets. By dissolving the tablets in
sterile water, or another appropriate vehicle, solutions may be
prepared in unit-dose form. Suitable excipients include, but are
not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding
agents, such as starch, gelatin, or acacia; or lubricating agents
such as magnesium stearate, stearic acid, or talc.
[0441] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving human
CGRP R antigen binding proteins in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art. See,
for example, International Patent Application No. PCT/US93/00829,
which is incorporated by reference and describes controlled release
of porous polymeric microparticles for delivery of pharmaceutical
compositions. Sustained-release preparations may include
semipermeable polymer matrices in the form of shaped articles,
e.g., films, or microcapsules. Sustained release matrices may
include polyesters, hydrogels, polylactides (as disclosed in U.S.
Pat. No. 3,773,919 and European Patent Application Publication No.
EP 058481, each of which is incorporated by reference), copolymers
of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,
1983, Biopolymers 2:547-556), poly (2-hydroxyethyl-inethacrylate)
(Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer,
1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et
al., 1981, supra) or poly-D(-)-3-hydroxybutyric acid (European
Patent Application Publication No. EP 133,988). Sustained release
compositions may also include liposomes that can be prepared by any
of several methods known in the art. See, e.g., Eppstein et al.,
1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent
Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949,
incorporated by reference.
[0442] Pharmaceutical compositions used for in viva administration
are typically provided as sterile preparations, Sterilization can
be accomplished by filtration through sterile filtration membranes.
When the composition is lyophilized, sterilization using this
method may be conducted either prior to or following lyophilization
and reconstitution. Compositions for parenteral administration can
be stored in lyophilized form or in a solution. Parenteral
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0443] In certain embodiments, cells expressing a recombinant
antigen binding protein as disclosed herein is encapsulated for
delivery (see, Invest. Ophthalmol Vis Sci 43:3292-3298, 2002 and
Proc. Natl. Acad. Sciences 103:3896-3901, 2006).
[0444] In certain formulations, an antigen binding protein has a
concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml,
50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml or 150
mg/ml. Some formulations contain a buffer, sucrose and polysorbate.
An example of a formulation is one containing 50-100 mg/ml of
antigen binding protein, 5-20 nM sodium acetate, 5-10% w/v sucrose,
and 0.002-0.008% w/v polysorbate. Certain, formulations, for
instance, contain 65-75 mg/ml of an antigen binding protein in 9-11
mM sodium acetate buffer, 8-10% w/v sucrose, and 0.005-0.006% w/v
polysorbate. The pH of certain such formulations is in the range of
4.5-6. Other formulations have a pH of 5.0-5.5 (e.g., pH of 5.0,
5.2 or 5.4).
[0445] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, crystal, or as a dehydrated or lyophilized powder.
Such formulations may be stored either in a ready-to-use form or in
a form (e.g., lyophilized) that is reconstituted prior to
administration. Kits for producing a single-dose administration
unit are also provided. Certain kits contain a first container
having a dried protein and a second container having an aqueous
formulation. In certain embodiments, kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes) are provided. The therapeutically effective amount of
a human CGRP R antigen binding protein-containing pharmaceutical
composition to be employed will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
vary depending, in part, upon the molecule delivered, the
indication for which the human CGRP R antigen binding protein is
being used, the route of administration, and the size (body weight,
body surface or organ size) and/or condition (the age and general
health) of the patient. In certain embodiments, the clinician may
titer the dosage and modify the route of administration to obtain
the optimal therapeutic effect.
[0446] A typical dosage may range from about 1 .mu.g/kg to up to
about 30 mg/kg or more, depending on the factors mentioned above.
In specific embodiments, the dosage may range from 10 .mu.g/kg up
to about 30 mg/kg, optionally from 0.1 mg/kg up to about 30 mg/kg,
alternatively from 0.3 mg/kg up to about 20 mg/kg. In some
applications, the dosage is from 0.5 mg/kg to 20 mg/kg. In some
instances, an antigen binding protein is dosed at 0.3 mg/kg,
0.5mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosage
schedule in some treatment regimes is at a dose of 0.3 mg/kg qW,
0.5 mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg
qW.
[0447] Dosing frequency will depend upon the pharmacokinetic
parameters of the particular human CGRP R antigen binding protein
in the formulation used. Typically, a clinician administers the
composition until a dosage is reached that achieves the desired
effect. The composition may therefore be administered as a single
dose, or as two or more doses (which may or may not contain the
same amount of the desired molecule over time, or as a continuous
infusion via an implantation device or catheter. Appropriate
dosages may be ascertained through use of appropriate dose-response
data. In certain embodiments, the antigen binding proteins can be
administered to patients throughout an extended time period.
Chronic administration of an antigen binding protein minimizes the
adverse immune or allergic response commonly associated with
antigen binding proteins that are not fully human, for example an
antibody raised against a human antigen in a non-human animal, for
example, a non-fully human antibody or non-human antibody produced
in a non-human species.
[0448] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally, through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intra-ocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems or by implantation devices. In certain
embodiments, the compositions may be administered by bolus
injection or continuously by infusion, or by implantation
device.
[0449] The composition also may be administered locally via
implantation of a membrane, sponge or another appropriate material
onto which the desired molecule has been absorbed or encapsulated.
In certain embodiments, where an implantation device is used, the
device may be implanted into any suitable tissue or organ, and
delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0450] It also may be desirable to use human CGRP R antigen binding
protein pharmaceutical compositions ex vivo. In such instances,
cells, tissues or organs that have been removed from the patient
are exposed to human CGRP R antigen binding protein pharmaceutical
compositions after which the cells, tissues and/or organs are
subsequently implanted back into the patient.
[0451] In particular, human CGRP R antigen binding proteins can be
delivered by implanting certain cells that have been genetically
engineered, using methods such as those described herein, to
express and secrete the polypeptide. In certain embodiments, such
cells may be animal or human cells, and may be autologous,
heterologous, or xenogeneic. In certain embodiments, the cells may
be immortalized. In other embodiments, in order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. In further
embodiments, the encapsulation materials are typically
biocompatible, semi-permeable polymeric enclosures or membranes
that allow the release of the protein product(s) but prevent the
destruction of the cells by the patient's immune system or by other
detrimental factors from the surrounding tissues.
[0452] The following examples, including the experiments conducted
and the results achieved, are provided for illustrative purposes
only and are not to be construed as limiting the scope of the
appended claims.
EXAMPLE 1
Generation of CGRP Receptor as Antigens
A. Molecular Cloning of Human CRLR and RAMP1
[0453] Human CRLR cDNA (GenBank Accession No. U17473; SEQ ID NO:1)
and RAMP1 cDNA (GenBank Accession No. AJ001014; SEQ ID NO:3) were
cloned into the mammalian cell expression vectors pcDNA3.1-Zeo and
pcDNA3.1-Hyg (Invitrogen, Carlsbad, Calif.), respectively, for
transfections of HEK 293EBNA cells (Invitrogen) as described below.
The hCRLR cDNA and hRAMP1 cDNA were also cloned into the
pDSR.alpha.24 vector (Kim, H. Y. et al. J. Inv. Derm. Symp. Proc.
(2007) 12: 48-49) for transfections of AM-1 CHO cells (U.S. Pat.
No. 6,210,924).
B. Stably-Transfected Cell Lines
[0454] 1. Stable Expression of Human CGRP R in 293EBNA Cells
[0455] HEK 293EBNA cells (available from ATCC or Invitrogen) were
seeded at a density of 1.5.times.10.sup.6 cells per 100 mm dish.
After 24 hours, the cells were co-transfected with 6 .mu.g
linearized DNAs of huRAMP1/pcDNA3.1-Hyg and huCRLR/pcDNA3.1-Zeo
with FuGene6 (Invitrogen, Carlsbad, Calif.) following instructions
supplied by Invitrogen. After two days, the cells were trypsinized
and subcultured into growth medium containing 400 .mu.g/ml
hygromycin +250 .mu.g/ml zeocin. After two weeks, the resulting
drug resistant colonies were trypsinized and combined into pools.
The pools were subjected to four rounds of FACS sorting an Alexa
647-labeled CGRP.sub.8-37peptide analog (described below). The
highest 5% of expressing cells were collected at each round.
[0456] 2. Stable Expression of Human CGRP R in AM-1 CHO Cells
[0457] AM-1 CHO cells (a serum-free growth media-adapted variant
from the CHO DHFR-deficient cell line described in Urlaub and
Chasin, Proc. Natl. Acad. Sci. 77, 4216 (1980), were seeded at
1.5.times.10.sup.6 cells per 100 mm. dish. After 24 hours, the
cells were co-transfected with linearized 4 .mu.g DNAs each of
pDSR.alpha.24/huRAMP1 and pDSR.alpha.24/huCRLR with FuGene6
(Invitrogen, Carlsbad, Calif.) following instructions supplied by
Invitrogen. The transfected cells were trypsinized 2 days after
transfection and seeded into CHO DHFR selective growth medium
containing 10% dialyzed FBS and without hypoxanthine/thymidine
supplement. After 2 weeks, the resulting transfected colonies were
trypsinized and pooled. The pools were subjected to FACS sorting
analysis.
[0458] 3. Stable Expression of Human Adrenomedullin (AM1) in HEK
293EBNA Cells
[0459] 293EBNA cells were seeded in 100 mm dishes at
1.5.times.10.sup.6 cells/dish DMEM (high glucose)+5% FBS+1% MEM
non-essential amino acids+1% sodium pyruvate. The following day the
cells were co-transfected using FuGENE 6 transfection reagent
(Roche) with pcDNA3.1/zeocin/huCRLR plus
pcDNA3.1/hygromycin/huRAMP2. Both DNA constructs were linearized
with FspI. After 48 hours the cells were subcultured into 100 mm
dishes at 3 cell densities (8.times.10.sup.5, 3.2.times.10.sup.5,
and 8.times.10.sup.4 cells/dish) in growth medium containing 200
.mu.g/ml zeocin. The medium was changed twice weekly. After one
week the plates were fed with medium containing 200 .mu.g/ml
hygromycin+200 .mu.g/ml zeocin. After two weeks, 96 colonies were
isolated with cloning rings. The remaining colonies were collected
into a single pool culture. The clones and pools were assayed for
their response to stimulation by receptor agonist or forskolin.
Several clones showed a good response, and one was selected for use
in subsequent experiments.
[0460] 4. Stable Expression of Cyno CGRP R HEK 293EBNA Cells
[0461] 293EBNA cells were seeded in 100 mm dishes at
1.5.times.10.sup.6 cells/dish in DMEM (high glucose)+5% FBS.+-.1%
MEM non-essential amino acids+1% sodium pyruvate. The following day
the cells were co-transfected using FuGENE 6 with
pcDNA3.1/zeocin/cynoCRLR plus pcDNA3.1/hygromycin/cynoRAMP1. Both
constructs were linearized with FspI. After 48 hours the cells were
subcultured into growth medium containing 200 .mu.g/ml zeocin+400
.mu.g/ml hygromycin at dilutions of 1:20, 1:40, 1:100, and 1:200.
The medium was changed twice weekly. After two weeks, 96
transfected colonies were isolated using cloning rings. The clones
were assayed for their response to stimulation by CGRP ligand.
Several clones showed similar high levels of response and one was
selected for use in subsequent experiments.
C. Isolation of High-expressing CGRP Receptor Cells
[0462] A CGRP.sub.8-37 peptide analog was synthesized (Midwest
Bio-Tech Inc. Fishers, Ind.) with the sequence below:
TABLE-US-00015 (SEQ ID NO: 9)
AC-WVTHRLAGLLSRSGGVVRCNFVPTDVGPFAF-.sub.NH2
[0463] The peptide was labeled with Alexa 647-NHS following the
manufacturer's instructions (Molecular Probes, Inc. Cat A 2006).
The Alexa 647-labeled CGRP.sub.8-37 showed specific staining of
CGRP receptor transfected cells and not the non-transfected
parental cells and was used as the FACS reagent.
[0464] The huCGRP receptor-transfected 293EBNA and AM-1 CHO cell
pools (generated as above) were sorted repeatedly up to four times
pools using with Alexa 647-labeled CGRP.sub.8-37 peptide. High
expressing cells were collected at each sort, expanded and after
the final sorting frozen into vials. The AM-1 CHO/huCGRP R cells
were used for immunization as described below, and the
293EBNA/huCGRP R cells were used for titering mouse sera after
immunization and in binding screens of the hybridoma
supernatants.
D. Generation of Soluble CGRP Receptor
[0465] Soluble CGRP receptor polypeptides containing the N-terminal
extracellular domains (ECDs) of human CRLR (SEQ ID NO:6) and human
RAMP1 (SEQ ID NO:8) were generated by transiently co-transfecting
293 6E cells (Durocher, et al., Nucleic Acids Res. 30:E9 (2002))
with vectors containing the corresponding cDNAs (SEQ ID NO:5 or SEQ
ID NO:7) as described below. Commonly used tags (polyHis, Flag, HA
and/or Fc) were employed to facilitate secretion and/or subsequent
purification.
[0466] A soluble heterodimeric CGRP R ECD fused to Fc was prepared
by PCR cloning with the appropriate primers into the transient
expression vector pTT5 (Durocher, et al., supra). The CRLR
N-terminal ECD-Fc consisted of the N-terminal extracellular domain
of CRLR (SEQ ID NO:6) fused to human IgG1 Fc. The RAMP1 ECD-Fc
contains the extracellular domain of RAMP1 (SEQ ID NO:8) fused to
human IgG1 Fc. In both cases, there was a linker consisting of five
consecutive glycines between the ECD domain and Fc.
[0467] The soluble heterodimeric CGRP receptor was expressed by
co-transfecting the two constructs as follows. 293-6E cells at
1.times.10.sup.6 cells/ml in shake flasks were transfected with 0.5
mg/L DNA (hCRLR N-ter ECD-Fc/pTT5 and huRAMP1 ECD-Fc/pTT5) with 3
ml PEI/mg DNA in FreeStyle 293 media (Invitrogen). Cells were grown
in suspension in FreeStyle 293 expression medium supplemented with
0.1% Pluronic F68 and 50 .mu.g/ml Geneticin for 7 days and
harvested for purification.
[0468] Purifications from conditioned media ("CM") were performed
by buffering the CM with the addition of 50 mM Tris, 400 mM sodium
citrate, and adjusting the pH to 8.5. The buffered CM was then
passed over a Protein A affinity column equilibrated in 50 mM Tris,
400 mM sodium citrate and pH adjusted to pH 8.5. The Protein A
column was washed with PBS and the Fc fusion protein eluted with
0.1 N HOAc. The eluted peak contained both CRLR and RAMP1
components when tested by western blot using individual antibodies
specific to either CRLR or RAMP1. Further LC-MS and N-terminal
sequencing confirmed the presence of both CRLR:RAMP1 heterodimer
and CRLR:CRLR homodimer in approximately (2:3) ratio. This "soluble
CGRP receptor" was shown to compete in Alexa647 labeled
CGRP.sub.8-37 binding to CGRP receptor expressing recombinant cells
in the FMAT analysis, although it failed to bind CGRP ligand as
determined using Biacore testing. The material was used as an
immunogen as described in Example, despite, inter alia, its
heterogeneity and lack of CGRP ligand binding.
E. Generation of Membrane Extracts from Recombinant CGRP Receptor
Expressing Cells
[0469] Membrane extracts were prepared from CGRP receptor
expressing cells using a method described by Bosse, R. et al.,
(Journal of Biomolecular Screening, 3(4): 285-292 (1998)). Briefly,
approximately 5 grams of cell paste were pelleted in 50 ml of PBS
at 3,000 rpm for 10 min. at 4.degree. C. and re-suspended in 30 ml
of cold lysis buffer (25 mM HEPES, pH 7.4, 3 mM MgCl.sub.2 plus one
Roche protease inhibitor cocktail tablet/50 mL). The lysate was
homogenized with Glas-Col (Teflon-glass homogenizer) with .about.20
strokes at 5,000 rpm and spun in a JA21 rotor at 20,000 rpm for 15
min at 4.degree. C. This process was repeated once more and the
final pellet was re-suspended in .about.1-5 ml `final pellet`
buffer (25 mM HEPES, pH 7.4, 3 mM MgCl.sub.2, 10% (w/v) sucrose
plus one Roche protease inhibitor cocktail tablet/50 mL). The
membrane extracts were sheared by passing through 16 G and 25 G
needles 2-3 times. Total membrane protein concentration was
determined with a Microplate BCA Protein Assay (Pierce).
EXAMPLE 2
Generation of Antibodies to CGRP Receptor
A. Immunization
[0470] Immunizations were conducted using the following forms of
CGRP receptor antigens, prepared as described in Example 1:
[0471] (i) AM-1 CHO transfectants expressing full length human CRLR
and RAMP1 at the cell surface, obtained by co-transfecting CHO
cells with human full length CRLR cDNA (SEQ ID NO:1) encoding a
polypeptide having the sequence SEQ ID NO:2, and RAMP1 cDNA (SEQ ID
NO:3) encoding a polypeptide having the sequence SEQ ID NO:4
[0472] (ii) membrane extract from the cells described in (i) above;
and
[0473] (iii) soluble CGRP receptor obtained by co-expressing and
purifying the N-terminal ECD of CRLR (SEQ ID NO:6) and the
extracellular domain (ECD) of RAMP1 (SEQ ID NO:8) as described in
Example 1.
[0474] XENOMOUSE animals were immunized with purified soluble CGRP
receptor protein and purified CGRP R membranes prepared from AM-1
CHO cells stably expressing CGRP R in the same manner using doses
of 10 .mu.g/mouse and 150 .mu.g/mouse respectively. CGRP membranes
were prepared using methods described above.
[0475] Subsequent boosts were administered at doses of ten
.mu.g/mouse of soluble CGRP R or 75 .mu.g of purified CGRP R
membranes. XENOMOUSE animals were also immunized with CGRP
receptor-expressing cells using doses of 3.4.times.10.sup.6 CGRP R
transfected cells/mouse and subsequent boosts were of
1.7.times.10.sup.6 CGRP R transfected cells/mouse. Injection sites
used were combinations of subcutaneous base-of-tail and
intraperitoneal. Immunizations were performed in accordance with
methods disclosed in U.S. Pat. No. 7,064,244, filed Feb. 19, 2002,
the disclosure of which is hereby incorporated by reference.
Adjuvants TiterMax Gold (Sigma; cat. # T2684), Alum (E. M. Sergent
Pulp and Chemical Co., Clifton, N.J., cat. #1.452-250) were
prepared according to manufacturers' instructions and mixed in a
1:1 ratio of adjuvant emulsion to antigen solution.
[0476] Sera were collected 4-6 weeks after the first injection and
specific titers were determined by FACs staining of recombinant
CGRP receptor-expressing 293EBNA cells.
[0477] Mice were immunized with either cells/membranes expressing
full length CGRP R cells or soluble CGRP R extracellular domain,
with a range of 11-17 immunizations over a period of approximately
one to three and one-half months. Mice with the highest sera titer
were identified and prepared for hybridoma generation. The
immunizations were performed in groups of multiple mice, typically
ten. Popliteal and inguinal lymph nodes and spleen tissues were
typically pooled from each group for generating fusions.
B. Preparation of Monoclonal Antibodies
[0478] Animals exhibiting suitable titers were identified, and
lymphocytes were obtained from draining lymph nodes and, if
necessary, pooled for each cohort. Lymphocytes were dissociated
from lymphoid tissue in a suitable medium (for example, Dulbecco's
Modified Eagle Medium; DMEM; obtainable from Invitrogen, Carlsbad,
Calif.) to release the cells from the tissues, and suspended in
DMEM. B cells were selected and/or expanded using a suitable
method, and fused with suitable fusion partner, for example,
nonsecretory myeloma P3X63Ag8.653 cells (American Type Culture
Collection CRL 1580; Kearney et al, J. Immunol. 123, 1979,
1548-1550).
[0479] Lymphocytes were mixed with fusion partner cells at a ratio
of 1:4. The cell mixture was gently pelleted by centrifugation at
400.times.g for 4 minutes, the supernatant decanted, and the cell
mixture gently mixed by using a 1 ml pipette. Fusion was induced
with PEG/DMSO (polyethylene glycol/dimethyl sulfoxide; obtained
from Sigma-Aldrich, St. Louis Mo.; 1 ml per million of
lymphocytes). PEG/DMSO was slowly added with gentle agitation over
one minute followed, by one minute of mixing. IDMEM (DMEM without
glutamine; 2 ml per million of B cells), was then added over 2
minutes with gentle agitation, followed by additional IDMEM (8 ml
per B-cells) which was added over 3 minutes.
[0480] The fused cells were gently pelleted (400.times.g 6 minutes)
and resuspended in 20 ml Selection media (for example, DMEM
containing Azaserine and Hypoxanthine [HA] and other supplemental
materials as necessary) per million B-cells. Cells were incubated
for 20-30 minutes at 37.degree. C. and then resuspended in 200 ml
Selection media and cultured for three to four days in T175 flasks
prior to 96-well plating.
[0481] Cells were distributed into 96-well plates using standard
techniques to maximize clonality of the resulting colonies. After
several days of culture, the hybridoma supernatants were collected
and subjected to screening assays as detailed in the examples
below, including confirmation of binding to human CGRP receptor,
identification of blocking antibodies by a ligand binding
competition assay and evaluation of cross-reactivity with other
receptors related to CGRP receptor (for example, human
Adrenomedullin receptor). Positive cells were further selected and
subjected to standard cloning and subcloning techniques. Clonal
lines were expanded in vitro, and the secreted human antibodies
obtained for analysis.
C. Sequence Analysis of Selected Monoclonal Antibodies
[0482] Selected subcloned monoclonal antibodies were sequenced
using standard RT-PCR methods. Table 2A shows the amino acid
sequences of the light chains of exemplary antibodies disclosed
herein. Table 2B shows the amino acid sequences of the heavy chains
of exemplary antibodies disclosed herein.
[0483] Amino acid sequences corresponding to CDR regions of
sequenced antibodies were aligned and the alignments were used to
group the clones by similarity.
[0484] Sequence alignments of light chain CDRs from clones having
kappa light chains, and certain corresponding consensus sequences,
are shown in FIGS. 3A and 3B.
[0485] Sequence alignments of light chain CDRs from clones having
lambda light chains, and certain corresponding consensus sequences,
are shown in FIG. 4.
[0486] Sequence alignments of heavy chain CDRs of exemplary
antibodies disclosed herein, and certain corresponding consensus
sequences, are shown in FIGS. 5A, 5B, 5C, 5D and 5E.
[0487] Certain consensus sequences of exemplary heavy chain CDRs
disclosed herein are shown in FIG. 5F.
EXAMPLE 3
Identification of CGRP Receptor Specific Antibodies
A. Selection of CGRP Receptor Specific Binding Antibodies by
FMAT
[0488] After 14 days of culture, hybridoma supernatants were
screened for CGRP R-specific monoclonal antibodies by Fluorometric
Microvolume Assay Technology (FMAT) (Applied Biosystems, Foster
City, Calif.). The supernatants were screened against either the
AM-1 CHO huCGRP R cells or recombinant HEK 293 cells that were
transfected with human CGRP R and counter-screened against parental
HEK293 cells (prepared as described in Example 1).
[0489] Briefly, the cells in Freestyle media (Invitrogen, Carlsbad,
Calif.) were seeded into 384-well FMAT plates in a volume of 50
.mu.l/well at a density of approximately 4000 cells/well for the
stable transfectants, and at a density of approximately 16,000
cells/well for the parental cells, and cells were incubated
overnight at 37.degree. C. Then, 10 .mu.L/well of supernatant was
added and plates were incubated for approximately one hour at
4.degree. C., after which 10 .mu.l/well of anti-human IgG-Cy5
secondary antibody (Jackson Immunoresearch, West Grove, Pa.) was
added at a concentration of 2.8 .mu.g/ml (400 ng/ml final
concentration). Plates were then incubated for one hour at
4.degree. C., and fluorescence was read using an FMAT macroconfocal
scanner (Applied Biosystems, Foster City, Calif.).
[0490] For counter screens, the parental AM-1 CHO cells or HEK 293
cells were seeded similarly and supernatants screened by FMAT on
these cells in parallel to differentiate and eliminate hybridomas
binding to cellular proteins, but not to the CGRP receptor.
B. Identification of Blocking Antibodies by Ligand Binding
Competition Assay Through FMAT
[0491] A ligand binding competition method was developed to
identify antibodies (in the hybridoma supernatants) that bind CGRP
receptor and block CGRP ligand binding. 384-wells plates (Corning
Costar, Cat: #3712) were prepared with 5,000 AM-1 huCGRP R Pool 2
cells and 20,000 untransfected CHO-S cells in each well. 20 .mu.l
of anti-CGRP R hybridoma supernatant were added to each well, and
the plates were incubated for 1 hr at room temperature. 10 .mu.l of
2.8 .mu.g/ml. Alexa647-CGRP.sub.8-37 peptide were then added to
each well and the plates were incubated for a further 3 hours at
room temperature. The amount of Alexa647-CGRP.sub.8-37 bound to the
cells was assayed on a FMAT 8200 Cellular Detection System (Applied
Biosystems). Output data were both a numerical FL1 value of signal
intensity (higher FL1 values indicate higher signal intensity) and
also an image of the cells.
[0492] The experiments included negative control hybridoma
supernatants. The average FL1 value observed in these negative
control experiments was adopted as the maximum possible signal for
the assay. Experimental supernatants were compared to this maximum
signal and a percent inhibition was calculated for each well (%
Inhibition=(1-(FL1 of the anti-CGRP R hybridoma supernatant/Maximum
FL1 signal)).
[0493] An overview of the data is shown in FIG. 6. In this
experiment, 1092 anti-CGRP R supernatants were tested using the
receptor ligand assay. The data were rank ordered using the average
percent inhibition. Ninety supernatants had >25% average
inhibition, 31 of these were >50% and 7 were >70% average
inhibition.
[0494] An abbreviated data set is shown in Table 10, below, Sample
ID Nos. 1-5 illustrate examples of anti-CGRP R hybridoma
supernatants which inhibited the binding Alexa647-CGRP.sub.8-37
peptide to CGRP receptor and Sample ID Nos 536-540 illustrate
examples of anti-CGRP R hybridoma supernatants which did not
inhibit the binding of the Alexa647-CGRP.sub.8-37 peptide to the
CGRP receptor.
TABLE-US-00016 TABLE 10 Exemplary data from FMAT ligand binding
competition assay Sam- ple Expt. #1 Expt. #2 ID FL1 Image %
Inhibition FL1 Image % Inhibition 1 2264 84% 2585 88% 2 3007 77%
2804 85% 3 3460 72% 2929 84% 4 3650 70% 3294 79% 5 3764 69% 3246
80% 536 10412 0% 11142 -17% 537 10413 0% 9388 5% 538 10414 0% 9420
4% 539 10415 0% 10943 -14% 540 10415 0% 10561 -10%
[0495] Based on the binding competition assays, approximately 30
supernatants were selected for further characterization.
EXAMPLE 4
Activity of CGRP Receptor Specific Blocking Monoclonal Antibodies
in a cAMP Functional Assay
A. CGRP Receptor Antibody Activity.
[0496] Selected CGRP receptor antibodies were screened in an in
vitro CGRP receptor mediated cAMP assay to determine intrinsic
potency. The in vitro cAMP assay employed a human
neuroblastoma-derived cell line (SK-N-MC; Spengler, et al., (1973)
In Vitro 8: 410) obtained from ATCC (ATCC Number HTB-10; "HTB-10
cells"). HTB-10 cells express CRLR and RAMP1, which form CGRP
receptor (L. M. McLatchie et al, 1998). A 293EBNA cell line
expressing recombinant cynomolgus CGRP R was generated as described
in Example 1, and a rat L6 cell line expressing rat CGRP receptor
was obtained from the ATCC (CRL-1458).
[0497] The LANCE cAMP assay kit (PerkinElmer, Boston, Mass.) was
used in the screening. The assays were performed in white 96-well
plates in a total volume of 60 .mu.L. Briefly, on the day of the
assay, the frozen HTB-10 cells were thawed at 37.degree. C., cells
were washed once with assay buffer and 12 .mu.L of cell suspension
containing 10000 cells mixed with Alexa-labeled anti-cAMP antibody
was added into 96 half-area white plates. After adding 12 .mu.L
CGRP receptor antibody, the mixture was incubated for 30 min at
room temperature. Then 12 .mu.L CGRP receptor agonist human
.alpha.-CGRP (1 nM final concentration) was added and further
incubated for 15 min at room temperature. After human .alpha.-CGRP
stimulation, 24 .mu.L of detection mix was added and incubated for
60 minutes at room temperature and the plates were red on EnVision
instrument (PerkinElmer, Boston, Mass.) at Em665nM. Data were
processed and analyzed by Prizm (GraphPad Software Inc.) or
ActivityBase (IDBS).
[0498] FIG. 7A shows exemplary data obtained as described above
using the hCGRP receptor-expressing cell line HTB-10 for three
antibodies--3C8, 13H2 and 1E11. The data are plotted as percentage
over control ("POC") as a function of antibody (3C8, 13H2 or 1E11)
concentration, and are fitted with standard nonlinear regression
curves to yield the IC50 values shown at the bottom of the
figure.
B. Lack of Antibody Activity in Related Receptors.
[0499] Cells expressing related receptors AM1 (HEK 293 cells
expressing hCRLR+hRAMP2; D. R. Poyner, et al, Pharmacological
review, 54;233-246, 2002), AM2 (CHO cells expressing hCRLR+hRAMP3;
D. R. Poyner, et al, Pharmacological review, 54:233-246, 2002) or
human amylin AMY1 receptor (MCF-7 cells hCTR+hRAMP1; Wen-Ji Chen,
et al, Molecular pharmacology, 52: 1164-1175, 1997) were used to
determine the selectivity of the tested antibodies. The
AM1-expressing HEK 293 cell line was generated as described in
Example 1, above. The AM2-expressing CHO cell line was purchased
from EuroScreen (now PerkinElmer, Inc.); and the human amylin AMY1
receptor-expressing MCF-7 cell line (Zimmermann, et al, Journal of
Endocrinology, 423-431, 1997), was obtained from the ATCC (HTB-22).
Exemplary results, plotted as described above, are shown in FIGS.
7B (hAM1-HEK cells), 7C (hAM2-CHO cells) and 7D (hAMY-MCF-7 cells).
Note that none of the tested antibodies had significant inhibitory
activity against hAM1, hAM2 or hAMY1 receptors over the range
tested.
[0500] Similar experiments were performed using recombinant HEK
cells expressing cynomolgus CGRP receptors and rat L6 cells
expressing rat CGRP receptor (ATCC). Data from these studies, as
well as additional IC50 data obtained as described in part A of
this Example, are shown in the "cAMP" columns in Table 11, below.
Note that the IC50 values against the human and cyno CGRP receptors
are in the nanomolar range, whereas activities against rat CGRP
receptor, and human AM1, AM2 and AMY1 receptors, as well as MCF7
cells expressing calcitonin (data not shown) are all greater than 1
micromolar. The difference in IC50 between human CGRP receptor and
human AM1, AM2, amylin and calcitonin receptors illustrates the
high selectivity of the these antibodies for the CGRP receptor over
related receptors formed in part of the same receptor components.
IC50 obtained using human and cynomolgus CGRP receptors were
similar, whereas the tested antibodies did not appear to
cross-react with rat CGRP receptor.
TABLE-US-00017 TABLE 11 cAMP assay .sup.125I assay hCGRP Cyno Rat
CGRP hAmylin1 hAM1 hAM2 Human R IC50 CGRP R R IC50 IC50 IC50 IC50
CGRP Ki Clone (nM) IC50 (nM) (nM) (nM) (nM) (nM) (nM) 01E11.2 1.77
2.79 >1000 >1000 >1000 >1000 0.030 01H7.2 3.27 4.74
>1000 >1000 >1000 >1000 0.079 02A10.1 11.81 17.6
>1000 >1000 >1000 >1000 0.291 02E7.2 6.30 5.51 >1000
>1000 >1000 >1000 0.117 03A5.1 9.89 28.9 >1000 >1000
>1000 >1000 0.093 03B6.2 2.74 2.22 >1000 >1000 >1000
>1000 0.033 03C8.2 6.66 5.32 >1000 >1000 >1000 >1000
0.044 03H8.2 10.84 10.6 >1000 >1000 >1000 >1000 0.111
04E4.2 2.38 3.52 >1000 >1000 >1000 >1000 0.015 04H6.1
3.78 5.59 >1000 >1000 >1000 >1000 0.052 05F5.1 4.79
4.78 >1000 >1000 >1000 >1000 0.147 07B2.1 8.96 27.7
>1000 >1000 >1000 >1000 0.116 07B3.1 10.2 14.1 >1000
>1000 >1000 >1000 0.127 07F1.1 8.92 10.5 >1000 >1000
>1000 >1000 0.140 08B11.2 10.7 17.0 >1000 >1000
>1000 >1000 0.118 09D4.2 1.40 2.46 >1000 >1000 >1000
>1000 0.023 09F5.2 3.06 4.44 >1000 >1000 >1000 >1000
0.043 10E4.2 3.08 3.23 >1000 >1000 >1000 >1000 0.100
11A9.1 16.1 47.8 >1000 >1000 >1000 >1000 0.157 11D11.1
4.93 3.85 >1000 >1000 >1000 >1000 0.044 11H9.1 4.56
5.07 >1000 >1000 >1000 >1000 0.057 12E8.2 2.93 4.13
>1000 >1000 >1000 >1000 0.097 12G8.2 2.14 2.74 >1000
>1000 >1000 >1000 0.017 13D6.2 8.23 11.8 >1000 >1000
>1000 >1000 0.055 13E2.2 18.3 49.2 >1000 >1000 >1000
>1000 0.128 13H2.2 1.95 8.41 >1000 >1000 >1000 >1000
0.033 32H7.1G 1.93 >1000 >1000 >1000
EXAMPLE 5
Radioligand CGRP Binding Assay for Ki Determination receptor
Blocking Antibodies
[0501] .sup.125I-labeled CGRP (Amersham Biosciences, Piscataway,
N.J.) and cell membranes from HTB-10 cells (PerkinElmer Inc.,
Waltham, Mass.) were used for radioligand binding experiment in the
presence of various concentrations of the test antibodies to
determine the corresponding Ki values. The CGRP binding assay was
set up at room temperature in 96-well plates containing: 110 .mu.l
binding buffer (20 nM Tris-HCl, pH7.5, 5.0 mM MgSO4, 0.2% BSA
(Sigma), 1 tablet of Comtplete.TM./50 ml buffer (a protease
inhibitor)); 20 .mu.l test compound (10.times.); 20 .mu.L
.sup.125I-h.alpha.CGRP (Amersham Biosciences; 10.times.); and 50
.mu.l human neuroblastoma cell (HTB-10) membrane suspension (10
.mu.g per well, PerkinElmer). The plates were incubated at room
temperature for 2 hours with shaking at 60 rpm, and then the
contents of each well were filtered over 0.5% polyethyleneimine
(PEI)-treated (for at least one hour) GF/C 96-well filter plates.
The GF/C filter plates were washed six times with ice-cold 50 mM
Tris, pH 7.5 and dried in an oven at 55.degree. C. for 1 hour. The
bottoms of the GF/C plates were then sealed. 40 .mu.l
Microscint.TM. 20 was added to each well, the tops of the GF/C
plates were sealed with TopSeal.TM.-A (a press-on adhesive sealing
film), and the GF/C plates were counted with TopCount NXT
(Packard). The data were analyzed using Prizm (GraphPad Software
Inc.)
[0502] Exemplary data and Ki values obtained using antibodies 3C8,
12H2 and 1E11 are shown in FIG. 8.
[0503] The right-most column in Table 11, above, lists the Ki
values of the indicated mAbs in the radiolabeled .sup.125-I-CGRP
competition binding assay to HTB-10 cell membranes. The data
demonstrate that the CGRP receptor antibodies were highly
competitive (all sub-nanomolar range) against CGRP binding.
EXAMPLE 6
FACS Binding Assay for KD Determination of CGRP Receptor Blocking
Antibodies
[0504] The affinities of anti-CGRP R mAbs for CGRP receptors
expressed on cells were determined using a FACS method. Briefly,
AM-1 CHO huCGRP R-expressing cells, prepared as described above,
were plated in 96-well plates at densities of 16,000 or 160,000
cells per well in DMEM medium containing 10% FBS, NEAA, PS, L Glut,
NaPyr and 0.05% sodium azide. CGRP receptor antibodies were
titrated in the same medium from 50 nM to 1 pM and incubated with
cells. After an overnight incubation at 4.degree. C. in a total
volume of 120 .mu.l, on a plate shaker, the cells were washed
2.times. with PBS+2% FBS, centrifuging and discarding supernatant
each time. 100 .mu.l/well of G anti-Hu Fc Cy5 (5 .mu.g/mL; Jackson
ImmunoResearch Laboratories Inc., West Grove, Pa., USA) containing
7AAD (5 .mu.l/well) was then added and incubated at 4.degree. C.
for 40 min. The cells were washed 2.times. with PBS+2% FBS,
centrifuging and discarding the supernatant each time. 100 .mu.l
PBS+2% FBS buffer was then added and analyzed by FACS to determined
the binding geomean. The Kd was calculated using KinExA software by
taking the negative geomean at each antibody concentration as the
amount of free Ab present Rathanaswami, et al., Biochemical and
Biophysical Research Communications 334 (2005) 1004-1013. The data
obtained at the two different cell concentrations were analyzed by
n-curve analysis to determine the Kd and the 95% confidence
interval as described in Rathanaswami, et al., Biochemical and
Biophysical Research Communications 334 (2005) 1004-1013.
[0505] Exemplary data with corresponding curve fits are shown in
FIG. 10 far antibody 12G8.2. The data of eight blocking antibodies
generated in support of the present disclosure are shown in Table
12. One of the antibodies (3B6) was analyzed on two different days.
The ratio of 0.9 obtained for the experiment with 16K cells
indicates that the antigen concentration is predicted as 0.9.times.
the Kd and hence the curve obtained by this experiment is a Kd
controlled curve. It can be appreciated that the Kd values obtained
in this manner were in the low single-digit nanomolar range for all
tested antibodies.
TABLE-US-00018 TABLE 12 N curve analysis Kd Low Kd High Ratio Kd
(nM) (nM) (nM) % error 16K 1H7 1.9 1.5 3 3.8 0.001 2E7 1.5 0.7 3.4
6.3 0.19 3B6 (a) 1.7 1.1 2.7 5.3 0.060 3B6 (b) 2.0 1.6 2.6 3.2 0.21
4E4 1.3 0.9 2.05 3.9 0.16 4H6 2.4 1.78 4.35 3.8 0.070 9D4 2.5 1.8
4.39 4.3 0.060 12E8 2.3 1.58 3.36 3.7 0.55 12G8 1.4 0.92 2.21 3.6
0.94
EXAMPLE 7
binning of CGRP Receptor Blocking Antibodies by Bicore Binding
Competition
[0506] Biacore analyses Karlsson, R. et al., Methods; A Companion
to Methods in Enzymology, 6: 99-110 (1994) were carried out as
follows. Immobilization of anti-CGRP receptor antibodies to the CM5
sensor chip surface was performed according to manufacturer's
instructions, using a continuous flow of 10 mM HEPES, 0.15M NaCl,
3.4 mM EDTA, 0.005% P-20, pH 7.4 (HBS-EP buffer). Carboxyl groups
on the sensor chip surfaces were activated by injecting 60 .mu.L of
a mixture containing 0.2 M N-ethyl-N'
(dimethylaminopropyl)carbodiimide (EDC) and 0.05 M
N-hydroxysuccinimide (NHS). Specific surfaces were obtained by
injecting 180 .mu.l of anti-CGRP receptor antibody diluted in 10 mM
acetate, pH 4.0 at a concentration of 30 .mu.g/mL. Excess reactive
groups on the surfaces were deactivated by injecting 60 .mu.L of 1
M ethanolamine. Final immobilized levels for the individual
antibodies were as follows:
TABLE-US-00019 Antibody Resonance Units (RU) 11D11 ~5.900 3B6
~7.200 4H6 ~8.000 12G8 ~7.800 9F5 ~6.600 34E3 ~3.700
A blank, mock-coupled reference surface was also prepared on the
sensor chip. Soluble huCGRP receptor at a concentration of 100 nM
was captured on sensor chips having one of the six immobilized
antibodies referenced above (11D11, 3B6, 4H6, 12G8, 9F5 or 34E3).
Each of the 20 test anti-CGRP R antibodies was then injected over
the captured huCGRP receptor. If the injected antibody recognized a
distinct epitope relative to that recognized by the immobilized
antibody, a second binding event would be observed. If the
antibodies recognize the same or very similar epitopes, only the
binding of the huCGRP receptor would be observed.
[0507] Exemplary data obtained using a sensor chip coated with
immobilized antibody 3B6 are shown in FIG. 9A. The four traces are
data obtained using antibodies 1E11, 4E4, 2E7 and 12G8 in the
injected solution. Events during the experiment are represented by
letters, with "A" corresponding to injection of huCGRP R-Fc, "B"
corresponding to end of the huCGRP R-Fc injection, "C"
corresponding to injection of second mAb, and "D" corresponding to
end second mAb injection and start of the buffer wash. Note that
there is no indication of any binding signal from any of the
injected antibody on the immobilized antibody surface, indicating
that the four injected antibodies apparently recognize the same or
very similar epitope(s)as the immobilized antibody. Essentially the
same results were observed with all tested blocking antibodies
washed over each the five immobilized neutralizing antibody
surfaces, indicating that all tested anti-huCGRP receptor blocking
antibodies recognize the same or very similar and strongly
overlapping epitope(s).
[0508] In contrast, as shown in part in FIGS. 9B, 9C and 9D, the
four tested non-blocking, CGRP receptor specific antibodies 32H8,
33B5, 33E4 and 34E3 failed to compete with 11D11 (data not shown),
3B6 (FIG. 9B), 12G8 (FIG. 9C) and 9F5 (data not shown) although
34E3 was able to compete with 4H6 (FIG. 9D) and weakly with 32H7
(data not shown). 32H8 failed to compete with 3B6, 4H6, 12G8, 9F5
or the non-blocking antibody 34E3, but 33B5 and 33E4 could compete
with the non-blocking antibody 34E3. The data for all blocking and
non-blocking antibodies are summarized in Table 13, below. "NB"
indicates no binding; "+" indicates significant binding; and "Weak"
indicates weak binding.
TABLE-US-00020 TABLE 13 Immobilized Antibodies Ab in Solution 11D11
3B6 4H6 12G8 9F5 34E3 1E11 NB NB NB NB NB + 1H7 NB NB NB NB NB +
2E7 NB NB NB NB NB + 3B6 NB NB NB NB NB + 3C8 NB NB NB NB NB + 4E4
NB NB NB NB NB + 4H6 NB NB NB NB NB NB 5F5 NB NB NB NB NB + 9D4 NB
NB NB NB NB + 9F5 NB NB NB NB NB + 10E4 NB NB NB NB NB + 11D11 NB
NB NB NB NB + 11H9 NB NB NB NB NB + 12E8 NB NB NB NB NB + 12G8 NB
NB NB NB NB + 13H2 NB NB NB NB NB + 32H7 NB NB NB NB NB Weak 32H8 +
+ + + + + 33B5 + + + + + NB 33E4 + + + + + NB
[0509] As can be appreciated from the data, all the tested blocking
or neutralizing antibodies bind to the same region as the five
immobilized blocking antibodies; i.e., all of the tested
neutralizing antibodies bind the same region of the CGRP R
molecule. On the other hand, the non-blocking antibodies did not
generally compete with the immobilized blocking antibodies,
indicating that the non-blocking antibodies primarily bind a
different region of CGRP R.
EXAMPLE 8
binding of CGRP Receptor Antibodies to Soluble CGRP Receptor in
Western Blot
[0510] Three representative CGRP receptor blocking antibodies were
tested using Western blots for binding to a soluble CGRP
receptor-muFc fusion protein.
[0511] 100 ng of purified CGRP R-muFc (produced and purified as
described above for the CGRP R-huFc except the mouse Fc was used
and the linker between RAMP1 or CRLR ECD and muFc was changed to
"GGGGGVDGGGGGV" (SEQ ID NO:213)) was diluted in PBS with PAGE
sample buffer with (reduced) or without (non-reduced)
beta-mercaptoethanol (.beta.ME) at 13.3% concentration. The sample
containing .beta.ME was then boiled for 4 min. Reduced and
non-reduced samples were loaded onto separate 4-20% Tris-glycine
gels (Invitrogen) with alternating lanes of CGRP R-Fc protein and
molecular weight markers (Invitrogen). Gels were electroblotted
onto 0.2 .mu.m nitrocellulose filters (Invitrogen). The blots were
washed with Tris-buffered saline+1% Tween 20 (TBST) and then
blocked with TBST+5% powered dry milk for 30 min. The blots were
cut into strips along the molecular weight marker lanes. One strip
each with reduced and non-reduced CGRP R-muFc were incubated with
purified huCGRP R antibodies 4E4, 9F5, or 3B6 (1:500 dilution
TBST+5% milk), goat anti-huRAMP1 N-20 (1:500; Santa Cruz
Biotechnology, Inc), rabbit anti-mouse IgG-Fc-HRP (1:10,000)
(Pierce), or goat anti-human IgG-Fc-HRP (1:10,000) (Pierce). Blots
were incubated with the antibodies for one hour followed by
3.times.10 min washes with TBST+1% milk. The blots treated with the
huCGRP R antibodies were then incubated with goat anti-mouse
IgG-Fc-HRP (1:10,000 in TBST+1% milk) and the blots treated with
anti-huRAMP1 (N-20 anti-RAMP1 goat polyclonal antibody, Santa Cruz
Biotech, CA) were incubated with rabbit anti-goat IgG-Fc-HRP
(1:10,000) for 20 min. Blots were washed 3.times.15min with TBST.
The huCGRP R and anti-huRAMP1 antibody blots were treated with
Pierce Supersignal West Pico Detection reagent, and the anti-mouse
and anti-human IgG-Fc-HRP blots were treated with Pierce standard
Detection Reagent (1 min). Blots were then exposed with Kodak
Biomax MS X-ray film.
[0512] All of the three CGRP receptor antibodies, 4E4, 9F5 and 3B6
were able to detect the soluble CGRP R-muFc (containing RAMP1-ECD
and CRLR ECD) under non-reduced condition but not under reduced
condition indicating that the binding epitope of these CGRP R
antibodies was conformational and sensitive to the disulfide
linkages (3 pairs in RAMP1-ECD and 3 pairs in CRLR N-ter ECD). In
contrast, the commercial anti-RAMP1 antibody N-20 (Santa Cruz
Biotech) bound RAMP1 under both reduced and no reduced conditions
indicating that the binding site for N-20 antibody was primarily
linear and not sensitive to disulfide linkages.
EXAMPLE 9
Binding of CGRP Receptor Blocking Antibodies to Chimeric
Receptors
[0513] CGRP receptors formed of either native RAMP1 with chimeric
CRLR, or native CRLR with chimeric RAMP1, were used to identify
CGRP receptor sequences involved in antibody binding. Since all of
the human CGRP receptor blocking antibodies tested failed to show
functional activity to the rat CGRP receptor, the chimeric
components contained regions of rat sequence in a human sequence
background. The following chimeras were generated for binding
analysis by FACS:
[0514] RAMP1 chimera#1 (Q28 to A34); SEQ ID NO:217
[0515] Amino acid residues Q28 to A34 in the human RAMP1 were
replaced with the corresponding sequences from rat RAMP1. This
stretch included five amino acid residues that are different
between human and rat RAMP1.
[0516] RAMP1 chimera#2 (Q43 to E53); SEQ ID NO:218
[0517] Amino acid residues Q43 to E53 in the human RAMP1 were
replaced with the corresponding sequences from rat RAMP1. This
stretch included six amino acid residues that are different between
human and rat RAMP1.
[0518] RAMP1 chimera#3 (R67 to E78); SEQ ID NO: 219
[0519] Amino acid residues R67 to E78 in the human RAMP1 were
replaced with the corresponding sequences from rat RAMP1, This
stretch included seven amino acid residues that are different
between human and rat RAMP1.
[0520] CRLR chimera#1 (L24 to Q33); SEQ ID NO:223
[0521] Amino acid residues L24 to Q33 in the human CRLR were
replaced with the corresponding sequences from rat CRLR. This
stretch included eight amino acid residues that are different
between human and rat CRLR.
[0522] FIG. 11 shows an alignment of RAMP1 amino acid sequences
from cynomolgus monkey (SEQ ID NO:215), human (SEQ ID NO:4), rat
(SEQ ID NO:214) and rhesus monkey (SEQ ID NO:216), together with
sequences of RAMP1 chimera #1 (SEQ ID NO:217), chimera #2 (SEQ ID
NO:218) and chimera #3 (SEQ ID NO:219). FIGS. 12A and 12B show an
alignment of CRLR amino acid sequences from human (SEQ ID NO:2),
cynomolgus monkey (SEQ ID NO:221), rhesus monkey (SEQ ID NO:222),
rat (SEQ ID NO:220) and, as well as the amino acid sequence of CRLR
chimera #1 (SEQ ID NO:223).
[0523] 293-6E cells were transiently transfected with CGRP R
chimera DNA constructs (CRLR wt+RAMP1 Q28-A34; CRLR wt+RAMP1
Q43-E53; CRLR wt+RAMP1 R67-E78; CRLR L24-Q33+RAMP wt; CRLR wt+RAMP1
wt; pTT5 vector control). Cells were harvested after 72 hr, washed
with PBS+0.5% BSA, and counted. Each transfected cell line was
resuspended at a dilution of 5.times.10.sup.5 cells per 100 .mu.l
PBS/BSA. 100 .mu.l of cell suspension was aliquot per well in a
96-well round-bottom plate (Falcon). The cells were pelleted at
1200 rpm for 5 min. The supernatant was removed and replaced with
100 .mu.l containing 0.5 .mu.g purified huCGRP R antibodies 1H17,
2E7, 3B6, 9F5, 4H6, 12G8, 3C8, 10E4, 11D11, 32H8, or 33B5. Control
wells were treated with anti-DNP huIgG2 (0.5 .mu.g), Alexa647-CGRP
peptide (0.5 .mu.g), or PBS/BSA alone. Cells were incubated on ice
for 1 hr. and then washed twice with PBS/BSA. The cells were
resuspended in 100 .mu.l/well PBS/BSA containing anti-hug-Fc-FITC
(0.5 .mu.g) (except for Alexa647-CGRP treated cells). Cells were
incubated on ice in the dark for 1 hr, and then washed twice with
PBS/BSA. Cells were resuspended in 200 .mu.l PBS/BSA and analyzed
using a FACS Calibur.
[0524] Ten representative blocking antibodies (3B6, 9F5, 4H6, 12G8,
3C8, 10E4, 32H7, 4E4, 11D11 and 1H7) and two non-blocking antibody
(32H8 and 33B5) were tested. Representative data (9F5 antibody) are
shown in FIGS. 13A, 13B and 13C. FIG. 13A shows binding to the
wild-type CGRP receptor; FIG. 13B show binding to CGRP receptors
containing the CRLR L24-Q33 chimera, and FIG. 13C show binding to
CGRP receptors containing the RAMP1 Q28-A34 chimera. The FACS
analysis showed that all 12 antibodies bind wild type human CGRP
receptor control as expected. All 12 antibodies showed
significantly reduced binding to any of the three RAMP1 chimera
RAMP1 (Q28-A34), (Q43-E53) and (R67-E78). This could result from
(1) the expression level of the chimera receptor was much lower,
(2) the RAMP1 chimera impaired the folding with human CRLR and
altered the conformation of the receptor complex, and/or (3) these
three selected regions on RAMP1 are directly involved in the
binding of these antibodies to CGRP receptor.
[0525] When the FACS tracing were gated to include only the very
small "expressing" cell populations, the non-blocking antibodies
33B5 and 32H8 appeared to consistently bind less well (lower Geo
Means) to the RAMP1 Q43-E53 chimera as compared to the blocking
antibodies, suggesting binding to the RAMP1 Q43-E53 region may be
more important for the non-blocking antibodies. On the other hand,
33B5 and 32H8 consistently bound better to the RAMP1 R67-E78
chimera than the blocking antibodies, suggesting the RAMP1 R67-E78
sequence may be more important for the blocking antibodies.
[0526] All CGRP receptor antibodies tested bound reasonably well to
the CRLR chimera (L24-Q33), suggesting this site is not essential
for binding of blocking antibodies.
[0527] In summary, the data show that three discontinuous regions
on RAMP1 --(Q28-A34), (Q43-E53) and (R67-E78)--could be involved in
CGRP receptor antibody binding, with (R67-E78) more important to
the blocking antibodies. The N-terminal sequences (L24-Q33) of CRLR
did not appear to be critically involved in binding for the CGRP
receptor antibodies as analyzed by this method. This approach does
not rule out additional binding sites that share identical or
similar sequences between human and rat CGRP receptors which were
not targeted in the analysis.
EXAMPLE 10
Identification of Human CGRP R Epitopes for Anti-CGRP R
Neutralizing Antibodies by Protease Protection Assay
[0528] The CRLR portion in the mature form of the CRLR-Fc fusion
molecule (with signal peptide removed; disclosed herein as SEQ ID
NO:10) contains 116 amino acids (preceding the glycine linker) and
has three large loop structures created by formation of three
disulfide bonds. The three disulfide bonds in CRLR are Cys1 at
sequence position 26 (all CRLR sequence positions listed in this
paragraph are with respect to the mature sequence presented as SEQ
ID NO:10) linked to Cys3 at sequence position 52 (referred to as
CRLR C1-C3), Cys2 sequence position 43 linked to Cys5 at sequence
position 83 (referred to as CRLR C2-C5), Cys4 at sequence position
66 linked to Cys6 at sequence position 105 (referred to as CRLR
C4-C6). RAMP1 portion in mature form of RAMP1 -Fc fusion molecule
contains 91 amino acids (SEQ ID NO:11) preceding the glycine
linker, which also forms three intramolecular disulfide bonds. The
three disulfide bonds in RAMP1 are Cys1 at sequence position 1 (all
RAMP1 sequence positions listed in this paragraph are with respect
to the mature sequence presented as SEQ ID NO:11) linked to Cys5 at
sequence position 56 (referred to as RAMP1 C1-C5), Cys2 at sequence
position 14 linked to Cys4 at sequence position 46 (referred to as
RAMP1 C2-C4), Cys3 at sequence position 31 linked to Cys6 at
sequence position 78 (referred to as RAMP1 C3-C6).
[0529] Regions of the human CGRP receptor protein bound by
anti-CGRP neutralizing monoclonal antibodies were identified by
fragmenting h CGRP R into peptides with specific proteases, and
determining the sequence of the resulting h CGRP R peptides (i.e.,
both disulfide- and non-disulfide-containing peptide fragments for
CRLR and RAMP1 portions). A protease protection assay was then
performed to determine the proteolytic digestion of hCGRP R in the
presence of binding monoclonal antibodies. The general principle of
this assay is that binding of a mAb to CGRP R can result in
protection of certain specific protease cleavage sites and this
information can be used to determine the region or portion of CGRP
R where the mAb binds to.
[0530] Briefly, the peptide digests were subjected to HPLC peptide
mapping; the individual peaks were collected, and the peptides
identified and mapped by on-line electrospray ionization LC-MS
(ESI-LC-MS) analyses and/or by N-terminal sequencing. All HPLC
analyses for these studies were performed using a narrow bore
reverse-phase C18 column (2.1 mm id..times.15 cm length; Zorbax
300SB, 5 .mu.m, Agilent Technologies) for off-line analysis and
using a capillary reverse phase C18 column (0.5 mm i.d..times.25 cm
Vydac C18 MS, 5 .mu.m; The Separation Group) for LC-MS. HPLC
peptide mapping was performed with a linear gradient from 0.05%
trifluoroacetic acid (mobile phase A) to 90% acetonitrile in 0.05%
trifluoroacetic acid. Columns were developed over 90 minutes at a
flow rate of 0.25 ml/min for narrow bore HPLC for off-line or
on-line LC-MS analyses, and 0.018 ml/min for capillary HPLC for
on-line LC-MS analyses.
[0531] Mature form human CGRP R was digested with AspN (which
cleaves after aspartic acid and some glutamic acid residues at the
amino end) by incubating about 100 .mu.g of CGRP R at 1.0 mg/ml in
0.1M sodium phosphate (pH 6.5) for 20 hrs at 37.degree. C. with 2
.mu.g of AspN.
[0532] HPLC chromatography of the AspN digests generated a peptide
profile as shown in FIG. 14 (each sample 30 .mu.g injected),
chromatogram labeled A for CGRP R alone (concentration 1 mg/ml),
while a control digestion with a similar amount of CGRP R
neutralizing antibody, clone 12G8, shows that the antibody is
essentially resistant to AspN endoproteinase (chromatogram labeled
B; CGRP R:antibody ratio, 100:2; 100:7; 100:20, weight by weight,
respectively). Sequence analyses were conducted by on-line LC-MS/MS
and by Edman sequencing on the peptide peaks recovered from HPLC.
On-line ESI LC-MS analyses of the peptide digest were performed to
determine the precise mass and sequence of the peptides that were
separated by HPLC. The identities of several peptides present in
the peptide peaks from the AspN digestion were thus determined
(indicated as numbered peaks in FIG. 14). Table 14, below, shows
the locations of these peptide sequences in the corresponding
component (CRLR or RAMP1) of the hCGRP R. A capital letter C
followed by a number or X represents a peptide identified as a CRLR
peptide; a capital letter R followed by a number or X is a RAMP1
peptide and "Fc" represents the large, undigested Fc fragment
released from the CRLR-Fc and RAMP1-Fc fusion molecules.
TABLE-US-00021 TABLE 14 CRLR and RAMP1 peptides identified by
peptide mapping of CGRP R AspN digestion Sequence Disulfide Peptide
location (#) Intact mass Origin C1 E111-V122 0 1059 CRLR C2 D33-A38
0 670 CRLR C3 D55-M63 0 880 CRLR C4 D68-Q71 0 571 CRLR C5
D55-P67/D86-H110 n.d. CRLR C6 D8-Y24 0 1938 CRLR C7 E25-Q32/D48-N54
1 1933 CRLR C-X 3 n.d. CRLR R1 D32-A44 0 1622 RAMP1 R2
E12-V20/D45-A51 1 1939 RAMP1 R-X C1-R86 3 10049 RAMP1 Fc 20500
RAMP1/CRLR
[0533] FIG. 15 shows a comparison of an AspN digestion experiment
(each sample 30 .mu.g injected) with CGRP R alone (chromatogram
labeled A) with one performed in the presence of neutralizing
antibody 12G8 (chromatogram labeled B). The weight ratio of CGRP
R:antibody was 1:1. Several peaks (C5, C6 and C7) show a decreased
in peak height in chromatogram B relative to chromatogram A, while
two other peaks (C-X and R-X) show an increase in peak height in
chromatogram B relative to chromatogram A. A similar peptide map
pattern was also observed if a different neutralizing anti-CGRP R
antibody (10E4, 3B6, 3C8 or 4E4) at a similar quantity was present
in the digestion sample as seen in the chromatogram labeled C. Both
C6 and C7 are disulfide linked peptides which cover a major portion
of the CRLR. molecule while C5 is a CRLR non-disulfide containing
peptide residing at the N-terminal end of the molecule and is
penultimate to the C7 disulfide peptide. Peak C-X contains three
CRLR disulfide bonds with multiple sequences, indicating at least
two to three peptides are linked together by disulfide bonds. The
fact that peak C-X has increased peak height in a CGRP R digest in
the presence of CGRP neutralizing antibody indicates that the
antibody has protected CGRP R from AspN digestion at several
cleavage sites related to Glu25 and Asp55. The antibody does not
appear to have a significant protective effect on Asp33 and Asp72
as peak intensity for peptides C2 and C4 did not decrease at all.
Therefore the antibody appears to bind to a region of CRLR which
includes the CRLR C1-C3 and CRLR C4-C6 disulfide region together
with the loop region between Cys53 and Cys66.
[0534] The AspN mapping of hCGRP R also identified a RAMP1
disulfide peptide (R2) and a RAMP1 non-disulfide peptide (R1) (see
Table 14 and FIG. 14). In the presence of any of the
above-mentioned neutralizing antibodies (12G8, 10E4, 4E4, 3B6 3C8),
peptide R-X was recovered at a significantly higher peak intensity
than what was obtained from the digestion with no antibody in the
sample. Mass and sequence analyses showed that R-x contains a
single polypeptide chain corresponding to the RAMP1 sequence
between Cys1 and Arg86. These experiments indicate that CGRP R
neutralizing antibody can protect a significant region of RAMP1
from AspN proteolytic digestion.
[0535] To assess whether the protective effect of CGRP R AspN
proteolysis is specific to CGRP R-neutralizing (blocking)
antibodies (as compared with anti-CGRP R non-neutralizing
antibodies), an AspN digestion of CGRP R was performed in the
presence of an unrelated control monoclonal antibody which does not
neutralize CGRP R activity. The results are shown in FIG. 15 in
chromatogram D. The non-neutralizing antibody does not show any
significant blocking effect on CGRP R AspN proteolysis; indeed, the
peptide map profile (chromatogram D) is nearly indistinguishable in
the relevant aspects to the profile derived from digestion of CGRP
R alone (chromatogram A).
[0536] The proteolysis protection effect was dependent on the
concentration added to the digestion sample. As seen in FIG. 16, a
fixed CGRP R quantity in the sample (100 .mu.g) with variable
amounts of anti-CGRP R neutralizing antibody 4E4 (CGRP R:antibody
ratio in micrograms, 100:2; 100:7; 100:20,weight by weight,
respectively) was performed for Aspen proteolysis. The protection
profile can be observed and the protection is antibody
concentration-dependent.
[0537] Taken together, these data demonstrate that blocking or
neutralizing anti-CGRP R antibodies disclosed herein can block CGRP
R (on both CRLR and RAMP1 components) from AspN proteolysis,
suggesting that the blocking antibodies bind to both CRLR and RAMP1
when these antibodies bind to the CGRP receptor. Further, the
protection effect is antibody-concentration dependent. These
results also indicate that CGRP R neutralizing antibodies bind to
common regions on human CGRP R which are close the Asp N cleavage
sites.
EXAMPLE 11
Commercially-available Anti-RAMP1 and Anti-CRLR Antibodies in a
cAMP functional Assay
[0538] Commercially-available antibodies directed against one or
the other components (RAMP1 or CRLR) of the human CGRP receptor
were screened in the CGRP receptor mediated cAMP assay using HTB-10
cells as described in Example 4, above, to determine whether the
antibodies had biological activity. The data are presented in Table
15, below. The antibodies had either no detectable ("ND"), very
weak ("VW") or weak ("W") biological activity over a concentration
range where the exemplary antibodies disclosed herein had strong
biological activity.
TABLE-US-00022 TABLE 15 Commercially-available antibody activity
Antigen or HTB-10 Name Source epitope Vendor activity CRLR Rabbit
N-terminal ECD Abcam Inc., antibody polyclonal of hCRLR Cambridge,
MA ND (ab13164) Ab CALCRL Rabbit N-terminal ECD GenWay ND antibody
polyclonal of hCRLR Biotech, Inc., Ab San Diego, CA CRLR Goat
epitope mapping Santa Cruz VW (N-18) polyclonal near N-terminus
Biotech Ab of hCRLR CRLR Rabbit aa23-64 of Santa Cruz ND (H-42)
polyclonal hCRLR Biotech Ab CALCRL Mouse aa23-133 of Novus VW
Antibody polyclonal hCRLR Biologicals, Inc. (A01) Ab RAMP1 Goat
epitope mapping Santa Cruz ND (N-20) polyclonal at N-terminus of
Biotech Ab hCRLR RAMP1 Mouse aa27-118 of Novus W Antibody
polyclonal hRAMP1 Biologicals, (M01) Ab Inc., Littleton, CO RAMP1
Mouse aa27-118 of Novus ND Antibody monoclonal hRAMP1 Biologicals,
Inc. (1F1) Ab RAMP1 Mouse full-length of Abcam, Inc. W antibody
polyclonal hRAMP1 (ab67151) Ab RAMP1 Rabbit full length Santa Cruz
ND (FL-148) polyclonal hRAMP1 Biotech, Santa Ab Cruz, CA
EXAMPLE 12
Immunohistochemistry Staining of Cells Expressing Different
Receptor Components
[0539] 2-4.times.10.sup.6 cells were injected per colla plug
(Integra LifeSciences Co., Plainsboro, N.J.). Colla plugs were
embedded in OCT medium (Sakura Finetek Inc., Torrance, Calif.),
frozen at -20.degree. C. and cut into 20 .mu.m sections using a
cryostat. Sections were fixed with 4% paraformaldehyde for 1 hour
at room temperature (RT) and subsequently washed in
phosphate-buffered saline (PBS). Endogenous peroxidase was blocked
with 3% H.sub.2O.sub.2/PBS for 15 min and sections were incubated
in blocking solution (PBS with 3% normal goat serum (Vector Labs,
Burlingame, Calif.) and 0.3% triton X-100) for 1 hour.
Subsequently, sections were incubated in human anti-CGRP receptor
primary antibody (32H17, 0.03-0.1 .mu.g/ml) at 4.degree. C. over
night, washed in PBS and incubated in secondary antibody
(biotinylated goat anti-human IgG Fe fragment, 1:800, Jackson
Immunoresearch, West Grove, Pa.) in 1% normal goat serum/PBS for 1
hour at RT. Immunoreactivity was amplified using the Vector Elite
Kit according to the manufacturer's instructions (Vector Labs,
Burlingame, Calif.) and staining was developed using
3,3'-diaminobenzidine-nickel as chromogen (Sigma-Aldrich, St.
Louis, Mo.). Sections were cleared with xylene and cover slipped
with Permount (Fisher Chemicals, Fair Lawn, N.J.). Immunoreactivity
was analyzed using a Nikon E-800 microscope and associated software
(Nikon, Melville, N.Y.).
[0540] Data from cells expressing different receptor components (as
identified below) using antibody 32H7 as described above revealed
pronounced staining of CHO cells expressing recombinant human CGRP
receptor (CRLR+RAMP1; "CHO/CGRP R cells") and weaker staining of
SK-N-MC cells that endogenously express CGRP receptors (due to much
lower receptor density). No staining was observed in the parent CHO
cell line, CHO cells expressing an unrelated recombinant protein
(TRPM8), CHO/CGRP R cells after preabsorption with the
corresponding 32H7 antigen, CHO cells expressing recombinant human
adrenomedullin receptor 2 (CRLR+RAMP3), MCF-7 cells endogenously
expressing amylin receptors, HEK cells expressing recombinant human
adrenomedullin receptor 1 (CRLR+RAMP2), or the parent HEK cells.
The data from these experiments are summarized in Table 16,
below.
TABLE-US-00023 TABLE 16 Immunohistochemical staining intensity of
indicated cells Cell line Staining intensity (visual score)
CGRP/CHO 4+ SK-N-MC 1+ CHO 0 TRPM8/CHO 0 CGRP/CHO preadsorbed 0
AM2/CHO 0 MCF-7 0 AM1/HEK 0 HEK 0
[0541] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the subject
matter disclosed herein. These publications are provided solely for
their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
Sequence CWU 1
1
26111419DNAHomo sapiens 1atgttataca gcatatttca ttttggctta
atgatggaga aaaagtgtac cctgtatttt 60ctggttctct tgcctttttt tatgattctt
gttacagcag aattagaaga gagtcctgag 120gactcaattc agttgggagt
tactagaaat aaaatcatga cagctcaata tgaatgttac 180caaaagatta
tgcaagaccc cattcaacaa gcagaaggcg tttactgcaa cagaacctgg
240gatggatggc tctgctggaa cgatgttgca gcaggaactg aatcaatgca
gctctgccct 300gattactttc aggactttga tccatcagaa aaagttacaa
agatctgtga ccaagatgga 360aactggttta gacatccagc aagcaacaga
acatggacaa attataccca gtgtaatgtt 420aacacccacg agaaagtgaa
gactgcacta aatttgtttt acctgaccat aattggacac 480ggattgtcta
ttgcatcact gcttatctcg cttggcatat tcttttattt caagagccta
540agttgccaaa ggattacctt acacaaaaat ctgttcttct catttgtttg
taactctgtt 600gtaacaatca ttcacctcac tgcagtggcc aacaaccagg
ccttagtagc cacaaatcct 660gttagttgca aagtgtccca gttcattcat
ctttacctga tgggctgtaa ttacttttgg 720atgctctgtg aaggcattta
cctacacaca ctcattgtgg tggccgtgtt tgcagagaag 780caacatttaa
tgtggtatta ttttcttggc tggggatttc cactgattcc tgcttgtata
840catgccattg ctagaagctt atattacaat gacaattgct ggatcagttc
tgatacccat 900ctcctctaca ttatccatgg cccaatttgt gctgctttac
tggtgaatct ttttttcttg 960ttaaatattg tacgcgttct catcaccaag
ttaaaagtta cacaccaagc ggaatccaat 1020ctgtacatga aagctgtgag
agctactctt atcttggtgc cattgcttgg cattgaattt 1080gtgctgattc
catggcgacc tgaaggaaag attgcagagg aggtatatga ctacatcatg
1140cacatcctta tgcacttcca gggtcttttg gtctctacca ttttctgctt
ctttaatgga 1200gaggttcaag caattctgag aagaaactgg aatcaataca
aaatccaatt tggaaacagc 1260ttttccaact cagaagctct tcgtagtgcg
tcttacacag tgtcaacaat cagtgatggt 1320ccaggttata gtcatgactg
tcctagtgaa cacttaaatg gaaaaagcat ccatgatatt 1380gaaaatgttc
tcttaaaacc agaaaattta tataattga 14192472PRTHomo sapiens 2Met Leu
Tyr Ser Ile Phe His Phe Gly Leu Met Met Glu Lys Lys Cys 1 5 10 15
Thr Leu Tyr Phe Leu Val Leu Leu Pro Phe Phe Met Ile Leu Val Thr 20
25 30 Ala Glu Leu Glu Glu Ser Pro Glu Asp Ser Ile Gln Leu Gly Val
Thr 35 40 45 Arg Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys Tyr Gln
Lys Ile Met 50 55 60 Gln Asp Pro Ile Gln Gln Ala Glu Gly Val Tyr
Cys Asn Arg Thr Trp 65 70 75 80 Asp Gly Trp Leu Cys Trp Asn Asp Val
Ala Ala Gly Thr Glu Ser Met 85 90 95 Gln Leu Cys Pro Asp Tyr Phe
Gln Asp Phe Asp Pro Ser Glu Lys Val 100 105 110 Thr Lys Ile Cys Asp
Gln Asp Gly Asn Trp Phe Arg His Pro Ala Ser 115 120 125 Asn Arg Thr
Trp Thr Asn Tyr Thr Gln Cys Asn Val Asn Thr His Glu 130 135 140 Lys
Val Lys Thr Ala Leu Asn Leu Phe Tyr Leu Thr Ile Ile Gly His 145 150
155 160 Gly Leu Ser Ile Ala Ser Leu Leu Ile Ser Leu Gly Ile Phe Phe
Tyr 165 170 175 Phe Lys Ser Leu Ser Cys Gln Arg Ile Thr Leu His Lys
Asn Leu Phe 180 185 190 Phe Ser Phe Val Cys Asn Ser Val Val Thr Ile
Ile His Leu Thr Ala 195 200 205 Val Ala Asn Asn Gln Ala Leu Val Ala
Thr Asn Pro Val Ser Cys Lys 210 215 220 Val Ser Gln Phe Ile His Leu
Tyr Leu Met Gly Cys Asn Tyr Phe Trp 225 230 235 240 Met Leu Cys Glu
Gly Ile Tyr Leu His Thr Leu Ile Val Val Ala Val 245 250 255 Phe Ala
Glu Lys Gln His Leu Met Trp Tyr Tyr Phe Leu Gly Trp Gly 260 265 270
Phe Pro Leu Ile Pro Ala Cys Ile His Ala Ile Ala Arg Ser Leu Tyr 275
280 285 Tyr Asn Asp Asn Cys Trp Ile Ser Ser Asp Thr His Leu Leu Tyr
Ile 290 295 300 Ile His Gly Pro Ile Cys Ala Ala Leu Leu Val Asn Leu
Phe Phe Leu 305 310 315 320 Leu Asn Ile Val Arg Val Leu Ile Thr Lys
Leu Lys Val Thr His Gln 325 330 335 Ala Glu Ser Asn Leu Tyr Met Lys
Ala Val Arg Ala Thr Leu Ile Leu 340 345 350 Val Pro Leu Leu Gly Ile
Glu Phe Val Leu Ile Pro Trp Arg Pro Glu 355 360 365 Gly Lys Ile Ala
Glu Glu Val Tyr Asp Tyr Ile Met His Ile Leu Met 370 375 380 His Phe
Gln Gly Leu Leu Val Ser Thr Ile Phe Cys Phe Phe Asn Gly 385 390 395
400 Glu Val Gln Ala Ile Leu Arg Arg Asn Trp Asn Gln Tyr Lys Ile Gln
405 410 415 Phe Gly Asn Ser Phe Ser Asn Ser Glu Ala Leu Arg Ser Ala
Ser Tyr 420 425 430 Thr Val Ser Thr Ile Ser Asp Gly Pro Gly Tyr Ser
His Asp Cys Pro 435 440 445 Ser Glu His Leu Asn Gly Lys Ser Ile His
Asp Ile Glu Asn Val Leu 450 455 460 Leu Lys Pro Glu Asn Leu Tyr Asn
465 470 3447DNAHomo sapiens 3atggcccggg ccctgtgccg cctcccgcgg
cgcggcctct ggctgctcct ggcccatcac 60ctcttcatga ccactgcctg ccaggaggct
aactacggtg ccctcctccg ggagctctgc 120ctcacccagt tccaggtaga
catggaggcc gtcggggaga cgctgtggtg tgactggggc 180aggaccatca
ggagctacag ggagctggcc gactgcacct ggcacatggc ggagaagctg
240ggctgcttct ggcccaatgc agaggtggac aggttcttcc tggcagtgca
tggccgctac 300ttcaggagct gccccatctc aggcagggcc gtgcgggacc
cgcccggcag catcctctac 360cccttcatcg tggtccccat cacggtgacc
ctgctggtga cggcactggt ggtctggcag 420agcaagcgca ctgagggcat tgtgtag
4474148PRTHomo sapiens 4Met Ala Arg Ala Leu Cys Arg Leu Pro Arg Arg
Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met Thr Thr
Ala Cys Gln Glu Ala Asn Tyr 20 25 30 Gly Ala Leu Leu Arg Glu Leu
Cys Leu Thr Gln Phe Gln Val Asp Met 35 40 45 Glu Ala Val Gly Glu
Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Arg 50 55 60 Ser Tyr Arg
Glu Leu Ala Asp Cys Thr Trp His Met Ala Glu Lys Leu 65 70 75 80 Gly
Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala Val 85 90
95 His Gly Arg Tyr Phe Arg Ser Cys Pro Ile Ser Gly Arg Ala Val Arg
100 105 110 Asp Pro Pro Gly Ser Ile Leu Tyr Pro Phe Ile Val Val Pro
Ile Thr 115 120 125 Val Thr Leu Leu Val Thr Ala Leu Val Val Trp Gln
Ser Lys Arg Thr 130 135 140 Glu Gly Ile Val 145 5414DNAHomo sapiens
5atggagaaaa agtgtaccct gtattttctg gttctcttgc ctttttttat gattcttgtt
60acagcagaat tagaagagag tcctgaggac tcaattcagt tgggagttac tagaaataaa
120atcatgacag ctcaatatga atgttaccaa aagattatgc aagaccccat
tcaacaagca 180gaaggcgttt actgcaacag aacctgggat ggatggctct
gctggaacga tgttgcagca 240ggaactgaat caatgcagct ctgccctgat
tactttcagg actttgatcc atcagaaaaa 300gttacaaaga tctgtgacca
agatggaaac tggtttagac atccagcaag caacagaaca 360tggacaaatt
atacccagtg taatgttaac acccacgaga aagtgaagac tgca 4146138PRTHomo
sapiens 6Met Glu Lys Lys Cys Thr Leu Tyr Phe Leu Val Leu Leu Pro
Phe Phe 1 5 10 15 Met Ile Leu Val Thr Ala Glu Leu Glu Glu Ser Pro
Glu Asp Ser Ile 20 25 30 Gln Leu Gly Val Thr Arg Asn Lys Ile Met
Thr Ala Gln Tyr Glu Cys 35 40 45 Tyr Gln Lys Ile Met Gln Asp Pro
Ile Gln Gln Ala Glu Gly Val Tyr 50 55 60 Cys Asn Arg Thr Trp Asp
Gly Trp Leu Cys Trp Asn Asp Val Ala Ala 65 70 75 80 Gly Thr Glu Ser
Met Gln Leu Cys Pro Asp Tyr Phe Gln Asp Phe Asp 85 90 95 Pro Ser
Glu Lys Val Thr Lys Ile Cys Asp Gln Asp Gly Asn Trp Phe 100 105 110
Arg His Pro Ala Ser Asn Arg Thr Trp Thr Asn Tyr Thr Gln Cys Asn 115
120 125 Val Asn Thr His Glu Lys Val Lys Thr Ala 130 135 7351DNAHomo
sapiens 7atggcccggg ccctgtgccg cctcccgcgg cgcggcctct ggctgctcct
ggcccatcac 60ctcttcatga ccactgcctg ccaggaggct aactacggtg ccctcctccg
ggagctctgc 120ctcacccagt tccaggtaga catggaggcc gtcggggaga
cgctgtggtg tgactggggc 180aggaccatca ggagctacag ggagctggcc
gactgcacct ggcacatggc ggagaagctg 240ggctgcttct ggcccaatgc
agaggtggac aggttcttcc tggcagtgca tggccgctac 300ttcaggagct
gccccatctc aggcagggcc gtgcgggacc cgcccggcag c 3518117PRTHomo
sapiens 8Met Ala Arg Ala Leu Cys Arg Leu Pro Arg Arg Gly Leu Trp
Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met Thr Thr Ala Cys Gln
Glu Ala Asn Tyr 20 25 30 Gly Ala Leu Leu Arg Glu Leu Cys Leu Thr
Gln Phe Gln Val Asp Met 35 40 45 Glu Ala Val Gly Glu Thr Leu Trp
Cys Asp Trp Gly Arg Thr Ile Arg 50 55 60 Ser Tyr Arg Glu Leu Ala
Asp Cys Thr Trp His Met Ala Glu Lys Leu 65 70 75 80 Gly Cys Phe Trp
Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala Val 85 90 95 His Gly
Arg Tyr Phe Arg Ser Cys Pro Ile Ser Gly Arg Ala Val Arg 100 105 110
Asp Pro Pro Gly Ser 115 931PRTHomo sapiens 9Trp Val Thr His Arg Leu
Ala Gly Leu Leu Ser Arg Ser Gly Gly Val 1 5 10 15 Val Arg Cys Asn
Phe Val Pro Thr Asp Val Gly Pro Phe Ala Phe 20 25 30 10116PRTHomo
sapiens 10Glu Leu Glu Glu Ser Pro Glu Asp Ser Ile Gln Leu Gly Val
Thr Arg 1 5 10 15 Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys Tyr Gln
Lys Ile Met Gln 20 25 30 Asp Pro Ile Gln Gln Ala Glu Gly Val Tyr
Cys Asn Arg Thr Trp Asp 35 40 45 Gly Trp Leu Cys Trp Asn Asp Val
Ala Ala Gly Thr Glu Ser Met Gln 50 55 60 Leu Cys Pro Asp Tyr Phe
Gln Asp Phe Asp Pro Ser Glu Lys Val Thr 65 70 75 80 Lys Ile Cys Asp
Gln Asp Gly Asn Trp Phe Arg His Pro Ala Ser Asn 85 90 95 Arg Thr
Trp Thr Asn Tyr Thr Gln Cys Asn Val Asn Thr His Glu Lys 100 105 110
Val Lys Thr Ala 115 1191PRTHomo sapiens 11Cys Gln Glu Ala Asn Tyr
Gly Ala Leu Leu Arg Glu Leu Cys Leu Thr 1 5 10 15 Gln Phe Gln Val
Asp Met Glu Ala Val Gly Glu Thr Leu Trp Cys Asp 20 25 30 Trp Gly
Arg Thr Ile Arg Ser Tyr Arg Glu Leu Ala Asp Cys Thr Trp 35 40 45
His Met Ala Glu Lys Leu Gly Cys Phe Trp Pro Asn Ala Glu Val Asp 50
55 60 Arg Phe Phe Leu Ala Val His Gly Arg Tyr Phe Arg Ser Cys Pro
Ile 65 70 75 80 Ser Gly Arg Ala Val Arg Asp Pro Pro Gly Ser 85 90
12238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ser Val
Leu Thr Gln Pro Pro Ser Val 20 25 30 Ser Glu Ala Pro Gly Gln Lys
Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45 Ser Asn Ile Gly Asn
Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly 50 55 60 Thr Ala Pro
Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly 65 70 75 80 Ile
Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu 85 90
95 Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly
100 105 110 Thr Trp Asp Ser Arg Leu Ser Ala Val Val Phe Gly Gly Gly
Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr
Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175 Asp Gly Ser Pro Val
Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180 185 190 Gln Ser Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 195 200 205 Glu
Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 210 215
220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230
235 13238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala 20 25 30 Ser Gly Thr Pro Gly Gln Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45 Ser Asn Ile Gly Ser
Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 50 55 60 Ala Ala Pro
Lys Leu Leu Ile Phe Arg Ser Asn Gln Arg Pro Ser Gly 65 70 75 80 Val
Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu 85 90
95 Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
100 105 110 Ala Trp Asp Asp Ser Leu Ser Gly Trp Val Phe Gly Gly Gly
Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr
Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175 Asp Gly Ser Pro Val
Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180 185 190 Gln Ser Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 195 200 205 Glu
Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 210 215
220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230
235 14236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Asn
Asp Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys
Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90
95 Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln
100 105 110 Tyr Asn Ile Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235 15236PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 15Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Ser Ser Glu Leu Thr Gln Asp Pro Thr Val 20 25 30 Ser Val Ala Leu
Gly Gln Thr Val Lys Ile Thr Cys Gln Gly Asp Ser 35 40 45 Leu Arg
Ser Phe Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60
Pro Val Leu Val Phe Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro 65
70 75 80 Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu
Thr Ile 85 90 95 Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
Cys Asn Ser Arg 100 105 110 Asp Ser Ser Val Tyr His Leu Val Leu Gly
Gly Gly Thr Lys Leu Thr 115 120 125 Val Leu Gly Gln Pro Lys Ala Asn
Pro Thr Val Thr Leu Phe Pro Pro 130 135 140 Ser Ser Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile 145 150 155 160 Ser Asp Phe
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly 165 170 175 Ser
Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser 180 185
190 Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
195 200 205 Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser 210 215 220 Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
225 230 235 16241PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 16Met Asp Met Arg Val Pro Ala Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys
Asp Ile Ile Leu Ala Gln Thr Pro Leu Ser 20 25 30 Leu Ser Val Thr
Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser 35 40 45 Gln Ser
Leu Leu His Ser Ala Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu 50 55 60
Gln Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn 65
70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp Val Gly Ile 100 105 110 Tyr Tyr Cys Met Gln Ser Phe Pro Leu Pro
Leu Thr Phe Gly Gly Gly 115 120 125 Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile 130 135 140 Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150 155 160 Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175 Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185
190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
195 200 205 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr 210 215 220 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu 225 230 235 240 Cys 17238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Gln Ser Val Leu Thr Gln Pro Pro Ser
Val 20 25 30 Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser
Gly Ser Ser 35 40 45 Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr
Gln Gln Leu Pro Gly 50 55 60 Thr Ala Pro Lys Leu Leu Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly 65 70 75 80 Ile Pro Asp Arg Phe Ser Gly
Ser Lys Ser Gly Thr Ser Thr Thr Leu 85 90 95 Gly Ile Thr Gly Leu
Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly 100 105 110 Thr Trp Asp
Ser Arg Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys 115 120 125 Leu
Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe 130 135
140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala
Trp Lys Ala 165 170 175 Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr
Thr Lys Pro Ser Lys 180 185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro 195 200 205 Glu Gln Trp Lys Ser His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu 210 215 220 Gly Ser Thr Val Glu
Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235 18241PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Leu
Ser 20 25 30 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser 35 40 45 Gln Ser Leu Leu His Ser Phe Gly Tyr Asn Tyr
Leu Asp Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu
Leu Ile Tyr Leu Gly Ser Asn 65 70 75 80 Arg Ala Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110 Tyr Tyr Cys
Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly 115 120 125 Thr
Lys Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135
140 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
145 150 155 160 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys 165 170 175 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu 195 200 205 Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220 His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240 Cys
19241PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Ile
Leu Thr Gln Thr Pro Leu Ser 20 25 30 Leu Ser Val Thr Pro Gly Gln
Pro Ala Ser Ile Ser Cys Lys Ser Ser 35 40 45 Gln Ser Leu Leu His
Ser Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu 50 55 60 Gln Lys Pro
Gly Gln Pro Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn 65 70 75 80 Arg
Phe Ser Gly Glu Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90
95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Thr
100 105 110 Tyr Tyr Cys Met Gln Ser Phe Pro Leu Pro Leu Thr Phe Gly
Gly Gly 115 120 125 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile 130 135 140 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val 145 150 155 160 Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175 Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190 Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205 Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215
220 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
225 230 235 240 Cys 20238PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Ser Val Leu Thr Gln Pro Pro Ser Val 20 25 30 Ser Ala
Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45
Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly 50
55 60 Thr Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly 65 70 75 80 Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Thr Leu 85 90 95 Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala
Asp Tyr Tyr Cys Gly 100 105 110 Thr Trp Asp Ser Arg Leu Ser Ala Val
Val Phe Gly Gly Gly Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro
Lys Ala Asn Pro Thr Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile
Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175
Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180
185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro 195 200 205 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu 210 215 220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 225 230 235 21238PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 21Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Ser Val Leu Thr Gln Ser Pro Ser Ala 20 25 30 Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45
Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 50
55 60 Ala Ala Pro Lys Leu Leu Ile Leu Arg Asn Asn Gln Arg Pro Ser
Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu 85 90 95 Thr Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala 100 105 110 Ala Trp Asp Asp Ser Leu Ser Gly Trp
Val Phe Gly Gly Gly Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro
Lys Ala Asn Pro Thr Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile
Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175
Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180
185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro 195 200 205 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu 210 215 220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 225 230 235 22238PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 22Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 20 25 30 Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45
Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly 50
55 60 Thr Ala Pro Lys Leu Leu Ile Tyr Thr Asn Asn Gln Arg Pro Ser
Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu 85 90 95 Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala
Asp Phe Tyr Cys Ala 100 105 110 Ala Arg Asp Glu Ser Leu Asn Gly Val
Val Phe Gly Gly Gly Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro
Lys Ala Asn Pro Thr Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile
Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175
Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180
185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro 195 200 205 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu 210 215 220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 225 230 235 23238PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 20 25 30 Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45
Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 50
55 60 Ala Ala Pro Lys Leu Leu Ile Phe Arg Asn Asn Gln Arg Pro Ser
Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu 85 90 95 Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala 100 105 110 Ala Trp Asp Asp Ser Leu Ser Gly Trp
Val Phe Gly Gly Gly Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro
Lys Ala Asn Pro Thr Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile
Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175
Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180
185 190 Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro 195 200 205 Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu 210 215 220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 225 230 235 24241PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Asp Ile Thr Leu Thr Gln Thr Pro Leu Ser 20
25 30 Leu Ser Val Ser Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser
Ser 35 40 45 Gln Ser Leu Leu His Ser Asp Gly Arg Asn Tyr Leu Tyr
Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile
Tyr Glu Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Leu Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Val Gly Ile 100 105 110 Tyr Tyr Cys Met Gln
Ser Phe Pro Leu Pro Leu Thr Phe Gly Gly Gly 115 120 125 Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135 140 Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150
155 160 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys 165 170 175 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu 195 200 205 Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr 210 215 220 His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240 Cys
25238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ser Val
Leu Thr Gln Pro Pro Ser Val 20 25 30 Ser Ala Ala Pro Gly Gln Lys
Val Thr Ile Ser Cys Ser Gly Ser Ser 35 40 45 Ser Asn Ile Gly Asn
Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly 50 55 60 Thr Ala Pro
Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly 65 70 75 80 Ile
Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu 85 90
95 Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly
100 105 110 Thr Trp Asp Ser Arg Leu Ser Ala Val Val Phe Gly Gly Gly
Thr Lys 115 120 125 Leu Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr
Val Thr Leu Phe 130 135 140 Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys 145 150 155 160 Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala 165 170 175 Asp Gly Ser Pro Val
Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys 180 185 190 Gln Ser Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 195 200 205 Glu
Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu 210 215
220 Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230
235 26236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Arg Lys
Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys
Arg Leu Ile Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90
95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
100 105 110 Tyr Asn Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
27235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu
Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser Ser Gly Tyr
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Leu
Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90
95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
100 105 110 Gly Asn Ser Leu Cys Arg Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215
220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
28235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu
Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser Ser Gly Tyr
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Leu
Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90
95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
100 105 110 Gly Asn Ser Leu Ser Arg Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215
220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
29478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser
Phe Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ala Val Ile Ser Phe Asp Gly Ser Ile Lys 65 70 75 80 Tyr
Ser Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90
95 Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Leu Asn Tyr Tyr
Asp Ser 115 120 125 Ser Gly Tyr Tyr His Tyr Lys Tyr Tyr Gly Met Ala
Val Trp Gly Gln 130 135 140 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 145 150 155 160 Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 165 170 175 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 180 185 190 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 195 200 205 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 210 215
220 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
225 230 235 240 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys
Cys Cys Val 245 250 255 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 325 330 335
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340
345 350 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 355 360 365 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 370 375 380 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
30479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Lys Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Asn
Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Gly Arg Ile Lys Ser Thr Thr Asp Gly Gly 65 70 75 80 Thr
Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90
95 Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Asp Arg Thr Gly
Tyr Ser 115 120 125 Ile Ser Trp Ser Ser Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly 130 135 140 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 145 150 155 160 Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 165 170 175 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 180 185 190 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 195 200 205 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 210 215
220 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
225 230 235 240 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys Cys 245 250 255 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 325 330 335
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340
345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile 355 360 365 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 370 375 380 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 420 425 430 Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
475 31478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Glu Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser
Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Arg Thr 65 70 75 80 Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90
95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100
105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Asp Gln Arg Glu Val Gly Pro
Tyr 115 120 125 Ser Ser Gly Trp Tyr Asp Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln 130 135 140 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 145 150 155 160 Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala 165 170 175 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 180 185 190 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 195 200 205 Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 210 215 220
Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 225
230 235 240 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val 245 250 255 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val 290 295 300 Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 325 330 335 Leu
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345
350 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
355 360 365 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 370 375 380 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
32478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Gly
Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu
Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90
95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
100 105 110 Thr Ala Val Tyr Phe Cys Ala Arg Asp Gln Met Ser Ile Ile
Met Leu 115 120 125 Arg Gly Val Phe Pro Pro Tyr Tyr Tyr Gly Met Asp
Val Trp Gly Gln 130 135 140 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 145 150 155 160 Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 165 170 175 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 180 185 190 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 195 200 205 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 210 215
220 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
225 230 235 240 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys
Cys Cys Val 245 250 255 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 325 330 335
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340
345 350 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 355 360 365 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 370 375 380 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
33477PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser
Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser His Glu 65 70 75 80 Ser
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile 85 90
95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100 105 110 Thr Ala Val Tyr Phe Cys Ala Arg Glu Arg Lys Arg Val Thr
Met Ser 115 120 125 Thr Leu Tyr Tyr Tyr Phe Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly 130 135 140 Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 145 150 155 160 Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu 165 170 175 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 180 185 190 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 195 200 205 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 210 215
220 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
225 230 235 240 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val Glu 245 250 255 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 275 280 285 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln 290 295 300 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320 Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 325 330 335
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 340
345 350 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 355 360 365 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 370 375 380 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 405 410 415 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 420 425 430 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
34469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 34Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Lys Pro Gly Arg Ser
Leu Arg Leu Ser Cys Thr Ala Ser Gly 35 40 45 Phe Thr Phe Gly Asp
Tyr Ala Met Ser Trp Phe Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Ile Gly Phe Ile Arg Ser Arg Ala Tyr Gly Gly 65 70 75 80 Thr
Pro Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90
95 Asp Asp Ser Lys Thr Ile Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr
100 105 110 Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Gly Arg Gly Ile
Ala Ala 115 120 125 Arg Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser 145 150 155 160 Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 210 215
220 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr
225 230 235 240 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro
Ala Pro Pro 245 250 255 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 305 310 315 320 Thr Phe
Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 340
345 350 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460
Leu Ser Pro Gly Lys 465 35479PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 35Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala
Arg Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val
Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45
Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 50
55 60 Lys Gly Leu Glu Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly
Gly 65 70 75 80 Thr Thr Asp Tyr Thr Ala Pro Val Lys Gly Arg Phe Thr
Ile Ser Arg 85 90 95 Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Lys Ala 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Thr Asp Arg Thr Gly Tyr Ser 115 120 125 Ile Ser Trp Ser Ser Tyr Tyr
Tyr Tyr Tyr Gly Met Asp Val Trp Gly 130 135 140 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 145 150 155 160 Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 165 170 175
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 180
185 190 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 195 200 205 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 210 215 220 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr
Cys Asn Val Asp His 225 230 235 240 Lys Pro Ser Asn Thr Lys Val Asp
Lys Thr Val Glu Arg Lys Cys Cys 245 250 255 Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 290 295 300
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305
310 315 320 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
Val Ser 325 330 335 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 340 345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
Pro Ile Glu Lys Thr Ile 355 360 365 Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro 370 375 380 Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 420 425
430 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
435 440 445 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
475 36475PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asp
Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu
Glu Trp Met Gly Trp Ile Ser Pro Asn Ser Gly Gly Thr 65 70 75 80 Asn
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 85 90
95 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
100 105 110 Thr Ala Val Tyr Tyr Cys Val Arg Gly Gly Tyr Ser Gly Tyr
Ala Gly 115 120 125 Leu Tyr Ser His Tyr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr 130 135 140 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 145 150 155 160 Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys 165 170 175 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 180 185 190 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 195 200 205 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 210 215
220 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
225 230 235 240 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro 245 250 255 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn 290 295 300 Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 325 330 335
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340
345 350 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 435 440 445 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
37479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Lys Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Gly Asn
Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly 65 70 75 80 Thr
Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90
95 Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
100 105 110 Glu Asp Thr Ala Val Tyr Phe Cys Thr Thr Asp Arg Thr Gly
Tyr Ser 115 120 125 Ile Ser Trp Ser Ser Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly 130 135 140 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 145 150 155 160 Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 165 170 175 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 180 185 190 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 195 200 205 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 210 215
220 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
225 230 235 240 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys Cys 245 250 255 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 325 330 335
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340
345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile 355 360 365 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 370 375 380 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 420 425 430 Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
475 38479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Lys Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Gly Asn
Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly 65 70 75 80 Thr
Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg 85 90
95 Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Asp Arg Thr Gly
Tyr Ser 115 120 125 Ile Ser Trp Ser Ser Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly 130 135 140 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 145 150 155 160 Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 165 170 175 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 180 185 190 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 195 200 205 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 210 215
220 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
225 230 235 240 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys Cys 245 250 255 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 325 330 335
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340
345 350 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile 355 360 365 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 370 375 380 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 420 425 430 Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
475 39478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser
Phe Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ala Val Ile Ser Phe Asp Gly Ser Ile Lys 65 70 75 80 Tyr
Ser Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90
95 Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Leu Asn Tyr Tyr
Asp Ser 115 120 125 Ser Gly Tyr Tyr His Tyr Lys Tyr Tyr Gly Leu Ala
Val Trp Gly Gln 130 135 140 Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 145 150 155 160 Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 165 170 175 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 180 185 190 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 195 200 205 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 210 215
220 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
225 230 235 240 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys
Cys Cys Val 245 250 255 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 325 330 335
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340
345 350 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 355 360 365 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 370 375 380 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
40479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 40Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Lys Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Tyr Thr Phe Ser Thr
Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Arg 65 70 75 80 Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90
95 Ala Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp
100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Val Ser Gly Ser
Ser Pro 115 120 125 Tyr Ser Ile Ser Trp Tyr Asp Tyr Tyr Tyr Gly Met
Asp Val Trp Gly 130 135 140 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 145 150 155 160 Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 165 170 175 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 180 185 190 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 195 200 205 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 210 215
220 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
225 230 235 240 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys Cys 245 250 255 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 290 295 300 Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys
Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 325 330 335 Val Leu
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 340 345 350
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 355
360 365 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro 370 375 380 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser 420 425 430 Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455 460 His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475
41474PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 41Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly
Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln
Leu Val Glu Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser
Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu
Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys 65 70 75 80 Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Lys 85 90
95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Gly Ile Ala Ala
Ala Gly 115 120 125 Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val 130 135 140 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala 145 150 155 160 Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu 165 170 175 Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 180 185 190 Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 195 200 205 Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe 210 215
220 Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
225 230 235 240 Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro 245 250 255 Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val 325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340
345 350 Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 4213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Ser
Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser 1 5 10
437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Asp Asn Asn Lys Arg Pro Ser 1 5
4411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gly Thr Trp Asp Ser Arg Leu Ser Ala Val Val 1 5
10 4513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val
Tyr 1 5 10 467PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 46Arg Ser Asn Gln Arg Pro Ser 1 5
4711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5
10 4811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5
10 497PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Ala Ala Ser Ser Leu Gln Ser 1 5
509PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Leu Gln Tyr Asn Ile Tyr Pro Trp Thr 1 5
5111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Gln Gly Asp Ser Leu Arg Ser Phe Tyr Ala Ser 1 5
10 527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gly Lys Asn Asn Arg Pro Ser 1 5
5311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Asn Ser Arg Asp Ser Ser Val Tyr His Leu Val 1 5
10 5416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Lys Ser Ser Gln Ser Leu Leu His Ser Ala Gly Lys
Thr Tyr Leu Tyr 1 5 10 15 557PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Glu Val Ser Asn Arg Phe Ser
1 5 569PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Met Gln Ser Phe Pro Leu Pro Leu Thr 1 5
5716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Arg Ser Ser Gln Ser Leu Leu His Ser Phe Gly Tyr
Asn Tyr Leu Asp 1 5 10 15 587PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Leu Gly Ser Asn Arg Ala Ser
1 5 599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Met Gln Ala Leu Gln Thr Pro Phe Thr 1 5
6016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Lys Ser Ser Gln Ser Leu Leu His Ser Asp Gly Lys
Thr Tyr Leu Tyr 1 5 10 15 617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Arg Asn Asn Gln Arg Pro Ser
1 5 6213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val
Asn 1 5 10 637PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Thr Asn Asn Gln Arg Pro Ser 1 5
6411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Ala Ala Arg Asp Glu Ser Leu Asn Gly Val Val 1 5
10 6516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Lys Ser Ser Gln Ser Leu Leu His Ser Asp Gly Arg
Asn Tyr Leu Tyr 1 5 10 15 6611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 66Arg Ala Ser Gln Gly Ile Arg
Lys Asp Leu Gly 1 5 10 677PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Gly Ala Ser Ser Leu Gln Ser
1 5 689PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Leu Gln Tyr Asn Ser Phe Pro Trp Thr 1 5
6912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 69Arg Ala Ser Gln Ser Val Ser Ser Gly Tyr Leu Thr
1 5 10 707PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Gly Ala Ser Ser Arg Ala Thr 1 5
719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 71Gln Gln Tyr Gly Asn Ser Leu Cys Arg 1 5
729PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Gln Gln Tyr Gly Asn Ser Leu Ser Arg 1 5
735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Ser Phe Gly Met His 1 5 7417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Val
Ile Ser Phe Asp Gly Ser Ile Lys Tyr Ser Val Asp Ser Val Lys 1 5 10
15 Gly 7521PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Asp Arg Leu Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr
His Tyr Lys Tyr 1 5 10 15 Tyr Gly Met Ala Val 20 765PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Asn
Ala Trp Met Ser 1 5 7719PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 77Arg Ile Lys Ser Thr Thr Asp
Gly Gly Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly
7820PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Asp Arg Thr Gly Tyr Ser Ile Ser Trp Ser Ser Tyr
Tyr Tyr Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 795PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Ser
Tyr Ala Met Ser 1 5 8017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 80Ala Ile Ser Gly Ser Gly Gly
Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 8121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Asp
Gln Arg Glu Val Gly Pro Tyr Ser Ser Gly Trp Tyr Asp Tyr Tyr 1 5 10
15 Tyr Gly Met Asp Val 20 825PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 82Gly Tyr Tyr Met His 1 5
8317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe Gln 1 5 10 15 Gly 8421PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 84Asp Gln Met Ser Ile Ile
Met Leu Arg Gly Val Phe Pro Pro Tyr Tyr 1 5 10 15 Tyr Gly Met Asp
Val 20 855PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Ser Tyr Gly Met His 1 5 8617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Val
Ile Ser Tyr Asp Gly Ser His Glu Ser Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 8720PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 87Glu Arg Lys Arg Val Thr Met Ser Thr Leu Tyr Tyr
Tyr Phe Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 885PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Asp
Tyr Ala Met Ser 1 5 8919PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Phe Ile Arg Ser Arg Ala Tyr
Gly Gly Thr Pro Glu Tyr Ala Ala Ser 1 5 10 15 Val Lys Gly
9010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Gly Arg Gly Ile Ala Ala Arg Trp Asp Tyr 1 5 10
9119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp
Tyr Thr Ala Pro 1 5 10 15 Val Lys Gly 925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Asp
Tyr Tyr Met Tyr 1 5 9317PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 93Trp Ile Ser Pro Asn Ser Gly
Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 9418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Gly
Gly Tyr Ser Gly Tyr Ala Gly Leu Tyr Ser His Tyr Tyr Gly Met 1 5 10
15 Asp Val 9519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 95Arg Ile Lys Ser Lys Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly 9621PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 96Asp
Arg Leu Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Tyr Lys Tyr 1 5 10
15 Tyr Gly Leu Ala Val 20 975PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 97Thr Tyr Ser Met Asn 1 5
9817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 98Ser Ile Ser Ser Ser Ser Ser Tyr Arg Tyr Tyr Ala
Asp Ser Val Lys 1 5 10 15 Gly 9922PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 99Glu Gly Val Ser Gly Ser
Ser Pro Tyr Ser Ile Ser Trp Tyr Asp Tyr 1 5 10 15 Tyr Tyr Gly Met
Asp Val 20 1005PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 100Ser Tyr Gly Met His 1 5
10117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 101Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 10217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Ala
Gly Gly Ile Ala Ala Ala Gly Leu Tyr Tyr Tyr Tyr Gly Met Asp 1 5 10
15 Val 10311PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 103Arg Ala Ser Gln Gly Ile Arg Xaa Asp
Leu Gly 1 5 10 1047PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 104Xaa Ala Ser Ser Leu Gln Ser 1 5
1059PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Leu Gln Tyr Asn Xaa Xaa Pro Trp Thr 1 5
1069PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 106Gln Gln Tyr Gly Asn Ser Leu Xaa Arg 1 5
10712PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Arg Ala Ser Gln Xaa Xaa Xaa Xaa Gly Xaa Leu
Xaa 1 5 10 1087PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 108Xaa Ala Ser Ser Xaa Xaa Xaa 1 5
1099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Xaa Gln Tyr Xaa Xaa Xaa Xaa Xaa Xaa 1 5
11016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Lys Ser Ser Gln Ser Leu Leu His Ser Xaa Gly
Xaa Xaa Tyr Leu Tyr 1 5 10 15 11116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Xaa
Ser Ser Gln Ser Leu Leu His Ser Xaa Gly Xaa Xaa Tyr Leu Xaa 1 5 10
15 1127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Xaa Xaa Ser Asn Arg Xaa Ser 1 5
1139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Met
Gln Xaa Xaa Xaa Xaa Pro Xaa Thr 1 5 1147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Arg
Xaa Asn Gln Arg Pro Ser 1 5 11513PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 115Ser Gly Ser Ser Ser Asn
Ile Gly Xaa Asn Xaa Val Xaa 1 5 10 1167PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 116Xaa
Xaa Asn Xaa Arg Pro Ser 1 5 11711PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 117Xaa Xaa Xaa Asp Xaa Xaa
Leu Xaa Xaa Val Val 1 5 10 11813PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 118Xaa Gly Xaa Xaa Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 1197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Xaa
Xaa Asn Xaa Arg Pro Ser 1 5 12011PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 120Xaa Xaa Xaa Asp Xaa Xaa
Xaa Xaa Xaa Xaa Val 1 5 10 1215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 121Xaa Tyr Tyr Met Xaa 1 5
12217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Trp Ile Xaa Pro Asn Ser Gly Gly Thr Asn Tyr
Ala Gln Lys Phe Gln 1 5 10 15 Gly 12321PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 123Xaa
Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Tyr Tyr 1 5 10
15 Xaa Gly Met Asp Val 20 12419PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 124Arg Ile Lys Ser Xaa Thr
Asp Gly Gly Thr Thr Asp Tyr Xaa Ala Pro 1 5 10 15 Val Lys Gly
1255PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Xaa Tyr Xaa Met Xaa 1 5 12617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 126Xaa
Ile Ser Xaa Ser Xaa Xaa Xaa Xaa Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 12722PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 127Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Tyr
Ser Xaa Xaa Trp Tyr Asp Tyr 1 5 10 15 Tyr Tyr Gly Met Asp Val 20
1285PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Ser Xaa Gly Met His 1 5 12917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 129Val
Ile Ser Xaa Asp Gly Ser Xaa Lys Tyr Xaa Xaa Asp Ser Val Lys 1 5 10
15 Gly 13021PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 130Xaa Arg Xaa Xaa Xaa Xaa Xaa Ser Xaa
Xaa Tyr Tyr Xaa Xaa Xaa Tyr 1 5 10 15 Tyr Gly Xaa Xaa Val 20
1315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Xaa Xaa Xaa Met Xaa 1 5 13219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 132Xaa
Ile Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Val Lys Gly 13321PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 133Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Gly Xaa Xaa Val 20
1345PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Xaa Xaa Xaa Xaa Xaa 1 5 13519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 135Xaa
Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Gly 13621PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 136Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Gly Xaa Xaa Val 20
137110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 137Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Glu Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn
Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu 85
90 95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 138110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 138Gln 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 Tyr Val Tyr Trp
Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Phe
Arg Ser 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 Arg 65
70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 139107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 139Asp 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 Gly Ile Arg Asn Asp 20 25 30 Leu
Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg 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 Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Tyr Asn
Ile Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 140108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 140Ser Ser Glu Leu Thr Gln Asp Pro
Thr Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Lys Ile Thr Cys
Gln Gly Asp Ser Leu Arg Ser Phe Tyr Ala 20 25 30 Ser Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Val Leu Val Phe Tyr 35 40 45 Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60
Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65
70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Val
Tyr His 85 90 95 Leu Val Leu Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 141112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 141Asp Ile Ile Leu Ala 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 Gln Ser Leu Leu His Ser 20 25 30 Ala Gly Lys Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln
Leu Leu Ile Tyr Glu 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 Ile Tyr Tyr Cys Met
Gln Ser 85 90 95 Phe Pro Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110 142110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 142Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr
Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Thr Thr Leu Gly Ile Thr Gly
Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp
Asp Ser Arg Leu 85 90 95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 143112PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 143Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Phe
Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys 100 105 110 144112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
144Asp Ile Ile Leu 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 Gln Ser Leu Leu
His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys
Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn
Arg Phe Ser Gly Glu 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 Thr Tyr Tyr Cys Met Gln Ser 85 90 95 Phe Pro Leu Pro
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
145110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 145Gln Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln
Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn
Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu 85
90 95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 146110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 146Gln Ser Val Leu Thr Gln Ser 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 Tyr Val Tyr Trp
Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Leu
Arg 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 Thr Ile Ser Gly Leu Arg 65
70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 147110PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 147Gln 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 Thr 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 Phe Tyr Cys Ala Ala Arg
Asp Glu Ser Leu 85 90 95 Asn Gly Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 148110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 148Gln 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 Tyr
Val Tyr Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40
45 Ile Phe Arg 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 Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
Asp Asp Ser Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 149112PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 149Asp Ile Thr Leu Thr
Gln Thr Pro Leu Ser Leu Ser Val Ser Pro Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asp
Gly Arg Asn Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40
45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Leu 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 Ile Tyr Tyr
Cys Met Gln Ser 85 90 95 Phe Pro Leu Pro Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 110 150110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
150Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln
1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp
Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu 85 90 95 Ser Ala Val Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
151107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
151Asp 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 Gly Ile Arg
Lys Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln Tyr Asn Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 152108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
152Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Gly 20 25 30 Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Leu 85 90 95 Cys Arg Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 153108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
153Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Gly 20 25 30 Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Leu 85 90 95 Ser Arg Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 154113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
154Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu
Asp Ser 20 25 30 Ser Asn Asn Asp Asn Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser
Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala
Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Thr
Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile 100 105 110 Lys
155107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 155Asp 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 Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Val Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asn Thr Tyr Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
156108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 156Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Arg Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 His Asp Ala Ser
Pro Arg Thr Ala Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Ser 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Tyr Trp Thr Pro 85
90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
157110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 157Gln Ser Val Leu Thr Gln Pro Pro Ser Met
Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn
Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80
Thr Gly Asp Glu Ala Asn Tyr Cys Cys Gly Thr Trp Asp Ile Gly Leu 85
90 95 Ser Val Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 158130PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 158Gln 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 Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val
Ile Ser Phe Asp Gly Ser Ile Lys Tyr Ser Val Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 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 Leu Asn Tyr Tyr Asp Ser Ser Gly
Tyr Tyr His Tyr 100 105 110 Lys Tyr Tyr Gly Met Ala Val Trp Gly Gln
Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130 159131PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
159Glu Val Gln Leu Val Glu Ser Gly Gly Gly 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 Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Thr Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr
Asp Arg Thr Gly Tyr Ser Ile Ser Trp Ser Ser Tyr 100 105 110 Tyr Tyr
Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 115 120 125
Val Ser Ser 130 160130PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 160Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu 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 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Ser Gly Ser Gly Gly Arg 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 Asp Gln Arg Glu Val Gly Pro Tyr
Ser Ser Gly Trp Tyr Asp 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val 115 120 125 Ser Ser 130
161130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 161Gln 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 Gly 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 Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85
90 95 Ala Arg Asp Gln Met Ser Ile Ile Met Leu Arg Gly Val Phe Pro
Pro 100 105 110 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val 115 120 125 Ser Ser 130 162129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
162Gln 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 Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Gly 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 His Glu
Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Ile Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Arg
Lys Arg Val Thr Met Ser Thr Leu Tyr Tyr Tyr Phe 100 105 110 Tyr Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125
Ser 163121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 163Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala
Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Ala Met Ser Trp Phe Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Arg
Ser Arg Ala Tyr Gly Gly Thr Pro Glu Tyr Ala Ala 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Thr Ile 65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95 Phe Cys Ala Arg Gly Arg Gly Ile Ala Ala Arg Trp Asp Tyr Trp
Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
164131PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 164Glu Val Gln Leu Val Glu Ser Gly Gly Gly
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 Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Thr Ala 50 55 60 Pro Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Thr Thr Asp Arg Thr Gly Tyr Ser Ile Ser Trp Ser Ser
Tyr 100 105 110 Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr 115 120 125 Val Ser Ser 130 165127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
165Gln 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 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Pro Asn Ser Gly Gly Thr
Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Gly
Tyr Ser Gly Tyr Ala Gly Leu Tyr Ser His Tyr Tyr 100 105 110 Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
166131PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 166Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Gly Asn Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys
Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85
90 95 Phe Cys Thr Thr Asp Arg Thr Gly Tyr Ser Ile Ser Trp Ser Ser
Tyr 100 105 110 Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr 115 120 125 Val Ser Ser 130 167131PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
167Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly
Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr
Asp Arg Thr Gly Tyr Ser Ile Ser Trp Ser Ser Tyr 100 105 110 Tyr Tyr
Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 115 120 125
Val Ser Ser 130 168130PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 168Gln 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 Ser Gly Phe Thr Phe
Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Phe Asp Gly Ser Ile
Lys Tyr Ser Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Phe 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 Leu Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Tyr 100 105 110 Lys
Tyr Tyr Gly Leu Ala Val Trp Gly Gln Gly Thr Thr Val Thr Val 115 120
125 Ser Ser 130 169131PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 169Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Thr Tyr 20 25 30 Ser
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Arg 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 Ser Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Val Ser Gly Ser Ser Pro
Tyr Ser Ile Ser Trp Tyr 100 105 110 Asp Tyr Tyr Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser 130
170126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 170Gln 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
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ile Ile Ser Arg Asp Lys 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 Ala Gly Gly Ile Ala Ala Ala Gly Leu Tyr Tyr Tyr Tyr
Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 125 171118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 171Gln 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 Ala Tyr 20 25 30 Tyr
Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Phe Tyr Cys 85 90 95 Ala Arg Gly Arg Gln Trp Leu Gly Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
172117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 172Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Ser Cys Ala Val
Tyr Gly Gly Ser Phe Gly Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
His Ser Gly Gly Thr Lys Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Ala 85
90 95 Arg Gly Asp Val Val Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 173120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
173Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Gly 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 Asn Ser Gly Gly Thr
Asn Tyr Val Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Glu
Tyr Ser Ser Ala Trp Pro Leu Gly Tyr Trp Gly Gln 100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120 174118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
174Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser
Thr Ser 20 25 30 Gly Val Gly Val Ala Trp Ile Arg Gln Pro Pro Gly
Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Thr Asp Asp
Lys Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr
Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Arg Met Thr Asn
Met Asp Pro Leu Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala His Arg
Pro Gly Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser 115 175321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 175gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gggcattaga aatgatttag gctggtttca gcagaaacca
120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
caatcagcag cctgcagcct 240gaagatttag caacttatta ctgtctacag
tataatattt acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa a
321176321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 176gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga
aaggatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct
gatctatgga gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct
240gaagattttg caacttatta ctgtctacag tataatagtt tcccgtggac
gttcggccaa 300gggaccaagg tggaaatcaa a 321177359DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
177aggtgcagct ggtgcagtct ggggctgagg tgaagaagtc tggggcctca
gtgaaggtct 60cctgcaaggc ttctggatac accttcaccg gctactatat gcactgggtg
cgacaggccc 120ctggacaagg gcttgagtgg atgggatgga tcaaccctaa
cagtggtggc acaaactatg 180tacagaagtt tcagggcagg gtcaccatga
ccagggacac gtccatcagc acagcctaca 240tggagctgag caggctgaga
tctgacgaca cggccgtgta ttactgtgcg agaaatgagt 300atagcagtgc
ctggcccttg gggtattggg gccagggaac cctggtcacc gtctctagt
359178336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 178gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
catagttttg ggtacaacta tttggattgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct
acaaactcca 300ttcactttcg gccctgggac caaagtggat atcaaa
336179336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 179gatattatac tggcccagac tccactttct
ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg
cacagtgctg gaaagaccta tttgtattgg 120tacctgcaga agccaggcca
gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagtgc
cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc
240agccgggtgg aggctgagga tgttgggatt tattactgca tgcaaagttt
tccgcttccg 300ctcactttcg gcggagggac caaggtggag atcaaa
336180336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 180gatattattc tgacccagac tccactttct
ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg
cacagtgatg gaaagaccta tttgtattgg 120tacctgcaga agcccggcca
gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagagc
cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc
240agccgggtgg aggctgagga tgttgggact tattattgca tgcaaagttt
tccgcttccg 300ctcactttcg gcggagggac caaggtggag atcaaa
336181336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 181gatattacac tgacccagac tccactttct
ctgtccgtct cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg
cacagtgatg gaaggaacta tctgtattgg 120tacctgcaga agccaggcca
gcctccacag ctcctgatct atgaagtgtc caaccggttc 180tctggactgc
cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc
240agccgggtgg aggctgagga tgttgggatt tattactgca tgcaaagttt
tccgcttccg 300ctcactttcg gcggagggac caaggtggag atcaaa
336182324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 182gaaattgtgt tgacgcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agcggctact taacctggta ccagcagaaa 120cctggccagg ctcccaggct
cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240cctgaagatt ttgcagtgta ttactgtcag cagtatggta actcactgtg
caggtttggc 300caggggacca agctggagat caaa 324183324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
183gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcggctact taacctggta
ccagcagaaa 120cctggccagg ctcccagact cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggacg
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta actcactgag caggtttggc 300caggggacca
agctggagat caaa 324184324DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 184gaaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgta
gggccagtca gagtgttcgc agcaatttag cctggtacca gcagaaacct
120ggccaggctc ccaggctcct cattcatgat gcatccccca ggaccgctgg
tatcccagcc 180aggttcagtg gcagtggatc tgggacagaa ttcactctca
ccatcaacag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag
tataattact ggactccgat caccttcggc 300caagggacac gactggagat taaa
324185339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 185gacatcgtga tgacccagtc tccagactcc
ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtatttta
gacagctcca acaatgataa ctacttagct 120tggtaccagc agaaaccagg
acagcctcct aaactgctca tttactgggc atctacccgg 180gaatccgggg
tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc
240atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata
ttataatact 300ccattcactt tcggccctgg gaccaaagtg gatatcaaa
339186330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 186cagtctgtgt tgacgcagcc gccctcagtg
tctgaggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattatg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gccgcctgag
tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
330187330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 187cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattatg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcaaccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gccgcctgag
tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
330188330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 188cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattatg tatcctggta ccagcagttc 120ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gccgcctgag
tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
330189330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 189cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattatg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gccgcctgag
tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
330190330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 190cagtctgtgt tgacgcagcc gccctcaatg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattatg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gacaataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccaatta ctgctgcgga acatgggata tcggcctgag
tgtttgggtg 300ttcggcggag ggaccaaact gaccgtccta
330191330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 191cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagttc caatatcgga
agtaatactg tgaactggta ccagcagctc 120ccaggaacgg cccccaaact
cctcatctat actaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tggactccag
240tctgaggatg aggctgattt ttactgtgca gcgcgggatg agagcctgaa
tggtgtggta 300ttcggcggag ggaccaagct gaccgtccta
330192330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 192cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag agtcaccatc 60tcttgttctg gaagcagctc caacatcggc
agtaattatg tatactggta ccagcagctc 120ccaggagcgg cccccaaact
cctcatcttt aggaataatc agcggccctc aggggtccct 180gaccgcttct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag
tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta
330193330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 193cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag agtcaccatc 60tcttgttctg gaagcagctc caacatcggc
agtaattatg tatactggta ccagcagctc 120ccaggagcgg cccccaaact
cctcatcttt aggagtaatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg
240tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag
tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta
330194330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 194cagtctgtgc tgactcagtc accctcagcg
tctgggaccc ccgggcagag agtcaccatc 60tcttgttctg gaagcagctc caacatcggc
agtaattatg tatactggta ccagcagctc 120ccaggagcgg cccccaaact
cctcatcctt aggaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tgaccatcag tgggctccgg
240tccgaggatg aggctgacta ttattgtgca gcatgggatg acagcctgag
tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta
330195324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide
195tcttctgagc tgactcagga ccctactgtg tctgtggcct tgggacagac
agtcaaaatc 60acatgccaag gagacagcct cagaagtttt tatgcaagct ggtaccagca
gaagccagga 120caggcccctg tacttgtctt ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccagctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattattg
taattcccgg gacagcagtg tttaccatct ggtactcggc 300ggagggacca
agctgaccgt ccta 324196390DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 196caggtgcagt
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg
cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt atttctgtgc gagagatcaa 300atgagtatta ttatgcttcg
gggagttttt cccccttact attacggtat ggacgtctgg 360ggccaaggga
ccacggtcac cgtctctagt 390197381DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 197caggtgcagc
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gactactata tgtactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcccta atagtggtgg
cacaaactat 180gcccagaagt ttcagggcag ggtcaccatg accagggaca
cgtctatcag cacagcctac 240atggagctga gtaggctgag atctgacgac
acggccgtgt attactgtgt gagaggagga 300tatagtggct acgctgggct
ctactcccac tactacggta tggacgtctg gggccaaggg 360accacggtca
ccgtctctag t 381198354DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 198caggtgcagc
tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggata caccttcacc gcctactatt tacactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcaaccctc acagtggtgg
cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg accagggaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt tctactgtgc gagaggaagg 300cagtggctgg gctttgacta
ctggggccag ggaaccctgg tcaccgtctc tagt 354199321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
199gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagttacc 60attacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca
gcagaaacca 120gggaaagccc ctaagcgcct gatctatgtt gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta
ctgtctacag tataacactt acccgctcac tttcggcgga 300gggaccaagg
tggagatcaa g 321200393DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 200gaggtacagc
tggtggagtc tgggggaggc ttggtaaagc ctggggggtc cctcagactc 60tcctgtgcag
cctctggatt cactttcggt aacgcctgga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggttggccgt attaaaagca aaactgatgg
tgggacaaca 180gactacgctg cacccgtgaa aggcagattc accatctcaa
gagatgattc aaaaaacacg 240ctgtatctgc aaatgaacag cctgaaaacc
gaggacacag ccgtgtattt ctgtaccaca 300gatcggaccg ggtatagcat
cagctggtct agttactact actactacgg tatggacgtc 360tggggccaag
ggaccacggt caccgtctct agt 393201393DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
201gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc
ccttagactc 60tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggttggccgt attaaaagca
aaactgatgg tgggacaaca 180gactacactg cacccgtgaa aggcagattc
accatctcaa gagatgattc aaaaaacacg 240ctgtatctgc aaatgaatag
cctgaaagcc gaggacacag ccgtgtatta ctgtaccaca 300gatcggaccg
ggtatagcat cagctggtct agttactact actactacgg tatggacgtc
360tggggccaag ggaccacggt caccgtctct agt 393202393DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
202gaggtacagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc
ccttagactc 60tcctgtgcag cctctggatt cactttcggt aacgcctgga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggttggccgt attaaaagca
aaactgatgg tgggacaaca 180gactacgctg cacccgtgaa aggcagattc
accatctcaa gagatgattc aaaaaacacg 240ctgtatctgc aaatgaacag
cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300gatcggaccg
ggtatagcat cagctggtct agttactact actactacgg tatggacgtc
360tggggccaag ggaccacggt caccgtctct agt 393203393DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
203gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc
ccttagactc 60tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggttggccgt attaaaagca
caactgatgg tgggacaaca 180gactacgctg cacccgtgaa aggcagattc
accatctcaa gagatgattc aaaaaacacg 240ctgtatctgc aaatgaacag
cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300gatcggaccg
gatatagcat cagctggtct agttactact actactacgg tatggacgtc
360tggggccaag ggaccacggt caccgtctct agt 393204393DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
204gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggata caccttcagt acctatagca tgaactgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcatcc attagtagta
gtagtagtta cagatattac 180gcagactcag tgaagggccg attcaccatc
tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga gtagcctgag
agccgaggac acggctgtgt attactgtgc gagagaaggg 300gtgtctggca
gttcgccgta tagcatcagc tggtacgact actattacgg tatggacgtc
360tggggccaag ggaccacggt caccgtctct agt 393205390DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
205gaggtgcagc tattggagtc tgggggaggc ttggtacagc ctggggagtc
cctgagactc 60tcctgtgcag cctctgggtt cacctttagc agctatgcca tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtcg cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga atagcctgag
agccgaggac acggccgtat attactgtgc gaaagatcaa 300agggaggtag
ggccgtatag cagtggctgg tacgactact actacggtat ggacgtctgg
360ggccaaggga ccacggtcac cgtctctagt 390206387DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
206caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atttcatatg
atggaagtca tgaatcctat 180gcagactccg tgaagggccg attcaccatc
tccagagaca tttccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agctgaggac acggctgtgt atttctgtgc gagagagagg 300aaacgggtta
cgatgtctac cttatattac tacttctact acggtatgga cgtctggggc
360caagggacca cggtcaccgt ctctagt 387207390DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
207caggtgcagc tggtggaatc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctttggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatttg
atggaagtat taagtattct 180gtagactccg tgaagggccg attcaccatc
tccagagaca attcaaagaa cacgctgttt 240ctgcaaatga acagcctgcg
agccgaggac acggctgtgt attactgtgc gagagatcgg 300ctcaattact
atgatagtag tggttattat cactacaaat actacggtat ggccgtctgg
360ggccaaggga ccacggtcac cgtctctagt 390208390DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
208caggtgcagc tggtggaatc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctttggca tgcattgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatcatttg
atggaagtat taagtactct 180gtagactccg tgaagggccg attcaccatc
tccagagaca attcaaagaa cacgctgttt 240ctgcaaatga acagcctgcg
agccgaggac acggctgtgt attactgtgc gagagatcgg 300ctcaattact
atgatagtag tggttattat cactacaaat actacggtct ggccgtctgg
360ggccaaggga ccacggtcac cgtctctagt 390209363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
209gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc
cctgagactc 60tcctgtacag cttctggatt cacctttggt gattatgcta tgagctggtt
ccgccaggct 120ccagggaagg ggctggagtg gataggtttc attagaagca
gagcttatgg tgggacacca 180gaatacgccg cgtctgtgaa aggcagattc
accatctcaa gagatgattc caaaaccatc 240gcctatctgc aaatgaacag
cctgaaaacc gaggacacag ccgtgtattt ctgtgctaga 300ggacggggta
ttgcagctcg ttgggactac tggggccagg gaaccctggt caccgtctct 360agt
363210378DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 210caggtgcagc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt
agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaatactat 180gcagactccg
tgaagggccg attcatcatc tccagagata aatccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagcgggg 300ggtatagcag cagctggcct ctactactac tacggtatgg
acgtctgggg ccaagggacc 360acggtcaccg tctctagt 378211351DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
211caggtgcagt tacagcagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60agctgcgctg tctatggtgg gtccttcggt ggttactact ggagctggat
ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atcaatcata
gtggaggcac caagtacaac 180ccgtccctca agagtcgagt caccatatca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctgtgtatt tctgtgcgag aggcgatgta 300gtaggtttct
ttgactattg gggccaggga accctggtca ccgtctctag t
351212354DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 212cagatcacct taaaggagtc tggtcctacg
ctggtgaaac ccacacagac cctcacgctg 60acctgcacct tctctgggtt ctcactcagc
actagtggtg tgggtgtggc ctggatccgt 120cagccccccg gaaaggccct
ggagtggctt gcactcattt attggactga tgataagcgc 180tacagtccat
ctctgaagag caggctcacc atcaccaagg acacctccaa gaaccaggtg
240gtccttagaa tgaccaacat ggaccctttg gacacagcca cttatttctg
tgcacacaga 300ccagggggct ggttcgaccc ctggggccag ggaaccctgg
tcaccgtctc tagt 35421313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 213Gly Gly Gly Gly Gly Val
Asp Gly Gly Gly Gly Gly Val 1 5 10 214148PRTRattus sp. 214Met Ala
Pro Gly Leu Arg Gly Leu Pro Arg Arg Gly Leu Trp Leu Leu 1 5 10 15
Leu Ala His His Leu Phe Met Val Thr Ala Cys Arg Asp Pro Asp Tyr 20
25 30 Gly Thr Leu Ile Gln Glu Leu Cys Leu Ser Arg Phe Lys Glu Asp
Met 35 40 45 Glu Thr Ile Gly Lys Thr Leu Trp Cys Asp Trp Gly Lys
Thr Ile Gly 50 55 60 Ser Tyr Gly Glu Leu Thr His Cys Thr Lys Leu
Val Ala Asn Lys Ile 65 70 75 80 Gly Cys Phe Trp Pro Asn Pro Glu Val
Asp Lys Phe Phe Ile Ala Val 85 90 95 His His Arg Tyr Phe Ser Lys
Cys Pro Val Ser Gly Arg Ala Leu Arg 100 105 110 Asp Pro Pro Asn Ser
Ile Leu Cys Pro Phe Ile Val Leu Pro Ile Thr 115 120 125 Val Thr Leu
Leu Met Thr Ala Leu Val Val Trp Arg Ser Lys Arg Thr 130 135 140 Glu
Gly Ile Val 145 215148PRTMacaca fascicularis 215Met Ala Arg Ala Leu
Cys Arg Leu Pro Gln Arg Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His
His Leu Phe Met Ala Thr Ala Cys Gln Glu Ala Asn Tyr 20 25 30 Gly
Ala Leu Leu Gln Glu Leu Cys Leu Thr Gln Phe Gln Val Asp Met 35 40
45 Glu Ala Val Gly Glu Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Gly
50 55 60 Ser Tyr Arg Glu Leu Ala Asp Cys Thr Trp His Met Ala Glu
Lys Leu 65 70 75 80 Gly Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe
Phe Leu Ala Val 85 90 95 His Gly His Tyr Phe Arg Ala Cys Pro Ile
Ser Gly Arg Ala Val Arg 100 105 110 Asp Pro Pro Gly Ser Val Leu Tyr
Pro Phe Ile Val Val Pro Ile Thr 115 120 125 Val Thr Leu Leu Val Thr
Ala Leu Val Val Trp Gln Ser Lys His Thr 130 135 140 Glu Gly Ile Val
145 216148PRTMacaca mulatta 216Met Ala Arg Ala Leu Cys Arg Leu Pro
Gln Arg Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met
Ala Thr Ala Cys Gln Glu Ala Asn Tyr 20 25 30 Gly Ala Leu Leu Gln
Glu Leu Cys Leu Thr Gln Phe Gln Val Asp Met 35 40 45 Glu Ala Val
Gly Glu Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Gly 50 55 60 Ser
Tyr Arg Glu Leu Ala Asp Cys Thr Trp His Met Ala Glu Lys Leu 65 70
75 80 Gly Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala
Val 85 90 95 His Gly His Tyr Phe Arg Ala Cys Pro Ile Ser Gly Arg
Ala Val Arg 100 105 110 Asp Pro Pro Gly Ser Val Leu Tyr Pro Phe Ile
Val Val Pro Ile Thr 115 120 125 Val Thr Leu Leu Val Thr Ala Leu Val
Val Trp Gln Ser Lys His Thr 130 135 140 Glu Gly Ile Val 145
217148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 217Met Ala Arg Ala Leu Cys Arg Leu Pro Arg
Arg Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met Thr
Thr Ala Cys Arg Asp Pro Asp Tyr 20 25 30 Gly Thr Leu Leu Arg Glu
Leu Cys Leu Thr Gln Phe Gln Val Asp Met 35 40 45 Glu Ala Val Gly
Glu Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Arg 50 55 60 Ser Tyr
Arg Glu Leu Ala Asp Cys Thr Trp His Met Ala Glu Lys Leu 65 70 75 80
Gly Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala Val 85
90 95 His Gly Arg Tyr Phe Arg Ser Cys Pro Ile Ser Gly Arg Ala Val
Arg 100 105 110 Asp Pro Pro Gly Ser Ile Leu Tyr Pro Phe Ile Val Val
Pro Ile Thr 115 120 125 Val Thr Leu Leu Val Thr Ala Leu Val Val Trp
Gln Ser Lys Arg Thr 130 135 140 Glu Gly Ile Val 145
218148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 218Met Ala Arg Ala Leu Cys Arg Leu Pro Arg
Arg Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met Thr
Thr Ala Cys Gln Glu Ala Asn Tyr 20 25 30 Gly Ala Leu Leu Arg Glu
Leu Cys Leu Thr Arg Phe Lys Glu Asp Met 35 40 45 Glu Thr Ile Gly
Lys Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Arg 50 55 60 Ser Tyr
Arg Glu Leu Ala Asp Cys Thr Trp His Met Ala Glu Lys Leu 65 70 75 80
Gly Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala Val 85
90 95 His Gly Arg Tyr Phe Arg Ser Cys Pro Ile Ser Gly Arg Ala Val
Arg 100 105 110 Asp Pro Pro Gly Ser Ile Leu Tyr Pro Phe Ile Val Val
Pro Ile Thr 115 120 125 Val Thr Leu Leu Val Thr Ala Leu Val Val Trp
Gln Ser Lys Arg Thr 130 135 140 Glu Gly Ile Val 145
219148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 219Met Ala Arg Ala Leu Cys Arg Leu Pro Arg
Arg Gly Leu Trp Leu Leu 1 5 10 15 Leu Ala His His Leu Phe Met Thr
Thr Ala Cys Gln Glu Ala Asn Tyr 20 25 30 Gly Ala Leu Leu Arg Glu
Leu Cys Leu Thr Gln Phe Gln Val Asp Met 35 40 45 Glu Ala Val Gly
Glu Thr Leu Trp Cys Asp Trp Gly Arg Thr Ile Arg 50 55 60 Ser Tyr
Gly Glu Leu Thr His Cys Thr Lys Leu Val Ala Asn Lys Leu 65 70 75 80
Gly Cys Phe Trp Pro Asn Ala Glu Val Asp Arg Phe Phe Leu Ala Val 85
90 95 His Gly Arg Tyr Phe Arg Ser Cys Pro Ile Ser Gly Arg Ala Val
Arg 100 105 110 Asp Pro Pro Gly Ser Ile Leu Tyr Pro Phe Ile Val Val
Pro Ile Thr 115 120 125 Val Thr Leu Leu Val Thr Ala Leu Val Val Trp
Gln Ser Lys Arg Thr 130 135 140
Glu Gly Ile Val 145 220464PRTRattus sp. 220Met Met Asp Lys Lys Cys
Thr Leu Cys Phe Leu Phe Leu Leu Leu Leu 1 5 10 15 Asn Met Ala Leu
Ile Ala Ala Glu Ser Glu Glu Gly Ala Asn Gln Thr 20 25 30 Asp Leu
Gly Val Thr Arg Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys 35 40 45
Tyr Gln Lys Ile Met Gln Asp Pro Ile Gln Gln Gly Glu Gly Leu Tyr 50
55 60 Cys Asn Arg Thr Trp Asp Gly Trp Leu Cys Trp Asn Asp Val Ala
Ala 65 70 75 80 Gly Thr Glu Ser Met Gln Tyr Cys Pro Asp Tyr Phe Gln
Asp Phe Asp 85 90 95 Pro Ser Glu Lys Val Thr Lys Ile Cys Asp Gln
Asp Gly Asn Trp Phe 100 105 110 Arg His Pro Asp Ser Asn Arg Thr Trp
Thr Asn Tyr Thr Leu Cys Asn 115 120 125 Asn Ser Thr His Glu Lys Val
Lys Thr Ala Leu Asn Leu Phe Tyr Leu 130 135 140 Thr Ile Ile Gly His
Gly Leu Ser Ile Ala Ser Leu Ile Ile Ser Leu 145 150 155 160 Ile Ile
Phe Phe Tyr Phe Lys Ser Leu Ser Cys Gln Arg Ile Thr Leu 165 170 175
His Lys Asn Leu Phe Phe Ser Phe Val Cys Asn Ser Ile Val Thr Ile 180
185 190 Ile His Leu Thr Ala Val Ala Asn Asn Gln Ala Leu Val Ala Thr
Asn 195 200 205 Pro Val Ser Cys Lys Val Ser Gln Phe Ile His Leu Tyr
Leu Met Gly 210 215 220 Cys Asn Tyr Phe Trp Met Leu Cys Glu Gly Ile
Tyr Leu His Thr Leu 225 230 235 240 Ile Val Val Ala Val Phe Ala Glu
Lys Gln His Leu Met Trp Tyr Tyr 245 250 255 Phe Leu Gly Trp Gly Phe
Pro Leu Leu Pro Ala Cys Ile His Ala Ile 260 265 270 Ala Arg Ser Leu
Tyr Tyr Asn Asp Asn Cys Trp Ile Ser Ser Asp Thr 275 280 285 His Leu
Leu Tyr Ile Ile His Gly Pro Ile Cys Ala Ala Leu Leu Val 290 295 300
Asn Leu Phe Phe Leu Leu Asn Ile Val Arg Val Leu Ile Thr Lys Leu 305
310 315 320 Lys Val Thr His Gln Ala Glu Ser Asn Leu Tyr Met Lys Ala
Val Arg 325 330 335 Ala Thr Leu Ile Leu Val Pro Leu Leu Gly Ile Glu
Phe Val Leu Phe 340 345 350 Pro Trp Arg Pro Glu Gly Lys Val Ala Glu
Glu Val Tyr Asp Tyr Val 355 360 365 Met His Ile Leu Met His Tyr Gln
Gly Leu Leu Val Ser Thr Ile Phe 370 375 380 Cys Phe Phe Asn Gly Glu
Val Gln Ala Ile Leu Arg Arg Asn Trp Asn 385 390 395 400 Gln Tyr Lys
Ile Gln Phe Gly Asn Gly Phe Ser His Ser Asp Ala Leu 405 410 415 Arg
Ser Ala Ser Tyr Thr Val Ser Thr Ile Ser Asp Val Gln Gly Tyr 420 425
430 Ser His Asp Cys Pro Thr Glu His Leu Asn Gly Lys Ser Ile Gln Asp
435 440 445 Ile Glu Asn Val Ala Leu Lys Pro Glu Lys Met Tyr Asp Leu
Val Met 450 455 460 221461PRTMacaca fascicularis 221Met Glu Lys Lys
Cys Thr Leu Tyr Phe Leu Val Leu Leu Pro Phe Phe 1 5 10 15 Met Ile
Phe Val Thr Ala Glu Leu Glu Glu Ser Pro Glu Asp Ser Ile 20 25 30
Gln Leu Gly Val Thr Arg Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys 35
40 45 Tyr Gln Lys Ile Met Gln Asp Pro Ile Gln Gln Ala Glu Gly Val
Tyr 50 55 60 Cys Asn Arg Thr Trp Asp Gly Trp Leu Cys Trp Asn Asn
Val Ala Ala 65 70 75 80 Gly Thr Glu Ser Met Gln Leu Cys Pro Asp Tyr
Phe Gln Asp Phe Asp 85 90 95 Pro Ser Glu Lys Val Thr Lys Ile Cys
Asp Gln Asp Gly Asn Trp Phe 100 105 110 Arg His Pro Ala Ser Asn Arg
Thr Trp Thr Asn Tyr Thr Gln Cys Asn 115 120 125 Val Asn Thr His Glu
Lys Val Lys Thr Ala Leu Asn Leu Phe Tyr Leu 130 135 140 Thr Ile Ile
Gly His Gly Leu Ser Ile Ala Ser Leu Leu Ile Ser Leu 145 150 155 160
Gly Ile Phe Phe Tyr Phe Lys Ser Leu Ser Cys Gln Arg Ile Thr Leu 165
170 175 His Lys Asn Leu Phe Phe Ser Phe Val Cys Asn Ser Val Val Thr
Ile 180 185 190 Ile His Leu Thr Ala Val Ala Asn Asn Gln Ala Leu Val
Ala Thr Asn 195 200 205 Pro Val Ser Cys Lys Val Ser Gln Phe Ile His
Leu Tyr Leu Met Gly 210 215 220 Cys Asn Tyr Phe Trp Met Leu Cys Glu
Gly Ile Tyr Leu His Thr Leu 225 230 235 240 Ile Val Val Ala Val Phe
Ala Glu Lys Gln His Leu Met Trp Tyr Tyr 245 250 255 Phe Leu Gly Trp
Gly Phe Pro Leu Ile Pro Ala Cys Ile His Ala Ile 260 265 270 Ala Arg
Ser Leu Tyr Tyr Asn Asp Asn Cys Trp Ile Ser Ser Asp Thr 275 280 285
His Leu Leu Tyr Ile Ile His Gly Pro Ile Cys Ala Ala Leu Leu Val 290
295 300 Asn Leu Phe Phe Leu Leu Asn Ile Val Arg Val Leu Ile Thr Lys
Leu 305 310 315 320 Lys Val Thr His Gln Ala Glu Ser Asn Leu Tyr Met
Lys Ala Val Arg 325 330 335 Ala Thr Leu Ile Leu Val Pro Leu Leu Gly
Ile Glu Phe Val Leu Ile 340 345 350 Pro Trp Arg Pro Glu Gly Lys Ile
Ala Glu Glu Val Tyr Asp Tyr Ile 355 360 365 Met His Ile Leu Met His
Phe Gln Gly Leu Leu Val Ser Thr Ile Phe 370 375 380 Cys Phe Phe Asn
Gly Glu Val Gln Ala Ile Leu Arg Arg Asn Trp Asn 385 390 395 400 Gln
Tyr Lys Ile Gln Phe Gly Asn Ser Phe Ser Asn Ser Glu Ala Leu 405 410
415 Arg Ser Ala Ser Tyr Thr Val Ser Thr Ile Ser Asp Gly Pro Gly Tyr
420 425 430 Ser His Asp Cys Pro Ser Glu His Leu Asn Gly Lys Ser Ile
His Asp 435 440 445 Ile Glu Asn Val Val Leu Lys Pro Glu Asn Leu Tyr
Asn 450 455 460 222461PRTMacaca mulatta 222Met Glu Lys Lys Cys Thr
Leu Tyr Phe Leu Val Leu Leu Pro Phe Phe 1 5 10 15 Met Ile Phe Val
Thr Ala Glu Leu Glu Glu Ser Pro Glu Asp Ser Ile 20 25 30 Gln Leu
Gly Val Thr Arg Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys 35 40 45
Tyr Gln Lys Ile Met Gln Asp Pro Ile Gln Gln Ala Glu Gly Val Tyr 50
55 60 Cys Asn Arg Thr Trp Asp Gly Trp Leu Cys Trp Asn Asn Val Ala
Ala 65 70 75 80 Gly Thr Glu Ser Met Gln Leu Cys Pro Asp Tyr Phe Gln
Asp Phe Asp 85 90 95 Pro Ser Glu Lys Val Thr Lys Ile Cys Asp Gln
Asp Gly Asn Trp Phe 100 105 110 Arg His Pro Ala Ser Asn Arg Thr Trp
Thr Asn Tyr Thr Gln Cys Asn 115 120 125 Val Asn Thr His Glu Lys Val
Lys Thr Ala Leu Asn Leu Phe Tyr Leu 130 135 140 Thr Ile Ile Gly His
Gly Leu Ser Ile Ala Ser Leu Leu Ile Ser Leu 145 150 155 160 Gly Ile
Phe Phe Tyr Phe Lys Ser Leu Ser Cys Gln Arg Ile Thr Leu 165 170 175
His Lys Asn Leu Phe Phe Ser Phe Val Cys Asn Ser Val Val Thr Ile 180
185 190 Ile His Leu Thr Ala Val Ala Asn Asn Gln Ala Leu Val Ala Thr
Asn 195 200 205 Pro Val Ser Cys Lys Val Ser Gln Phe Ile His Leu Tyr
Leu Met Gly 210 215 220 Cys Asn Tyr Phe Trp Met Leu Cys Glu Gly Ile
Tyr Leu His Thr Leu 225 230 235 240 Ile Val Val Ala Val Phe Ala Glu
Lys Gln His Leu Met Trp Tyr Tyr 245 250 255 Phe Leu Gly Trp Gly Phe
Pro Leu Ile Pro Ala Cys Ile His Ala Ile 260 265 270 Ala Arg Ser Leu
Tyr Tyr Asn Asp Asn Cys Trp Ile Ser Ser Asp Thr 275 280 285 His Leu
Leu Tyr Ile Ile His Gly Pro Ile Cys Ala Ala Leu Leu Val 290 295 300
Asn Leu Phe Phe Leu Leu Asn Ile Val Arg Val Leu Ile Thr Lys Leu 305
310 315 320 Lys Val Thr His Gln Ala Glu Ser Asn Leu Tyr Met Lys Ala
Val Arg 325 330 335 Ala Thr Leu Ile Leu Val Pro Leu Leu Gly Ile Glu
Phe Val Leu Ile 340 345 350 Pro Trp Arg Pro Glu Gly Lys Ile Ala Glu
Glu Val Tyr Asp Tyr Ile 355 360 365 Met His Ile Leu Met His Phe Gln
Gly Leu Leu Val Ser Thr Ile Phe 370 375 380 Cys Phe Phe Asn Gly Glu
Val Gln Ala Ile Leu Arg Arg Asn Trp Asn 385 390 395 400 Gln Tyr Lys
Ile Gln Phe Gly Asn Ser Phe Ser Asn Ser Glu Ala Leu 405 410 415 Arg
Ser Ala Ser Tyr Thr Val Ser Thr Ile Ser Asp Gly Pro Gly Tyr 420 425
430 Ser His Asp Cys Pro Ser Glu His Leu Asn Gly Lys Ser Ile His Asp
435 440 445 Ile Glu Asn Val Val Leu Lys Pro Glu Asn Leu Tyr Asn 450
455 460 223460PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 223Met Glu Lys Lys Cys Thr Leu Tyr
Phe Leu Val Leu Leu Pro Phe Phe 1 5 10 15 Met Ile Leu Val Thr Ala
Glu Ser Glu Glu Gly Ala Asn Gln Thr Asp 20 25 30 Leu Gly Val Thr
Arg Asn Lys Ile Met Thr Ala Gln Tyr Glu Cys Tyr 35 40 45 Gln Lys
Ile Met Gln Asp Pro Ile Gln Gln Ala Glu Gly Val Tyr Cys 50 55 60
Asn Arg Thr Trp Asp Gly Trp Leu Cys Trp Asn Asp Val Ala Ala Gly 65
70 75 80 Thr Glu Ser Met Gln Leu Cys Pro Asp Tyr Phe Gln Asp Phe
Asp Pro 85 90 95 Ser Glu Lys Val Thr Lys Ile Cys Asp Gln Asp Gly
Asn Trp Phe Arg 100 105 110 His Pro Ala Ser Asn Arg Thr Trp Thr Asn
Tyr Thr Gln Cys Asn Val 115 120 125 Asn Thr His Glu Lys Val Lys Thr
Ala Leu Asn Leu Phe Tyr Leu Thr 130 135 140 Ile Ile Gly His Gly Leu
Ser Ile Ala Ser Leu Leu Ile Ser Leu Gly 145 150 155 160 Ile Phe Phe
Tyr Phe Lys Ser Leu Ser Cys Gln Arg Ile Thr Leu His 165 170 175 Lys
Asn Leu Phe Phe Ser Phe Val Cys Asn Ser Val Val Thr Ile Ile 180 185
190 His Leu Thr Ala Val Ala Asn Asn Gln Ala Leu Val Ala Thr Asn Pro
195 200 205 Val Ser Cys Lys Val Ser Gln Phe Ile His Leu Tyr Leu Met
Gly Cys 210 215 220 Asn Tyr Phe Trp Met Leu Cys Glu Gly Ile Tyr Leu
His Thr Leu Ile 225 230 235 240 Val Val Ala Val Phe Ala Glu Lys Gln
His Leu Met Trp Tyr Tyr Phe 245 250 255 Leu Gly Trp Gly Phe Pro Leu
Ile Pro Ala Cys Ile His Ala Ile Ala 260 265 270 Arg Ser Leu Tyr Tyr
Asn Asp Asn Cys Trp Ile Ser Ser Asp Thr His 275 280 285 Leu Leu Tyr
Ile Ile His Gly Pro Ile Cys Ala Ala Leu Leu Val Asn 290 295 300 Leu
Phe Phe Leu Leu Asn Ile Val Arg Val Leu Ile Thr Lys Leu Lys 305 310
315 320 Val Thr His Gln Ala Glu Ser Asn Leu Tyr Met Lys Ala Val Arg
Ala 325 330 335 Thr Leu Ile Leu Val Pro Leu Leu Gly Ile Glu Phe Val
Leu Ile Pro 340 345 350 Trp Arg Pro Glu Gly Lys Ile Ala Glu Glu Val
Tyr Asp Tyr Ile Met 355 360 365 His Ile Leu Met His Phe Gln Gly Leu
Leu Val Ser Thr Ile Phe Cys 370 375 380 Phe Phe Asn Gly Glu Val Gln
Ala Ile Leu Arg Arg Asn Trp Asn Gln 385 390 395 400 Tyr Lys Ile Gln
Phe Gly Asn Ser Phe Ser Asn Ser Glu Ala Leu Arg 405 410 415 Ser Ala
Ser Tyr Thr Val Ser Thr Ile Ser Asp Gly Pro Gly Tyr Ser 420 425 430
His Asp Cys Pro Ser Glu His Leu Asn Gly Lys Ser Ile His Asp Ile 435
440 445 Glu Asn Val Leu Leu Lys Pro Glu Asn Leu Tyr Asn 450 455 460
224714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 224atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagt ctgtgttgac gcagccgccc
tcagtgtctg aggccccagg acagaaggtc 120accatctcct gctctggaag
cagctccaac attgggaata attatgtatc ctggtaccag 180cagctcccag
gaacagcccc caaactcctc atttatgaca ataataagcg accctcaggg
240attcctgacc gattctctgg ctccaagtct ggcacgtcag ccaccctggg
catcaccgga 300ctccagactg gggacgaggc cgattattac tgcggaacat
gggatagccg cctgagtgct 360gtggttttcg gcggagggac caagctgacc
gtcctaggtc agcccaaggc caaccccact 420gtcactctgt tcccgccctc
ctctgaggag ctccaagcca acaaggccac actagtgtgt 480ctgatcagtg
acttctaccc gggagctgtg acagtggcct ggaaggcaga tggcagcccc
540gtcaaggcgg gagtggagac caccaaaccc tccaaacaga gcaacaacaa
gtacgcggcc 600agcagctacc tgagcctgac gcccgagcag tggaagtccc
acagaagcta cagctgccag 660gtcacgcatg aagggagcac cgtggagaag
acagtggccc ctacagaatg ttca 714225714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
225atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg
gcagagagtc 120accatctctt gttctggaag cagctccaac atcggcagta
attatgtata ctggtaccag 180cagctcccag gagcggcccc caaactcctc
atctttagga gtaatcagcg gccctcaggg 240gtccctgacc gattctctgg
ctccaagtct ggcacctcag cctccctggc catcagtggg 300ctccggtccg
aggatgaggc tgattattac tgtgcagcat gggatgacag cctgagtggt
360tgggtgttcg gcggagggac caagctgacc gtcctaggtc agcccaaggc
caaccccact 420gtcactctgt tcccgccctc ctctgaggag ctccaagcca
acaaggccac actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg
acagtggcct ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac
caccaaaccc tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc
tgagcctgac gcccgagcag tggaagtccc acagaagcta cagctgccag
660gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714226708DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 226atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgaca tccagatgac ccagtctcca
tcctccctgt ctgcatctgt aggagacaga 120gtcaccatca cttgccgggc
aagtcagggc attagaaatg atttaggctg gtttcagcag 180aaaccaggga
aagcccctaa gcgcctgatc tatgctgcat ccagtttgca aagtggggtc
240ccatcaaggt tcagcggcag tggatctggg acagaattca ctctcacaat
cagcagcctg 300cagcctgaag atttagcaac ttattactgt ctacagtata
atatttaccc gtggacgttc 360ggccaaggga ccaaggtgga aatcaaacgt
acggtggctg caccatctgt cttcatcttc 420ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
540tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
cagcagcacc 600ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 660cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgt 708227708DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 227atggacatga
gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60cgctgttctt
ctgagctgac tcaggaccct actgtgtctg tggccttggg acagacagtc
120aaaatcacat gccaaggaga cagcctcaga agtttttatg caagctggta
ccagcagaag 180ccaggacagg cccctgtact tgtcttctat ggtaaaaaca
accggccctc agggatccca 240gaccgattct ctggctccag ctcaggaaac
acagcttcct tgaccatcac tggggctcag 300gcggaagatg aggctgacta
ttattgtaat
tcccgggaca gcagtgttta ccatctggta 360ctcggcggag ggaccaagct
gaccgtccta ggtcagccca aggccaaccc cactgtcact 420ctgttcccgc
cctcctctga ggagctccaa gccaacaagg ccacactagt gtgtctgatc
480agtgacttct acccgggagc tgtgacagtg gcctggaagg cagatggcag
ccccgtcaag 540gcgggagtgg agaccaccaa accctccaaa cagagcaaca
acaagtacgc ggccagcagc 600tacctgagcc tgacgcccga gcagtggaag
tcccacagaa gctacagctg ccaggtcacg 660catgaaggga gcaccgtgga
gaagacagtg gcccctacag aatgttca 708228723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
228atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtgata ttatactggc ccagactcca ctttctctgt ccgtcacccc
tggacagccg 120gcctccatct cctgcaagtc tagtcagagc ctcctgcaca
gtgctggaaa gacctatttg 180tattggtacc tgcagaagcc aggccagcct
ccacagctcc tgatctatga agtttccaac 240cggttctctg gagtgccaga
taggttcagt ggcagcgggt cagggacaga tttcacactg 300aaaatcagcc
gggtggaggc tgaggatgtt gggatttatt actgcatgca aagttttccg
360cttccgctca ctttcggcgg agggaccaag gtggagatca aacgtacggt
ggctgcacca 420tctgtcttca tcttcccgcc atctgatgag cagttgaaat
ctggaactgc ctctgttgtg 480tgcctgctga ataacttcta tcccagagag
gccaaagtac agtggaaggt ggataacgcc 540ctccaatcgg gtaactccca
ggagagtgtc acagagcagg acagcaagga cagcacctac 600agcctcagca
gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc
660tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa
caggggagag 720tgt 723229714DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 229atggacatga
gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagt
ctgtgttgac gcagccgccc tcagtgtctg cggccccagg acagaaggtc
120accatctcct gctctggaag cagctccaac attgggaata attatgtatc
ctggtaccag 180cagctcccag gaacagcccc caaactcctc atttatgaca
ataataagcg accctcaggg 240attcctgacc gattctctgg ctccaagtct
ggcacgtcaa ccaccctggg catcaccgga 300ctccagactg gggacgaggc
cgattattac tgcggaacat gggatagccg cctgagtgct 360gtggttttcg
gcggagggac caagctgacc gtcctaggtc agcccaaggc caaccccact
420gtcactctgt tcccgccctc ctctgaggag ctccaagcca acaaggccac
actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg acagtggcct
ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac caccaaaccc
tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc tgagcctgac
gcccgagcag tggaagtccc acagaagcta cagctgccag 660gtcacgcatg
aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714230723DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 230atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgata ttgtgatgac tcagtctcca
ctctccctgc ccgtcacccc tggagagccg 120gcctccatct cctgcaggtc
tagtcagagc ctcctgcata gttttgggta caactatttg 180gattggtacc
tgcagaagcc agggcagtct ccacagctcc tgatctattt gggttctaat
240cgggcctccg gggtccctga caggttcagt ggcagtggat caggcacaga
ttttacactg 300aaaatcagca gagtggaggc tgaggatgtt ggggtttatt
actgcatgca agctctacaa 360actccattca ctttcggccc tgggaccaaa
gtggatatca aacgtacggt ggctgcacca 420tctgtcttca tcttcccgcc
atctgatgag cagttgaaat ctggaactgc ctctgttgtg 480tgcctgctga
ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc
540ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga
cagcacctac 600agcctcagca gcaccctgac gctgagcaaa gcagactacg
agaaacacaa agtctacgcc 660tgcgaagtca cccatcaggg cctgagctcg
cccgtcacaa agagcttcaa caggggagag 720tgt 723231723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
231atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtgata ttattctgac ccagactcca ctttctctgt ccgtcacccc
tggacagccg 120gcctccatct cctgcaagtc tagtcagagc ctcctgcaca
gtgatggaaa gacctatttg 180tattggtacc tgcagaagcc cggccagcct
ccacagctcc tgatctatga agtttccaac 240cggttctctg gagagccaga
taggttcagt ggcagcgggt cagggacaga tttcacactg 300aaaatcagcc
gggtggaggc tgaggatgtt gggacttatt attgcatgca aagttttccg
360cttccgctca ctttcggcgg agggaccaag gtggagatca aacgtacggt
ggctgcacca 420tctgtcttca tcttcccgcc atctgatgag cagttgaaat
ctggaactgc ctctgttgtg 480tgcctgctga ataacttcta tcccagagag
gccaaagtac agtggaaggt ggataacgcc 540ctccaatcgg gtaactccca
ggagagtgtc acagagcagg acagcaagga cagcacctac 600agcctcagca
gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc
660tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa
caggggagag 720tgt 723232714DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 232atggacatga
gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagt
ctgtgttgac gcagccgccc tcagtgtctg cggccccagg acagaaggtc
120accatctcct gctctggaag cagctccaac attgggaata attatgtatc
ctggtaccag 180cagttcccag gaacagcccc caaactcctc atttatgaca
ataataagcg accctcaggg 240attcctgacc gattctctgg ctccaagtct
ggcacgtcag ccaccctggg catcaccgga 300ctccagactg gggacgaggc
cgattattac tgcggaacat gggatagccg cctgagtgct 360gtggttttcg
gcggagggac caagctgacc gtcctaggtc agcccaaggc caaccccact
420gtcactctgt tcccgccctc ctctgaggag ctccaagcca acaaggccac
actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg acagtggcct
ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac caccaaaccc
tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc tgagcctgac
gcccgagcag tggaagtccc acagaagcta cagctgccag 660gtcacgcatg
aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714233714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 233atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagt ctgtgctgac tcagtcaccc
tcagcgtctg ggacccccgg gcagagagtc 120accatctctt gttctggaag
cagctccaac atcggcagta attatgtata ctggtaccag 180cagctcccag
gagcggcccc caaactcctc atccttagga ataatcagcg gccctcaggg
240gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctgac
catcagtggg 300ctccggtccg aggatgaggc tgactattat tgtgcagcat
gggatgacag cctgagtggt 360tgggtgttcg gcggagggac caagctgacc
gtcctaggtc agcccaaggc caaccccact 420gtcactctgt tcccgccctc
ctctgaggag ctccaagcca acaaggccac actagtgtgt 480ctgatcagtg
acttctaccc gggagctgtg acagtggcct ggaaggcaga tggcagcccc
540gtcaaggcgg gagtggagac caccaaaccc tccaaacaga gcaacaacaa
gtacgcggcc 600agcagctacc tgagcctgac gcccgagcag tggaagtccc
acagaagcta cagctgccag 660gtcacgcatg aagggagcac cgtggagaag
acagtggccc ctacagaatg ttca 714234714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
234atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg
gcagagggtc 120accatctctt gttctggaag cagttccaat atcggaagta
atactgtgaa ctggtaccag 180cagctcccag gaacggcccc caaactcctc
atctatacta ataatcagcg gccctcaggg 240gtccctgacc gattctctgg
ctccaagtct ggcacctcag cctccctggc catcagtgga 300ctccagtctg
aggatgaggc tgatttttac tgtgcagcgc gggatgagag cctgaatggt
360gtggtattcg gcggagggac caagctgacc gtcctaggtc agcccaaggc
caaccccact 420gtcactctgt tcccgccctc ctctgaggag ctccaagcca
acaaggccac actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg
acagtggcct ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac
caccaaaccc tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc
tgagcctgac gcccgagcag tggaagtccc acagaagcta cagctgccag
660gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714235714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 235atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagt ctgtgctgac tcagccaccc
tcagcgtctg ggacccccgg gcagagagtc 120accatctctt gttctggaag
cagctccaac atcggcagta attatgtata ctggtaccag 180cagctcccag
gagcggcccc caaactcctc atctttagga ataatcagcg gccctcaggg
240gtccctgacc gcttctctgg ctccaagtct ggcacctcag cctccctggc
catcagtggg 300ctccggtccg aggatgaggc tgattattac tgtgcagcat
gggatgacag cctgagtggt 360tgggtgttcg gcggagggac caagctgacc
gtcctaggtc agcccaaggc caaccccact 420gtcactctgt tcccgccctc
ctctgaggag ctccaagcca acaaggccac actagtgtgt 480ctgatcagtg
acttctaccc gggagctgtg acagtggcct ggaaggcaga tggcagcccc
540gtcaaggcgg gagtggagac caccaaaccc tccaaacaga gcaacaacaa
gtacgcggcc 600agcagctacc tgagcctgac gcccgagcag tggaagtccc
acagaagcta cagctgccag 660gtcacgcatg aagggagcac cgtggagaag
acagtggccc ctacagaatg ttca 714236714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
236atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg
gcagagagtc 120accatctctt gttctggaag cagctccaac atcggcagta
attatgtata ctggtaccag 180cagctcccag gagcggcccc caaactcctc
atctttagga ataatcagcg gccctcaggg 240gtccctgacc gcttctctgg
ctccaagtct ggcacctcag cctccctggc catcagtggg 300ctccggtccg
aggatgaggc tgattattac tgtgcagcat gggatgacag cctgagtggt
360tgggtgttcg gcggagggac caagctgacc gtcctaggtc agcccaaggc
caaccccact 420gtcactctgt tcccgccctc ctctgaggag ctccaagcca
acaaggccac actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg
acagtggcct ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac
caccaaaccc tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc
tgagcctgac gcccgagcag tggaagtccc acagaagcta cagctgccag
660gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714237723DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 237atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgata ttacactgac ccagactcca
ctttctctgt ccgtctcccc tggacagccg 120gcctccatct cctgcaagtc
tagtcagagc ctcctgcaca gtgatggaag gaactatctg 180tattggtacc
tgcagaagcc aggccagcct ccacagctcc tgatctatga agtgtccaac
240cggttctctg gactgccaga taggttcagt ggcagcgggt cagggacaga
tttcacactg 300aaaatcagcc gggtggaggc tgaggatgtt gggatttatt
actgcatgca aagttttccg 360cttccgctca ctttcggcgg agggaccaag
gtggagatca aacgtacggt ggctgcacca 420tctgtcttca tcttcccgcc
atctgatgag cagttgaaat ctggaactgc ctctgttgtg 480tgcctgctga
ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc
540ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga
cagcacctac 600agcctcagca gcaccctgac gctgagcaaa gcagactacg
agaaacacaa agtctacgcc 660tgcgaagtca cccatcaggg cctgagctcg
cccgtcacaa agagcttcaa caggggagag 720tgt 723238714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
238atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagt ctgtgttgac gcagccgccc tcagtgtctg cggccccagg
acagaaggtc 120accatctcct gctctggaag cagctccaac attgggaata
attatgtatc ctggtaccag 180cagctcccag gaacagcccc caaactcctc
atttatgaca ataataagcg accctcaggg 240attcctgacc gattctctgg
ctccaagtct ggcacgtcag ccaccctggg catcaccgga 300ctccagactg
gggacgaggc cgattattac tgcggaacat gggatagccg cctgagtgct
360gtggttttcg gcggagggac caagctgacc gtcctaggtc agcccaaggc
caaccccact 420gtcactctgt tcccgccctc ctctgaggag ctccaagcca
acaaggccac actagtgtgt 480ctgatcagtg acttctaccc gggagctgtg
acagtggcct ggaaggcaga tggcagcccc 540gtcaaggcgg gagtggagac
caccaaaccc tccaaacaga gcaacaacaa gtacgcggcc 600agcagctacc
tgagcctgac gcccgagcag tggaagtccc acagaagcta cagctgccag
660gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttca
714239708DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 239atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgaca tccagatgac ccagtctcca
tcctccctgt ctgcatctgt aggagacaga 120gtcaccatca cttgccgggc
aagtcagggc attagaaagg atttaggctg gtatcagcag 180aaaccaggga
aagcccctaa gcgcctgatc tatggagcat ccagtttgca aagtggggtc
240ccatcaaggt tcagcggcag tggatctggg acagaattca ctctcacaat
cagcagcctg 300cagcctgaag attttgcaac ttattactgt ctacagtata
atagtttccc gtggacgttc 360ggccaaggga ccaaggtgga aatcaaacgt
acggtggctg caccatctgt cttcatcttc 420ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac
540tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
cagcagcacc 600ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga agtcacccat 660cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgt 708240705DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 240atggaaaccc
cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
120ctctcctgca gggccagtca gagtgttagc agcggctact taacctggta
ccagcagaaa 180cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 240gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 300cctgaagatt ttgcagtgta
ttactgtcag cagtatggta actcactgtg caggtttggc 360caggggacca
agctggagat caaacgtacg gtggctgcac catctgtctt catcttcccg
420ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 480tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 540caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 600acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 705241705DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
241atggaaaccc cagctcagct tctcttcctc ctgctactct ggctcccaga
taccaccgga 60gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 120ctctcctgca gggccagtca gagtgttagc agcggctact
taacctggta ccagcagaaa 180cctggccagg ctcccagact cctcatctat
ggtgcatcca gcagggccac tggcatccca 240gacaggttca gtggcagtgg
gtctgggacg gacttcactc tcaccatcag cagactggag 300cctgaagatt
ttgcagtgta ttactgtcag cagtatggta actcactgag caggtttggc
360caggggacca agctggagat caaacgtacg gtggctgcac catctgtctt
catcttcccg 420ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 480tatcccagag aggccaaagt acagtggaag
gtggataacg ccctccaatc gggtaactcc 540caggagagtg tcacagagca
ggacagcaag gacagcacct acagcctcag cagcaccctg 600acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
660ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt
7052421434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 242atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggaatctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcctc
tggattcacc ttcagtagct ttggcatgca ctgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttatat catttgatgg aagtattaag
240tattctgtag actccgtgaa gggccgattc accatctcca gagacaattc
aaagaacacg 300ctgtttctgc aaatgaacag cctgcgagcc gaggacacgg
ctgtgtatta ctgtgcgaga 360gatcggctca attactatga tagtagtggt
tattatcact acaaatacta cggtatggcc 420gtctggggcc aagggaccac
ggtcaccgtc tctagtgcct ccaccaaggg cccatcggtc 480ttccccctgg
cgccctgctc caggagcacc tccgagagca cagcggccct gggctgcctg
540gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc
tctgaccagc 600ggcgtgcaca ccttcccagc tgtcctacag tcctcaggac
tctactccct cagcagcgtg 660gtgaccgtgc cctccagcaa cttcggcacc
cagacctaca cctgcaacgt agatcacaag 720cccagcaaca ccaaggtgga
caagacagtt gagcgcaaat gttgtgtcga gtgcccaccg 780tgcccagcac
cacctgtggc aggaccgtca gtcttcctct tccccccaaa acccaaggac
840accctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt
gagccacgaa 900gaccccgagg tccagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca 960aagccacggg aggagcagtt caacagcacg
ttccgtgtgg tcagcgtcct caccgttgtg 1020caccaggact ggctgaacgg
caaggagtac aagtgcaagg tctccaacaa aggcctccca 1080gcccccatcg
agaaaaccat ctccaaaacc aaagggcagc cccgagaacc acaggtgtac
1140accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac
ctgcctggtc 1200aaaggcttct accccagcga catcgccgtg gagtgggaga
gcaatgggca gccggagaac 1260aactacaaga ccacacctcc catgctggac
tccgacggct ccttcttcct ctacagcaag 1320ctcaccgtgg acaagagcag
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1380gaggctctgc
acaaccacta cacgcagaag agcctctccc tgtctccggg taaa
14342431437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 243atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgagg tgcagctggt ggagtctggg
ggaggcttgg taaagcctgg ggggtccctt 120agactctcct gtgcagcctc
tggattcact ttcagtaacg cctggatgag ctgggtccgc 180caggctccag
ggaaggggct ggagtgggtt ggccgtatta aaagcacaac tgatggtggg
240acaacagact acgctgcacc cgtgaaaggc agattcacca tctcaagaga
tgattcaaaa 300aacacgctgt atctgcaaat gaacagcctg aaaaccgagg
acacagccgt gtattactgt 360accacagatc ggaccggata tagcatcagc
tggtctagtt actactacta ctacggtatg 420gacgtctggg gccaagggac
cacggtcacc gtctctagtg cctccaccaa gggcccatcg 480gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagcggc cctgggctgc
540ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgctctgacc 600agcggcgtgc acaccttccc agctgtccta cagtcctcag
gactctactc cctcagcagc 660gtggtgaccg tgccctccag caacttcggc
acccagacct acacctgcaa cgtagatcac 720aagcccagca acaccaaggt
ggacaagaca gttgagcgca aatgttgtgt cgagtgccca 780ccgtgcccag
caccacctgt ggcaggaccg tcagtcttcc tcttcccccc aaaacccaag
840gacaccctca tgatctcccg gacccctgag gtcacgtgcg tggtggtgga
cgtgagccac 900gaagaccccg aggtccagtt caactggtac gtggacggcg
tggaggtgca taatgccaag 960acaaagccac gggaggagca gttcaacagc
acgttccgtg tggtcagcgt cctcaccgtt 1020gtgcaccagg actggctgaa
cggcaaggag tacaagtgca aggtctccaa caaaggcctc 1080ccagccccca
tcgagaaaac catctccaaa accaaagggc agccccgaga accacaggtg
1140tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg 1200gtcaaaggct tctaccccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag 1260aacaactaca agaccacacc tcccatgctg
gactccgacg gctccttctt cctctacagc 1320aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc
tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
14372441434DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
244atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtgagg tgcagctatt ggagtctggg ggaggcttgg tacagcctgg
ggagtccctg 120agactctcct gtgcagcctc tgggttcacc tttagcagct
atgccatgag ctgggtccgc 180caggctccag ggaaggggct ggagtgggtc
tcagctatta gtggtagtgg tggtcgcaca 240tactacgcag actccgtgaa
gggccggttc accatctcca gagacaattc caagaacacg 300ctgtatctgc
aaatgaatag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaaa
360gatcaaaggg aggtagggcc gtatagcagt ggctggtacg actactacta
cggtatggac 420gtctggggcc aagggaccac ggtcaccgtc tctagtgcct
ccaccaaggg cccatcggtc 480ttccccctgg cgccctgctc caggagcacc
tccgagagca cagcggccct gggctgcctg 540gtcaaggact acttccccga
accggtgacg gtgtcgtgga actcaggcgc tctgaccagc 600ggcgtgcaca
ccttcccagc tgtcctacag tcctcaggac tctactccct cagcagcgtg
660gtgaccgtgc cctccagcaa cttcggcacc cagacctaca cctgcaacgt
agatcacaag 720cccagcaaca ccaaggtgga caagacagtt gagcgcaaat
gttgtgtcga gtgcccaccg 780tgcccagcac cacctgtggc aggaccgtca
gtcttcctct tccccccaaa acccaaggac 840accctcatga tctcccggac
ccctgaggtc acgtgcgtgg tggtggacgt gagccacgaa 900gaccccgagg
tccagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
960aagccacggg aggagcagtt caacagcacg ttccgtgtgg tcagcgtcct
caccgttgtg 1020caccaggact ggctgaacgg caaggagtac aagtgcaagg
tctccaacaa aggcctccca 1080gcccccatcg agaaaaccat ctccaaaacc
aaagggcagc cccgagaacc acaggtgtac 1140accctgcccc catcccggga
ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1200aaaggcttct
accccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac
1260aactacaaga ccacacctcc catgctggac tccgacggct ccttcttcct
ctacagcaag 1320ctcaccgtgg acaagagcag gtggcagcag gggaacgtct
tctcatgctc cgtgatgcat 1380gaggctctgc acaaccacta cacgcagaag
agcctctccc tgtctccggg taaa 14342451434DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
245atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagg tgcagttggt gcagtctggg gctgaggtga agaagcctgg
ggcctcagtg 120aaggtctcct gcaaggcttc tggatacacc ttcaccggct
actatatgca ctgggtgcga 180caggcccctg gacaagggct tgagtggatg
ggatggatca accctaacag tggtggcaca 240aactatgcac agaagtttca
gggcagggtc accatgacca gggacacgtc catcagcaca 300gcctacatgg
agctgagcag gctgagatct gacgacacgg ccgtgtattt ctgtgcgaga
360gatcaaatga gtattattat gcttcgggga gtttttcccc cttactatta
cggtatggac 420gtctggggcc aagggaccac ggtcaccgtc tctagtgcct
ccaccaaggg cccatcggtc 480ttccccctgg cgccctgctc caggagcacc
tccgagagca cagcggccct gggctgcctg 540gtcaaggact acttccccga
accggtgacg gtgtcgtgga actcaggcgc tctgaccagc 600ggcgtgcaca
ccttcccagc tgtcctacag tcctcaggac tctactccct cagcagcgtg
660gtgaccgtgc cctccagcaa cttcggcacc cagacctaca cctgcaacgt
agatcacaag 720cccagcaaca ccaaggtgga caagacagtt gagcgcaaat
gttgtgtcga gtgcccaccg 780tgcccagcac cacctgtggc aggaccgtca
gtcttcctct tccccccaaa acccaaggac 840accctcatga tctcccggac
ccctgaggtc acgtgcgtgg tggtggacgt gagccacgaa 900gaccccgagg
tccagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
960aagccacggg aggagcagtt caacagcacg ttccgtgtgg tcagcgtcct
caccgttgtg 1020caccaggact ggctgaacgg caaggagtac aagtgcaagg
tctccaacaa aggcctccca 1080gcccccatcg agaaaaccat ctccaaaacc
aaagggcagc cccgagaacc acaggtgtac 1140accctgcccc catcccggga
ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1200aaaggcttct
accccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac
1260aactacaaga ccacacctcc catgctggac tccgacggct ccttcttcct
ctacagcaag 1320ctcaccgtgg acaagagcag gtggcagcag gggaacgtct
tctcatgctc cgtgatgcat 1380gaggctctgc acaaccacta cacgcagaag
agcctctccc tgtctccggg taaa 14342461431DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
246atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagg tgcagctggt ggagtctggg ggaggcgtgg tccagcctgg
gaggtccctg 120agactctcct gtgcagcctc tggattcacc ttcagtagct
atggcatgca ctgggtccgc 180caggctccag gcaaggggct ggagtgggtg
gcagttattt catatgatgg aagtcatgaa 240tcctatgcag actccgtgaa
gggccgattc accatctcca gagacatttc caagaacacg 300ctgtatctgc
aaatgaacag cctgagagct gaggacacgg ctgtgtattt ctgtgcgaga
360gagaggaaac gggttacgat gtctacctta tattactact tctactacgg
tatggacgtc 420tggggccaag ggaccacggt caccgtctct agtgcctcca
ccaagggccc atcggtcttc 480cccctggcgc cctgctccag gagcacctcc
gagagcacag cggccctggg ctgcctggtc 540aaggactact tccccgaacc
ggtgacggtg tcgtggaact caggcgctct gaccagcggc 600gtgcacacct
tcccagctgt cctacagtcc tcaggactct actccctcag cagcgtggtg
660accgtgccct ccagcaactt cggcacccag acctacacct gcaacgtaga
tcacaagccc 720agcaacacca aggtggacaa gacagttgag cgcaaatgtt
gtgtcgagtg cccaccgtgc 780ccagcaccac ctgtggcagg accgtcagtc
ttcctcttcc ccccaaaacc caaggacacc 840ctcatgatct cccggacccc
tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac 900cccgaggtcc
agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag
960ccacgggagg agcagttcaa cagcacgttc cgtgtggtca gcgtcctcac
cgttgtgcac 1020caggactggc tgaacggcaa ggagtacaag tgcaaggtct
ccaacaaagg cctcccagcc 1080cccatcgaga aaaccatctc caaaaccaaa
gggcagcccc gagaaccaca ggtgtacacc 1140ctgcccccat cccgggagga
gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1200ggcttctacc
ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac
1260tacaagacca cacctcccat gctggactcc gacggctcct tcttcctcta
cagcaagctc 1320accgtggaca agagcaggtg gcagcagggg aacgtcttct
catgctccgt gatgcatgag 1380gctctgcaca accactacac gcagaagagc
ctctccctgt ctccgggtaa a 14312471434DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
247atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtcagg tgcagctggt ggaatctggg ggaggcgtgg tccagcctgg
gaggtccctg 120agactctcct gtgcagcctc tggattcacc ttcagtagct
ttggcatgca ctgggtccgc 180caggctccag gcaaggggct ggagtgggtg
gcagttatat catttgatgg aagtattaag 240tattctgtag actccgtgaa
gggccgattc accatctcca gagacaattc aaagaacacg 300ctgtttctgc
aaatgaacag cctgcgagcc gaggacacgg ctgtgtatta ctgtgcgaga
360gatcggctca attactatga tagtagtggt tattatcact acaaatacta
cggtatggcc 420gtctggggcc aagggaccac ggtcaccgtc tctagtgcct
ccaccaaggg cccatcggtc 480ttccccctgg cgccctgctc caggagcacc
tccgagagca cagcggccct gggctgcctg 540gtcaaggact acttccccga
accggtgacg gtgtcgtgga actcaggcgc tctgaccagc 600ggcgtgcaca
ccttcccagc tgtcctacag tcctcaggac tctactccct cagcagcgtg
660gtgaccgtgc cctccagcaa cttcggcacc cagacctaca cctgcaacgt
agatcacaag 720cccagcaaca ccaaggtgga caagacagtt gagcgcaaat
gttgtgtcga gtgcccaccg 780tgcccagcac cacctgtggc aggaccgtca
gtcttcctct tccccccaaa acccaaggac 840accctcatga tctcccggac
ccctgaggtc acgtgcgtgg tggtggacgt gagccacgaa 900gaccccgagg
tccagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
960aagccacggg aggagcagtt caacagcacg ttccgtgtgg tcagcgtcct
caccgttgtg 1020caccaggact ggctgaacgg caaggagtac aagtgcaagg
tctccaacaa aggcctccca 1080gcccccatcg agaaaaccat ctccaaaacc
aaagggcagc cccgagaacc acaggtgtac 1140accctgcccc catcccggga
ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1200aaaggcttct
accccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac
1260aactacaaga ccacacctcc catgctggac tccgacggct ccttcttcct
ctacagcaag 1320ctcaccgtgg acaagagcag gtggcagcag gggaacgtct
tctcatgctc cgtgatgcat 1380gaggctctgc acaaccacta cacgcagaag
agcctctccc tgtctccggg taaa 14342481407DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
248atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtgagg tgcagctggt ggagtctggg ggaggcttgg taaagccagg
gcggtccctg 120agactctcct gtacagcttc tggattcacc tttggtgatt
atgctatgag ctggttccgc 180caggctccag ggaaggggct ggagtggata
ggtttcatta gaagcagagc ttatggtggg 240acaccagaat acgccgcgtc
tgtgaaaggc agattcacca tctcaagaga tgattccaaa 300accatcgcct
atctgcaaat gaacagcctg aaaaccgagg acacagccgt gtatttctgt
360gctagaggac ggggtattgc agctcgttgg gactactggg gccagggaac
cctggtcacc 420gtctctagtg cctccaccaa gggcccatcg gtcttccccc
tggcgccctg ctccaggagc 480acctccgaga gcacagcggc cctgggctgc
ctggtcaagg actacttccc cgaaccggtg 540acggtgtcgt ggaactcagg
cgctctgacc agcggcgtgc acaccttccc agctgtccta 600cagtcctcag
gactctactc cctcagcagc gtggtgaccg tgccctccag caacttcggc
660acccagacct acacctgcaa cgtagatcac aagcccagca acaccaaggt
ggacaagaca 720gttgagcgca aatgttgtgt cgagtgccca ccgtgcccag
caccacctgt ggcaggaccg 780tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 840gtcacgtgcg tggtggtgga
cgtgagccac gaagaccccg aggtccagtt caactggtac 900gtggacggcg
tggaggtgca taatgccaag acaaagccac gggaggagca gttcaacagc
960acgttccgtg tggtcagcgt cctcaccgtt gtgcaccagg actggctgaa
cggcaaggag 1020tacaagtgca aggtctccaa caaaggcctc ccagccccca
tcgagaaaac catctccaaa 1080accaaagggc agccccgaga accacaggtg
tacaccctgc ccccatcccg ggaggagatg 1140accaagaacc aggtcagcct
gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1200gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacacc tcccatgctg
1260gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1320caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 1380aagagcctct ccctgtctcc gggtaaa
14072491431DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 249atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggagtctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcctc
tggattcacc ttcagtagct atggcatgca ctgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttattt catatgatgg aagtcatgaa
240tcctatgcag actccgtgaa gggccgattc accatctcca gagacatttc
caagaacacg 300ctgtatctgc aaatgaacag cctgagagct gaggacacgg
ctgtgtattt ctgtgcgaga 360gagaggaaac gggttacgat gtctacctta
tattactact tctactacgg tatggacgtc 420tggggccaag ggaccacggt
caccgtctct agtgcctcca ccaagggccc atcggtcttc 480cccctggcgc
cctgctccag gagcacctcc gagagcacag cggccctggg ctgcctggtc
540aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgctct
gaccagcggc 600gtgcacacct tcccagctgt cctacagtcc tcaggactct
actccctcag cagcgtggtg 660accgtgccct ccagcaactt cggcacccag
acctacacct gcaacgtaga tcacaagccc 720agcaacacca aggtggacaa
gacagttgag cgcaaatgtt gtgtcgagtg cccaccgtgc 780ccagcaccac
ctgtggcagg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc
840ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag
ccacgaagac 900cccgaggtcc agttcaactg gtacgtggac ggcgtggagg
tgcataatgc caagacaaag 960ccacgggagg agcagttcaa cagcacgttc
cgtgtggtca gcgtcctcac cgttgtgcac 1020caggactggc tgaacggcaa
ggagtacaag tgcaaggtct ccaacaaagg cctcccagcc 1080cccatcgaga
aaaccatctc caaaaccaaa gggcagcccc gagaaccaca ggtgtacacc
1140ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg
cctggtcaaa 1200ggcttctacc ccagcgacat cgccgtggag tgggagagca
atgggcagcc ggagaacaac 1260tacaagacca cacctcccat gctggactcc
gacggctcct tcttcctcta cagcaagctc 1320accgtggaca agagcaggtg
gcagcagggg aacgtcttct catgctccgt gatgcatgag 1380gctctgcaca
accactacac gcagaagagc ctctccctgt ctccgggtaa a
14312501434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 250atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggaatctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcctc
tggattcacc ttcagtagct ttggcatgca ctgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttatat catttgatgg aagtattaag
240tattctgtag actccgtgaa gggccgattc accatctcca gagacaattc
aaagaacacg 300ctgtttctgc aaatgaacag cctgcgagcc gaggacacgg
ctgtgtatta ctgtgcgaga 360gatcggctca attactatga tagtagtggt
tattatcact acaaatacta cggtatggcc 420gtctggggcc aagggaccac
ggtcaccgtc tctagtgcct ccaccaaggg cccatcggtc 480ttccccctgg
cgccctgctc caggagcacc tccgagagca cagcggccct gggctgcctg
540gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc
tctgaccagc 600ggcgtgcaca ccttcccagc tgtcctacag tcctcaggac
tctactccct cagcagcgtg 660gtgaccgtgc cctccagcaa cttcggcacc
cagacctaca cctgcaacgt agatcacaag 720cccagcaaca ccaaggtgga
caagacagtt gagcgcaaat gttgtgtcga gtgcccaccg 780tgcccagcac
cacctgtggc aggaccgtca gtcttcctct tccccccaaa acccaaggac
840accctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt
gagccacgaa 900gaccccgagg tccagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca 960aagccacggg aggagcagtt caacagcacg
ttccgtgtgg tcagcgtcct caccgttgtg 1020caccaggact ggctgaacgg
caaggagtac aagtgcaagg tctccaacaa aggcctccca 1080gcccccatcg
agaaaaccat ctccaaaacc aaagggcagc cccgagaacc acaggtgtac
1140accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac
ctgcctggtc 1200aaaggcttct accccagcga catcgccgtg gagtgggaga
gcaatgggca gccggagaac 1260aactacaaga ccacacctcc catgctggac
tccgacggct ccttcttcct ctacagcaag 1320ctcaccgtgg acaagagcag
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1380gaggctctgc
acaaccacta cacgcagaag agcctctccc tgtctccggg taaa
14342511437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 251atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgagg tgcagctggt ggagtctggg
ggaggcttgg taaagcctgg ggggtccctt 120agactctcct gtgcagcctc
tggattcact ttcagtaacg cctggatgag ctgggtccgc 180caggctccag
ggaaggggct ggagtgggtt ggccgtatta aaagcaaaac tgatggtggg
240acaacagact acactgcacc cgtgaaaggc agattcacca tctcaagaga
tgattcaaaa 300aacacgctgt atctgcaaat gaatagcctg aaagccgagg
acacagccgt gtattactgt 360accacagatc ggaccgggta tagcatcagc
tggtctagtt actactacta ctacggtatg 420gacgtctggg gccaagggac
cacggtcacc gtctctagtg cctccaccaa gggcccatcg 480gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagcggc cctgggctgc
540ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgctctgacc 600agcggcgtgc acaccttccc agctgtccta cagtcctcag
gactctactc cctcagcagc 660gtggtgaccg tgccctccag caacttcggc
acccagacct acacctgcaa cgtagatcac 720aagcccagca acaccaaggt
ggacaagaca gttgagcgca aatgttgtgt cgagtgccca 780ccgtgcccag
caccacctgt ggcaggaccg tcagtcttcc tcttcccccc aaaacccaag
840gacaccctca tgatctcccg gacccctgag gtcacgtgcg tggtggtgga
cgtgagccac 900gaagaccccg aggtccagtt caactggtac gtggacggcg
tggaggtgca taatgccaag 960acaaagccac gggaggagca gttcaacagc
acgttccgtg tggtcagcgt cctcaccgtt 1020gtgcaccagg actggctgaa
cggcaaggag tacaagtgca aggtctccaa caaaggcctc 1080ccagccccca
tcgagaaaac catctccaaa accaaagggc agccccgaga accacaggtg
1140tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg 1200gtcaaaggct tctaccccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag 1260aacaactaca agaccacacc tcccatgctg
gactccgacg gctccttctt cctctacagc 1320aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc
tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
14372521425DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 252atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt gcagtctggg
gctgaggtga agaagcctgg ggcctcagtg 120aaggtctcct gcaaggcttc
tggatacacc ttcaccgact actatatgta ctgggtgcga 180caggcccctg
gacaagggct tgagtggatg ggatggatca gccctaatag tggtggcaca
240aactatgccc agaagtttca gggcagggtc accatgacca gggacacgtc
tatcagcaca 300gcctacatgg agctgagtag gctgagatct gacgacacgg
ccgtgtatta ctgtgtgaga 360ggaggatata gtggctacgc tgggctctac
tcccactact acggtatgga cgtctggggc 420caagggacca cggtcaccgt
ctctagtgcc tccaccaagg gcccatcggt cttccccctg 480gcgccctgct
ccaggagcac ctccgagagc acagcggccc tgggctgcct ggtcaaggac
540tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ctctgaccag
cggcgtgcac 600accttcccag ctgtcctaca gtcctcagga ctctactccc
tcagcagcgt ggtgaccgtg 660ccctccagca acttcggcac ccagacctac
acctgcaacg tagatcacaa gcccagcaac 720accaaggtgg acaagacagt
tgagcgcaaa tgttgtgtcg agtgcccacc gtgcccagca 780ccacctgtgg
caggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg
840atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccacga
agaccccgag 900gtccagttca actggtacgt ggacggcgtg gaggtgcata
atgccaagac aaagccacgg 960gaggagcagt tcaacagcac gttccgtgtg
gtcagcgtcc tcaccgttgt gcaccaggac 1020tggctgaacg gcaaggagta
caagtgcaag gtctccaaca aaggcctccc agcccccatc 1080gagaaaacca
tctccaaaac caaagggcag ccccgagaac cacaggtgta caccctgccc
1140ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt
caaaggcttc 1200taccccagcg acatcgccgt ggagtgggag agcaatgggc
agccggagaa caactacaag 1260accacacctc ccatgctgga ctccgacggc
tccttcttcc tctacagcaa gctcaccgtg 1320gacaagagca ggtggcagca
ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380cacaaccact
acacgcagaa gagcctctcc ctgtctccgg gtaaa 14252531437DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
253atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct
gagaggtgcg 60cgctgtgagg tacagctggt ggagtctggg ggaggcttgg taaagcctgg
ggggtccctc 120agactctcct gtgcagcctc tggattcact ttcggtaacg
cctggatgag ctgggtccgc 180caggctccag ggaaggggct ggagtgggtt
ggccgtatta aaagcaaaac tgatggtggg 240acaacagact acgctgcacc
cgtgaaaggc agattcacca tctcaagaga tgattcaaaa 300aacacgctgt
atctgcaaat gaacagcctg aaaaccgagg acacagccgt gtatttctgt
360accacagatc ggaccgggta tagcatcagc tggtctagtt actactacta
ctacggtatg 420gacgtctggg gccaagggac cacggtcacc gtctctagtg
cctccaccaa gggcccatcg 480gtcttccccc tggcgccctg ctccaggagc
acctccgaga gcacagcggc cctgggctgc 540ctggtcaagg actacttccc
cgaaccggtg acggtgtcgt ggaactcagg cgctctgacc 600agcggcgtgc
acaccttccc agctgtccta cagtcctcag gactctactc cctcagcagc
660gtggtgaccg tgccctccag caacttcggc acccagacct acacctgcaa
cgtagatcac 720aagcccagca acaccaaggt ggacaagaca gttgagcgca
aatgttgtgt cgagtgccca 780ccgtgcccag caccacctgt ggcaggaccg
tcagtcttcc tcttcccccc aaaacccaag 840gacaccctca tgatctcccg
gacccctgag gtcacgtgcg tggtggtgga cgtgagccac 900gaagaccccg
aggtccagtt caactggtac gtggacggcg tggaggtgca taatgccaag
960acaaagccac gggaggagca gttcaacagc acgttccgtg tggtcagcgt
cctcaccgtt 1020gtgcaccagg actggctgaa cggcaaggag tacaagtgca
aggtctccaa caaaggcctc 1080ccagccccca tcgagaaaac catctccaaa
accaaagggc agccccgaga accacaggtg 1140tacaccctgc ccccatcccg
ggaggagatg accaagaacc aggtcagcct gacctgcctg 1200gtcaaaggct
tctaccccag cgacatcgcc
gtggagtggg agagcaatgg gcagccggag 1260aacaactaca agaccacacc
tcccatgctg gactccgacg gctccttctt cctctacagc 1320aagctcaccg
tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg
1380catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
14372541437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 254atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgagg tacagctggt ggagtctggg
ggaggcttgg taaagcctgg ggggtccctt 120agactctcct gtgcagcctc
tggattcact ttcggtaacg cctggatgag ctgggtccgc 180caggctccag
ggaaggggct ggagtgggtt ggccgtatta aaagcaaaac tgatggtggg
240acaacagact acgctgcacc cgtgaaaggc agattcacca tctcaagaga
tgattcaaaa 300aacacgctgt atctgcaaat gaacagcctg aaaaccgagg
acacagccgt gtattactgt 360accacagatc ggaccgggta tagcatcagc
tggtctagtt actactacta ctacggtatg 420gacgtctggg gccaagggac
cacggtcacc gtctctagtg cctccaccaa gggcccatcg 480gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagcggc cctgggctgc
540ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgctctgacc 600agcggcgtgc acaccttccc agctgtccta cagtcctcag
gactctactc cctcagcagc 660gtggtgaccg tgccctccag caacttcggc
acccagacct acacctgcaa cgtagatcac 720aagcccagca acaccaaggt
ggacaagaca gttgagcgca aatgttgtgt cgagtgccca 780ccgtgcccag
caccacctgt ggcaggaccg tcagtcttcc tcttcccccc aaaacccaag
840gacaccctca tgatctcccg gacccctgag gtcacgtgcg tggtggtgga
cgtgagccac 900gaagaccccg aggtccagtt caactggtac gtggacggcg
tggaggtgca taatgccaag 960acaaagccac gggaggagca gttcaacagc
acgttccgtg tggtcagcgt cctcaccgtt 1020gtgcaccagg actggctgaa
cggcaaggag tacaagtgca aggtctccaa caaaggcctc 1080ccagccccca
tcgagaaaac catctccaaa accaaagggc agccccgaga accacaggtg
1140tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg 1200gtcaaaggct tctaccccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag 1260aacaactaca agaccacacc tcccatgctg
gactccgacg gctccttctt cctctacagc 1320aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc
tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
14372551431DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 255atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggagtctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcctc
tggattcacc ttcagtagct atggcatgca ctgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttattt catatgatgg aagtcatgaa
240tcctatgcag actccgtgaa gggccgattc accatctcca gagacatttc
caagaacacg 300ctgtatctgc aaatgaacag cctgagagct gaggacacgg
ctgtgtattt ctgtgcgaga 360gagaggaaac gggttacgat gtctacctta
tattactact tctactacgg tatggacgtc 420tggggccaag ggaccacggt
caccgtctct agtgcctcca ccaagggccc atcggtcttc 480cccctggcgc
cctgctccag gagcacctcc gagagcacag cggccctggg ctgcctggtc
540aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgctct
gaccagcggc 600gtgcacacct tcccagctgt cctacagtcc tcaggactct
actccctcag cagcgtggtg 660accgtgccct ccagcaactt cggcacccag
acctacacct gcaacgtaga tcacaagccc 720agcaacacca aggtggacaa
gacagttgag cgcaaatgtt gtgtcgagtg cccaccgtgc 780ccagcaccac
ctgtggcagg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc
840ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag
ccacgaagac 900cccgaggtcc agttcaactg gtacgtggac ggcgtggagg
tgcataatgc caagacaaag 960ccacgggagg agcagttcaa cagcacgttc
cgtgtggtca gcgtcctcac cgttgtgcac 1020caggactggc tgaacggcaa
ggagtacaag tgcaaggtct ccaacaaagg cctcccagcc 1080cccatcgaga
aaaccatctc caaaaccaaa gggcagcccc gagaaccaca ggtgtacacc
1140ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg
cctggtcaaa 1200ggcttctacc ccagcgacat cgccgtggag tgggagagca
atgggcagcc ggagaacaac 1260tacaagacca cacctcccat gctggactcc
gacggctcct tcttcctcta cagcaagctc 1320accgtggaca agagcaggtg
gcagcagggg aacgtcttct catgctccgt gatgcatgag 1380gctctgcaca
accactacac gcagaagagc ctctccctgt ctccgggtaa a
14312561434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 256atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggaatctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcctc
tggattcacc ttcagtagct ttggcatgca ttgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttatat catttgatgg aagtattaag
240tactctgtag actccgtgaa gggccgattc accatctcca gagacaattc
aaagaacacg 300ctgtttctgc aaatgaacag cctgcgagcc gaggacacgg
ctgtgtatta ctgtgcgaga 360gatcggctca attactatga tagtagtggt
tattatcact acaaatacta cggtctggcc 420gtctggggcc aagggaccac
ggtcaccgtc tctagtgcct ccaccaaggg cccatcggtc 480ttccccctgg
cgccctgctc caggagcacc tccgagagca cagcggccct gggctgcctg
540gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc
tctgaccagc 600ggcgtgcaca ccttcccagc tgtcctacag tcctcaggac
tctactccct cagcagcgtg 660gtgaccgtgc cctccagcaa cttcggcacc
cagacctaca cctgcaacgt agatcacaag 720cccagcaaca ccaaggtgga
caagacagtt gagcgcaaat gttgtgtcga gtgcccaccg 780tgcccagcac
cacctgtggc aggaccgtca gtcttcctct tccccccaaa acccaaggac
840accctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt
gagccacgaa 900gaccccgagg tccagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca 960aagccacggg aggagcagtt caacagcacg
ttccgtgtgg tcagcgtcct caccgttgtg 1020caccaggact ggctgaacgg
caaggagtac aagtgcaagg tctccaacaa aggcctccca 1080gcccccatcg
agaaaaccat ctccaaaacc aaagggcagc cccgagaacc acaggtgtac
1140accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac
ctgcctggtc 1200aaaggcttct accccagcga catcgccgtg gagtgggaga
gcaatgggca gccggagaac 1260aactacaaga ccacacctcc catgctggac
tccgacggct ccttcttcct ctacagcaag 1320ctcaccgtgg acaagagcag
gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1380gaggctctgc
acaaccacta cacgcagaag agcctctccc tgtctccggg taaa
14342571437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 257atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtgagg tgcagctggt ggagtctggg
ggaggcctgg tcaagcctgg ggggtccctg 120agactctcct gtgcagcctc
tggatacacc ttcagtacct atagcatgaa ctgggtccgc 180caggctccag
ggaaggggct ggagtgggtc tcatccatta gtagtagtag tagttacaga
240tattacgcag actcagtgaa gggccgattc accatctcca gagacaacgc
caagaactca 300ctgtatctgc aaatgagtag cctgagagcc gaggacacgg
ctgtgtatta ctgtgcgaga 360gaaggggtgt ctggcagttc gccgtatagc
atcagctggt acgactacta ttacggtatg 420gacgtctggg gccaagggac
cacggtcacc gtctctagtg cctccaccaa gggcccatcg 480gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagcggc cctgggctgc
540ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgctctgacc 600agcggcgtgc acaccttccc agctgtccta cagtcctcag
gactctactc cctcagcagc 660gtggtgaccg tgccctccag caacttcggc
acccagacct acacctgcaa cgtagatcac 720aagcccagca acaccaaggt
ggacaagaca gttgagcgca aatgttgtgt cgagtgccca 780ccgtgcccag
caccacctgt ggcaggaccg tcagtcttcc tcttcccccc aaaacccaag
840gacaccctca tgatctcccg gacccctgag gtcacgtgcg tggtggtgga
cgtgagccac 900gaagaccccg aggtccagtt caactggtac gtggacggcg
tggaggtgca taatgccaag 960acaaagccac gggaggagca gttcaacagc
acgttccgtg tggtcagcgt cctcaccgtt 1020gtgcaccagg actggctgaa
cggcaaggag tacaagtgca aggtctccaa caaaggcctc 1080ccagccccca
tcgagaaaac catctccaaa accaaagggc agccccgaga accacaggtg
1140tacaccctgc ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg 1200gtcaaaggct tctaccccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag 1260aacaactaca agaccacacc tcccatgctg
gactccgacg gctccttctt cctctacagc 1320aagctcaccg tggacaagag
caggtggcag caggggaacg tcttctcatg ctccgtgatg 1380catgaggctc
tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa
14372581422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 258atggacatga gggtgcccgc tcagctcctg
gggctcctgc tgctgtggct gagaggtgcg 60cgctgtcagg tgcagctggt ggagtctggg
ggaggcgtgg tccagcctgg gaggtccctg 120agactctcct gtgcagcgtc
tggattcacc ttcagtagct atggcatgca ctgggtccgc 180caggctccag
gcaaggggct ggagtgggtg gcagttatat ggtatgatgg aagtaataaa
240tactatgcag actccgtgaa gggccgattc atcatctcca gagataaatc
caagaacacg 300ctgtatctgc aaatgaacag cctgagagcc gaggacacgg
ctgtgtatta ctgtgcgaga 360gcggggggta tagcagcagc tggcctctac
tactactacg gtatggacgt ctggggccaa 420gggaccacgg tcaccgtctc
tagtgcctcc accaagggcc catcggtctt ccccctggcg 480ccctgctcca
ggagcacctc cgagagcaca gcggccctgg gctgcctggt caaggactac
540ttccccgaac cggtgacggt gtcgtggaac tcaggcgctc tgaccagcgg
cgtgcacacc 600ttcccagctg tcctacagtc ctcaggactc tactccctca
gcagcgtggt gaccgtgccc 660tccagcaact tcggcaccca gacctacacc
tgcaacgtag atcacaagcc cagcaacacc 720aaggtggaca agacagttga
gcgcaaatgt tgtgtcgagt gcccaccgtg cccagcacca 780cctgtggcag
gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
840tcccggaccc ctgaggtcac gtgcgtggtg gtggacgtga gccacgaaga
ccccgaggtc 900cagttcaact ggtacgtgga cggcgtggag gtgcataatg
ccaagacaaa gccacgggag 960gagcagttca acagcacgtt ccgtgtggtc
agcgtcctca ccgttgtgca ccaggactgg 1020ctgaacggca aggagtacaa
gtgcaaggtc tccaacaaag gcctcccagc ccccatcgag 1080aaaaccatct
ccaaaaccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
1140tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa
aggcttctac 1200cccagcgaca tcgccgtgga gtgggagagc aatgggcagc
cggagaacaa ctacaagacc 1260acacctccca tgctggactc cgacggctcc
ttcttcctct acagcaagct caccgtggac 1320aagagcaggt ggcagcaggg
gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380aaccactaca
cgcagaagag cctctccctg tctccgggta aa 1422259981DNAHomo sapiens
259gctagcacca agggcccatc ggtcttcccc ctggcgccct gctccaggag
cacctccgag 60agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 120tggaactcag gcgctctgac cagcggcgtg cacaccttcc
cagctgtcct acagtcctca 180ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcaacttcgg cacccagacc 240tacacctgca acgtagatca
caagcccagc aacaccaagg tggacaagac agttgagcgc 300aaatgttgtg
tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc
360ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga
ggtcacgtgc 420gtggtggtgg acgtgagcca cgaagacccc gaggtccagt
tcaactggta cgtggacggc 480gtggaggtgc ataatgccaa gacaaagcca
cgggaggagc agttcaacag cacgttccgt 540gtggtcagcg tcctcaccgt
tgtgcaccag gactggctga acggcaagga gtacaagtgc 600aaggtctcca
acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg
660cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat
gaccaagaac 720caggtcagcc tgacctgcct ggtcaaaggc ttctacccca
gcgacatcgc cgtggagtgg 780gagagcaatg ggcagccgga gaacaactac
aagaccacac ctcccatgct ggactccgac 840ggctccttct tcctctacag
caagctcacc gtggacaaga gcaggtggca gcaggggaac 900gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc
960tccctgtctc cgggtaaatg a 981260324DNAHomo sapiens 260cgtacggtgg
ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60ggaactgcct
ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag
120tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac
agagcaggac 180agcaaggaca gcacctacag cctcagcagc accctgacgc
tgagcaaagc agactacgag 240aaacacaaag tctacgcctg cgaagtcacc
catcagggcc tgagctcgcc cgtcacaaag 300agcttcaaca ggggagagtg ttag
324261321DNAHomo sapiens 261ggtcagccca aggccaaccc cactgtcact
ctgttcccgc cctcctctga ggagctccaa 60gccaacaagg ccacactagt gtgtctgatc
agtgacttct acccgggagc tgtgacagtg 120gcctggaagg cagatggcag
ccccgtcaag gcgggagtgg agaccaccaa accctccaaa 180cagagcaaca
acaagtacgc ggccagcagc tacctgagcc tgacgcccga gcagtggaag
240tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga
gaagacagtg 300gcccctacag aatgttcata g 321
* * * * *