U.S. patent application number 14/755301 was filed with the patent office on 2015-10-29 for antibodies to receptor for advanced glycation end products (rage) and uses thereof.
The applicant listed for this patent is ABBVIE INC.. Invention is credited to Jill-Desiree Brederson, Marina I. Strakhova.
Application Number | 20150307610 14/755301 |
Document ID | / |
Family ID | 43922539 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150307610 |
Kind Code |
A1 |
Strakhova; Marina I. ; et
al. |
October 29, 2015 |
ANTIBODIES TO RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE)
AND USES THEREOF
Abstract
The present application relates to antibodies, such as
monoclonal antibodies, and in particular CDR grafted, humanized
versions thereof, for the treatment and diagnosis of pain and other
neuroinflammatory conditions associated with the Receptor for
Advanced Glycation End Products (RAGE).
Inventors: |
Strakhova; Marina I.;
(Vernon Hills, IL) ; Brederson; Jill-Desiree;
(Evanston, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE INC. |
North Chicago |
IL |
US |
|
|
Family ID: |
43922539 |
Appl. No.: |
14/755301 |
Filed: |
June 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13834269 |
Mar 15, 2013 |
9067996 |
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14755301 |
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12914426 |
Oct 28, 2010 |
8420083 |
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13834269 |
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61256949 |
Oct 31, 2009 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 530/387.9 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 29/00 20180101; C07K 16/2803 20130101; C07K 2317/90 20130101;
C07K 2317/34 20130101; C07K 2317/92 20130101; C07K 2317/24
20130101; C07K 2317/565 20130101; C07K 16/28 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A method for treating pain, comprising the step of administering
to a mammal in need thereof an effective amount of a composition
comprising an isolated monoclonal antibody comprising an antigen
binding domain, said antibody capable of binding an epitope of a
human RAGE molecule, said antigen binding domain comprising at
least one CDR comprising an amino acid sequence selected from the
group consisting of: a) the CDR-H3 group of amino acid sequences
consisting of SEQ ID NOs: 12 and 20; modified CDR amino acid
sequences having a sequence identity of at least 50% to one of said
sequences; and b) the CDR-L3 group of amino acid sequences
consisting of SEQ ID NOs: 16 and 24; and modified CDR amino acid
sequences having a sequence identity of at least 50% to one of said
sequences.
2. The method according to claim 1, wherein the antibody further
comprises at least one CDR comprising an amino acid sequence
selected from the CDR-H1 group consisting of SEQ ID NOs: 10 and 18;
or selected from the CDR-H2 group consisting of SEQ ID NOs: 11, 19;
or selected from the CDR-L1 group consisting of SEQ ID NOs: 14 and
22; or selected from the CDR-L2 group consisting of SEQ ID NOs: 15
and 23; and modified CDR amino acid sequences having a sequence
identity of at least 50% to one of said sequences.
3. The method according to claim 2, wherein the antibody further
comprises at least 3 CDRs which are selected from a variable domain
CDR set comprising: TABLE-US-00016 VH 11E6 set VH 11E6 CDR-H1
Residues 31-35 of SEQ ID NO : 9 SEQ ID NO: 10 VH 11E6 CDR-H2
Residues 50-66 of SEQ ID NO : 9 SEQ ID NO: 11 VH 11E6 CDR-H3
Residues 99-109 of SEQ ID NO: 9 SEQ ID NO: 12 VL 11E6 set VL 11E6
CDR-L1 Residues 24-34 of SEQ ID NO: 13 SEQ ID NO: 14 VL 11E6 CDR-L2
Residues 50-56 of SEQ ID NO: 13 SEQ ID NO: 15 VL 11E6 CDR-L3
Residues 89-97 of SEQ ID NO: 13 SEQ ID NO: 16 VH 4E5 set VH 4E5
CDR-H1 Residues 31-35 of SEQ ID NO: 17 SEQ ID NO: 18 VH 4E5 CDR-H2
Residues 50-66 of SEQ ID NO: 17 SEQ ID NO: 19 VH 4E5 CDR-H3
Residues 99-109 of SEQ ID NO: 17 SEQ ID NO: 20 VL 4E5 set VL 4E5
CDR-L1 Residues 24-34 of SEQ ID NO: 21 SEQ ID NO: 22 VL 4E5 CDR-L2
Residues 50-56 of SEQ ID NO: 21 SEQ ID NO: 23 VL 4E5 CDR-L3
Residues 89-97 of SEQ ID NO: 21 SEQ ID NO: 24
or a variable domain set wherein at least one of said 3 CDRs is a
modified CDR amino acid sequence having a sequence identity of at
least 50% to the parent sequence.
4. The method according to claim 1, wherein the antibody further
comprises a human acceptor framework.
5. The method of claim 1, wherein the antibody comprises at least
one heavy chain variable domain selected from the group consisting
of: SEQ ID NOs: 56 and 57, at least one light chain variable domain
selected from the group consisting of: SEQ ID NOs: 58 and 59 or
combinations thereof.
6. The method according to claim 5, wherein said binding protein
comprises (a) at least one heavy chain framework mutation at a
position selected from the group consisting of: 1, 2, 68, 70, 72,
76, 85, 89 and 95; (b) at least one light chain framework mutation
at a position selected from the group consisting of: 11, 13, 43,
49, 58, 70 and 87; or (c) combinations of (a) and (b).
7. The method of claim 1, wherein the antibody comprises (a) at
least one framework mutation at a position selected from the group
consisting of: 62, 67, 68 and 69; (b) at least one light chain
framework mutation at a position selected from the group consisting
of: 63, 64, 65 and 66; or (c) combinations of (a) and (b).
8. The method of claim 1, wherein the antibody is selected from the
group consisting of antibodies 11E6 and 4E5.
9. The method of claim 1, wherein the composition comprises a
formulation, wherein said formulation comprises the antibody of
claim 1 in crystallized form as an ingredient; and at least one
polymeric carrier.
10. The method of claim 8, wherein the antibody is 11E6.
11. The method of claim 1, wherein said pain is selected from the
group consisting of chronic and acute pain.
12. The method of claim 1, wherein said pain is selected from the
group consisting of: peripheral or central neuropathic pain,
nociceptive pain; and psychogenic pain.
13. The method of claim 1, wherein said pain is nociceptive pain or
psychogenic pain.
14. The method of claim 1, wherein said pain is neuropathic
pain.
15. The method of claim 14, wherein said neuropathic pain is
peripheral or central neuropathic pain.
16. The method of claim 1, wherein said pain is inflammatory
pain.
17. A monoclonal antibody as defined in claim 1 for use in treating
pain.
Description
RELATED APPLICATION INFORMATION
[0001] This application claims the benefit of U.S. Application No.
61/256,949 filed on Oct. 31, 2009, the contents of which are herein
incorporated by reference.
TECHNICAL FIELD
[0002] The present application relates to antibodies, particularly
monoclonal antibodies, and in particular CDR grafted, humanized
versions thereof, for the treatment and diagnosis of pain and other
neuroinflammatory conditions associated with the Receptor for
Advanced Glycation End Products (RAGE).
BRIEF DESCRIPTION OF SEQUENCE LISTING
[0003] A sequence listing in accordance with 37 C.F.R.
.sctn..sctn.1.821-1.825 is attached to the present invention and
contained in a file named "10216USO1.txt" (119 Kb, created Oct. 20,
2010) and is hereby incorporated herein by reference.
BACKGROUND INFORMATION
[0004] Chronic pathological pain remains an area of significant
unmet medical need. Current treatments, such as duloxetine, are
efficacious only in a limited subset of patients and are associated
with dose-limiting side effects, such as nausea and somnolence.
Advanced Glycation End products (AGEs) and one of their receptors
(RAGE) have been implicated in the ethiology of chronic pain
syndromes, including those of diabetic neuropathy and
fibromyalgia.
[0005] The Receptor for Advanced Glycation Endproducts (RAGE) is a
multi-ligand receptor of the immunoglobulin superfamily.
Extracellular domain of RAGE includes one N-terminal V-type and two
C.sub.2-type immunoglobulin domains usually termed C1 and C2.
Ligands of RAGE include AGEs (the products of nonenzymatic
glycation and oxidation of proteins and lipids such as
carboxymethyl lysine (CML) adducts), A.beta., S100/calgranulins and
amphoterin. Different ligands have been shown to bind to different
domains within RAGE. For example, S100b, HMGB1 and amyloid A.beta.
are reported to bind to the V-domain, as evidenced by various
groups (Ostendorp et al. EMBO J. 2007; 26(16): 3868-3878; Leclerc E
et al, J Biol Chem. 2007; 282 (43): 31317-31331; WO
2007/109749(A2). Ligands of C1 (first C.sub.2-like) domain and C2
(second C.sub.2-like) domain were reported to include S100A12 (also
called ENRAGE or Calgranulin C) and A.beta. in case of C1 (Hofmann
M A et al. Cell, 1999; 97: 889-901), and S100A6 (Leclerc E et al, J
Biol Chem. 2007; 282 (43): 31317-31331). In some cases, the precise
location of binding of a ligand to RAGE remains unclear.
SUMMARY OF THE INVENTION
[0006] The present invention provides a method of treatment of
chronic pain using anti-RAGE antibodies; representative anti-RAGE
antibodies of the invention may comprise at least one of the
antibody variable region amino acid sequences shown in SEQ ID NOs:
9, 13, 17 and 21, or individual CDRs thereof or related CDR
sequences, as specified in more detail below.
[0007] Included in the present invention are anti-RAGE antibodies
that bind specifically to RAGE and comprise a light chain variable
region having an amino acid sequence that is at least 90% identical
to any of SEQ ID NOs: 13 and 21, or is a RAGE-binding fragment of
an antibody comprised in said sequences.
[0008] Also included are anti-RAGE antibodies that bind
specifically to RAGE and comprise a heavy chain variable region
having an amino acid sequence that is at least 90% identical to any
of SEQ ID NOs: 9 and 17, or is a RAGE-binding fragment of an
antibody comprised in said sequences.
[0009] A particular embodiment of the present invention is
represented by several monoclonal antibodies that are able to bind
to the C-domain of RAGE, and to block the binding of
A.beta.-globulomers. More specifically the present invention
describes monoclonal antibody 11E6, which binds to the C-2 domain
of RAGE, does not bind to peptides with amino acid sequences used
to generate polyclonal antibodies, and is able to neutralize in
vivo the effect of A.beta.1-40 on cerebral vasculature in mice.
[0010] The anti-RAGE antibodies of the invention include antibodies
that bind specifically to the C-domain of RAGE.
[0011] The anti-RAGE antibodies of the invention include an
anti-RAGE antibody or a RAGE-binding fragment as described above,
which is selected from the group consisting of chimeric antibody, a
CDR-grafted or humanized antibody, a single chain antibody, a
fusion protein, and a human antibody.
[0012] In various embodiments, the antibodies of the invention are
recombinant antibodies or monoclonal antibodies. Particular
neutralizing antibodies of the present application are referred to
herein as mAb7F9, mAb11E6, and mAb4E5 and functional antibody
fragments thereof, and other antibodies and functional antibody
fragments with equivalent properties to mAb7F9, mAb11E6, and
mAb4E5, such as high affinity binding to RAGE with low dissociation
kinetics and high neutralizing capacity, are intended as part of
the present invention. The human antibodies of the present
application, however, may include amino acid residues not encoded
by human germline immunoglobulin immunogenic RAGE polypeptide or
fragment thereof that may be determined by any method known in the
art. For example, the binding affinity can be measured by
competitive ELISAs, RIAs, BIAcore or KinExA technology. The
dissociation rate also can be measured by BIAcore or KinExA
technology. The binding affinity and dissociation rate are measured
by surface plasmon resonance using, e.g., BIAcore.
[0013] One of the monoclonal antibodies of the present application,
the mAb11E6 antibody, has at least 90% amino acid sequence identity
with a sequence comprising a heavy chain variable region (VH
region) comprising the sequence of SEQ ID NO: 9; and SEQ ID NOs:
10, 11, and 12 which are residues 31-35, 50-66, and 99-109 of SEQ
ID NO: 9, respectively. The mAb11E6 antibody of the present
invention has at least 90% amino acid sequence identity with a
sequence comprising a light chain variable region (VL region)
comprising the sequence of SEQ ID NO: 13, and SEQ ID NOs: 14, 15,
and 16 which are residues 24-34, 50-56, 89-97 of SEQ ID NO: 13,
respectively. The mAb11E6 binds to the C-2 domain of RAGE, does not
bind to peptides with amino acid sequences used to generate
polyclonal antibodies, and is able to neutralize in vivo the effect
of A.beta.1-40 on cerebral vasculature in mice.
[0014] Another of the monoclonal antibodies of the present
application, the mAb4E5 antibody, has at least 90% amino acid
sequence identity with a sequence comprising a heavy chain variable
region (VH region) comprising the sequence of SEQ ID NO: 17; and
SEQ ID NOs: 18, 19, and 20 which are residues 31-35, 50-66, and
99-109 of SEQ ID NO: 17, respectively. The mAb4E5 antibody of the
present invention has at least 90% amino acid sequence identity
with a sequence comprising a light chain variable region (VL
region) comprising the sequence of SEQ ID NO: 21, and SEQ ID NOs:
22, 23, and 24 which are residues 24-34, 50-56, 89-97 of SEQ ID NO:
21, respectively.
[0015] It is also intended that the isolated monoclonal antibodies
that interact with RAGE of the present application may be a
glycosylated binding protein wherein the antibody or
antigen-binding portion thereof comprises one or more carbohydrate
residues. Nascent in vivo protein production may undergo further
processing, known as post-translational modification. In
particular, sugar (glycosyl) residues may be added enzymatically, a
process known as glycosylation. The resulting proteins bearing
covalently linked oligosaccharide side chains are known as
glycosylated proteins or glycoproteins. Protein glycosylation
depends on the amino acid sequence of the protein of interest, as
well as the host cell in which the protein is expressed. Different
organisms may produce different glycosylation enzymes (eg.,
glycosyltransferases and glycosidases), and have different
substrates (nucleotide sugars) available. Due to such factors,
protein glycosylation pattern, and composition of glycosyl
residues, may differ depending on the host system in which the
particular protein is expressed. Glycosyl residues useful in the
invention may include, but are not limited to, glucose, galactose,
mannose, fucose, n-acetylglucosamine and sialic acid.
[0016] The antibodies of the present application comprise a heavy
chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE,
IgM or IgD constant region. Furthermore, the antibody can comprise
a light chain constant region, either a kappa light chain constant
region or a lambda light chain constant region. Particularly, the
antibody comprises a kappa light chain constant region.
Alternatively, the antibody portion can be, for example, a Fab
fragment or a single chain Fv fragment. Replacements of amino acid
residues in the Fc portion to alter antibody effector function are
known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260;
5,624,821). The Fc portion of an antibody mediates several
important effector functions, e.g. cytokine induction, ADCC,
phagocytosis, complement dependent cytotoxicity (CDC) and
half-life/clearance rate of antibody and antigen-antibody
complexes. In some cases these effector functions are desirable for
therapeutic antibodies but in other cases might be unnecessary or
even deleterious, depending on the therapeutic objectives. Certain
human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and
CDC via binding to Fc.gamma. Rs and complement C1q, respectively.
Neonatal Fc receptors (FcRn) are the critical components
determining the circulating half-life of antibodies. In still
another embodiment at least one amino acid residue is replaced in
the constant region of the antibody, for example the Fc region of
the antibody, such that effector functions of the antibody are
altered.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A-1C show ELISA binding of recombinant and
hybridoma-derived anti-human RAGE monoclonal antibodies 7F9, 11E6,
and 4E5 to recombinant human RAGE.
[0018] FIG. 2 shows characterization of monoclonal antibodies 7F9,
11E6, and 4E5 by dot blot binding.
[0019] FIGS. 3A-3C show ELISA binding experiments of monoclonal
antibodies of the invention to different RAGE fragments.
[0020] FIGS. 4A-4C show ELISA binding experiments of monoclonal
antibodies of the invention to different RAGE fragments.
[0021] FIG. 5 shows effects of RAGE antibody on Chronic
Constriction Injury Model of neuropathic pain in mice. In this FIG.
5, CONTRA refers to contralateral paw tested. BL refers to baseline
test.
[0022] FIG. 6 shows the effects of RAGE antibody in a mouse model
of inflammatory pain as described in Example 13. Specifically, as
shown in this figure, RAGE mAB ML39-11E6 was dosed at 30, 100 and
300 .mu.g per mouse (C57BL/6), ip; IgG-1 (KLH mAB H35C206) at 100
.mu.g per mouse (C57BL/6), intraperitoneal (ip) Animals were dosed
on day 1 post CFA and tested 60 minutes post dose. *p<0.05;
**p<0.01 versus vehicle (n=10 per group).
[0023] FIG. 7 shows the time course effects of human and mouse
anti-RAGE antibodies in a mouse model of neuropathic pain as
described in Example 14. Specifically, chronic constriction injury
(Bennett model) surgeries were performed 7 days before this
experiment was conducted. RAGE mAB 11E6 (which is also referred to
herein as ms11E6, 11E6.50, A-992401.0 and ML39-11E6)), hu11E6 (also
referred to herein as 11E6.12hu and A-1175708.0) and human IgG were
dosed at 300 ug in 200 .mu.l/mouse once per mouse (C57BL/6), ip and
tested 24 hours, following treatment. **p<0.01, versus vehicle,
unpaired t-test (n=5; 24 hours). In this FIG. 7, CONTRA refers to
contralateral paw tested.
SEQUENCE LISTING
[0024] SEQ ID NO: 1: amino acid sequence of mAb VH 7F9 SEQ ID NO:
2: amino acid sequence of mAb VH 7F9 CDR-H1 SEQ ID NO: 3: amino
acid sequence of mAb VH 7F9 CDR-H2 SEQ ID NO: 4: amino acid
sequence of mAb VH 7F9 CDR-H3 SEQ ID NO: 5: amino acid sequence of
mAb VL 7F9 SEQ ID NO: 6: amino acid sequence of mAb VL 7F9 CDR-L1
SEQ ID NO: 7: amino acid sequence of mAb VL 7F9 CDR-L2 SEQ ID NO:
8: amino acid sequence of mAb VL 7F9 CDR-L3 SEQ ID NO: 9: amino
acid sequence of mAb VH 11E6 SEQ ID NO: 10: amino acid sequence of
mAb VH 11E6 CDR-H1 SEQ ID NO: 11: amino acid sequence of mAb VH
11E6 CDR-H2 SEQ ID NO: 12: amino acid sequence of mAb VH 11E6
CDR-H3 SEQ ID NO: 13: amino acid sequence of mAb VL 11E6 SEQ ID NO:
14: amino acid sequence of mAb VL 11E6 CDR-L1 SEQ ID NO: 15: amino
acid sequence of mAb VL 11E6 CDR-L2 SEQ ID NO: 16: amino acid
sequence of mAb VL 11E6 CDR-L3 SEQ ID NO: 17: amino acid sequence
of mAb VH 4E5 SEQ ID NO: 18: amino acid sequence of mAb VH 4E5
CDR-H1 SEQ ID NO: 19: amino acid sequence of mAb VH 4E5 CDR-H2 SEQ
ID NO: 20: amino acid sequence of mAb VH 4E5 CDR-H3 SEQ ID NO: 21:
amino acid sequence of mAb VL 4E5 SEQ ID NO: 22: amino acid
sequence of mAb VL 4E5 CDR-L1 SEQ ID NO: 23: amino acid sequence of
mAb VL 4E5 CDR-L2 SEQ ID NO: 24: amino acid sequence of mAb VL 4E5
CDR-L3 SEQ ID NO: 25: amino acid sequence of human Ig gamma1 heavy
chain constant region SEQ ID NO: 26: amino acid sequence of human
Ig kappa light chain constant region SEQ ID NO: 27: Full plasmid
nucleotide sequence of Construct #1 (bold) encoding OmpA-[RAGE
(23-340)]-6His SEQ ID NO: 28: Full plasmid nucleotide sequence of
Construct #2 (bold) encoding 6His-(Thr)-[RAGE (24-129)] SEQ ID NO:
29: Full plasmid nucleotide sequence of Construct #3 (bold)
encoding 6His-(Thr)-[RAGE (24-234)] SEQ ID NO: 30: Full plasmid
nucleotide sequence of Construct #4 (bold) encoding
6His-(Thr)-[RAGE (24-336)] SEQ ID NO: 31: Full plasmid nucleotide
sequence of Construct #5 (bold) encoding 6His-(Thr)-[RAGE
(130-234)] SEQ ID NO: 32: Full plasmid nucleotide sequence of
Construct #6 (bold) encoding 6His-(Thr)-[RAGE (130-336)] SEQ ID NO:
33: Full plasmid nucleotide sequence of Construct #7 (bold)
encoding 6His-(Thr)-[RAGE (235-336)] SEQ ID NO: 34: encoded amino
acid sequence RAGE protein #1 SEQ ID NO: 35: encoded amino acid
sequence RAGE protein #2 SEQ ID NO: 36: encoded amino acid sequence
RAGE protein #3 SEQ ID NO: 37: encoded amino acid sequence RAGE
protein #4 SEQ ID NO: 38: encoded amino acid sequence RAGE protein
#5 SEQ ID NO: 39: encoded amino acid sequence RAGE protein #6 SEQ
ID NO: 40: encoded amino acid sequence RAGE protein #7 SEQ ID
NO:41: Ig gamma-1 constant region mutant amino acid sequence SEQ ID
NO:42: Ig gamma-2 constant region amino acid sequence SEQ ID NO:43:
framework amino acid sequence VH7-4.1/JH6FR1 SEQ ID NO:44:
framework amino acid sequence VH7-4.1/JH6 FR2 and VH1-2/JH6 FR2 SEQ
ID NO:45: framework amino acid sequence VH7-4.1/JH6 FR3 SEQ ID
NO:46: framework amino acid sequence VH7-4.1/JH6 FR4 and VH1-2/JH6
FR4 SEQ ID NO:47: framework amino acid sequence VH1-2/JH6 FR1 SEQ
ID NO:48: framework amino acid sequence VH1-2/JH6 FR3 SEQ ID NO:49:
framework amino acid sequence 1-12/L5/JK2 FR1 SEQ ID NO:50:
framework amino acid sequence 1-12/L5/JK2 FR2 SEQ ID NO:51:
framework amino acid sequence 1-12/L5/JK2 FR3 SEQ ID NO:52:
framework amino acid sequence 1-12/L5/JK2 FR4 and 3-15/L2/JK2 FR4
SEQ ID NO:53: framework amino acid sequence 3-15/L2/JK2 FR1 SEQ ID
NO:54: framework amino acid sequence 3-15/L2/JK2 FR2 SEQ ID NO:55:
framework amino acid sequence 3-15/L2/JK2 FR3 SEQ ID NO:56:
CDR-grafted amino acid sequence VH 11E6.1-GL SEQ ID NO:57:
CDR-grafted amino acid sequence VH 11E6.2-GL SEQ ID NO:58:
CDR-grafted amino acid sequence VL 11E6.1-GL SEQ ID NO:59:
CDR-grafted amino acid sequence VL 11E6.2-GL SEQ ID NO: 60: amino
acid sequence of hRAGE SEQ ID NO: 61: amino acid sequence of a
husRAGE fragment SEQ ID NO: 62: humanized antibody sequence VH
h11E6.1 SEQ ID NO: 63: humanized antibody sequence VL h11E6.1 SEQ
ID NO: 64: humanized antibody sequence VL h11E6.2 SEQ ID NO: 65:
humanized antibody sequence VL h11E6.3 SEQ ID NO: 66: humanized
antibody sequence VL h11E6.4 SEQ ID NO: 67: humanized antibody
sequence VH h11E6.5 SEQ ID NO: 68: humanized antibody sequence VH
h11E6.9 SEQ ID NO: 69: humanized antibody sequence VH h11E6.16 SEQ
ID NO:70: amino acid sequence of RAGE-derived peptide NtermR31 SEQ
ID NO:71: amino acid sequence of RAGE-derived peptide 1 SEQ ID
NO:72: amino acid sequence of RAGE-derived peptide 2 SEQ ID NO:73:
amino acid sequence of RAGE-derived peptide 3 SEQ ID NO:74: amino
acid sequence of RAGE-derived peptide 4 SEQ ID NO:75: amino acid
sequence of RAGE-derived peptide 5 SEQ ID NO:76: amino acid
sequence of RAGE-derived peptide 6 SEQ ID NO:77: amino acid
sequence of RAGE-derived peptide 7 SEQ ID NO:78: amino acid
sequence of RAGE-derived peptide 8 SEQ ID NO:79: amino acid
sequence of RAGE-derived peptide 9 SEQ ID NO:80: amino acid
sequence of RAGE-derived peptide 10 SEQ ID NO:81: nucleotide
sequence of oligonucleotide primers SEQ ID NO:82: nucleotide
sequence of oligonucleotide primers SEQ ID NO:83: nucleotide
sequence of oligonucleotide primers SEQ ID NO:84: nucleotide
sequence of oligonucleotide primers SEQ ID NO:85: nucleotide
sequence of oligonucleotide primers SEQ ID NO:86: nucleotide
sequence of oligonucleotide primers SEQ ID NO:87: nucleotide
sequence of oligonucleotide primers SEQ ID NO:88: nucleotide
sequence of oligonucleotide primers SEQ ID NO:89: nucleotide
sequence of oligonucleotide primers SEQ ID NO:90: nucleotide
sequence of oligonucleotide primers SEQ ID NO:91: nucleotide
sequence of oligonucleotide primers SEQ ID NO:92: nucleotide
sequence of oligonucleotide primers SEQ ID NO:93: nucleotide
sequence of oligonucleotide primers SEQ ID NO:94: nucleotide
sequence of oligonucleotide primers SEQ ID NO:95: amino acid
sequence of a His tag (His His His His His His) SEQ ID NO:96: amino
acid sequence of a His tag (His His His His His His Thr) SEQ ID
NO:97: amino acid sequence--Gly Gly Gly Gly Ser
DETAILED DESCRIPTION
1. General Definitions
[0025] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0026] 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 invention 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.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
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
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0027] That the present invention may be more readily understood,
selected terms are defined below.
[0028] The term "polypeptide" as used herein, refers to any
polymeric chain of amino acids. The terms "peptide" and "protein"
are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide"
encompasses native or artificial proteins, protein fragments and
polypeptide analogs of a protein sequence. A polypeptide may be
monomeric or polymeric.
[0029] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0030] The term "recovering" as used herein, refers to the process
of rendering a chemical species such as a polypeptide substantially
free of naturally associated components by isolation, e.g., using
protein purification techniques well known in the art.
[0031] The term "Receptor for Advanced Glycation Endproducts
(RAGE)" designates a multiligand receptor in the immunoglobulin
family, which binds soluble A.beta. peptide, S100b, and HMGB1 (also
known as amphoterin) among others. RAGE mediates
patho-physiologically relevant cellular changes in response to
binding to these ligands. Transgenic animals overexpressing RAGE
and human APP display early abnormalities in spatial learning and
memory, indicating that RAGE is a cofactor for A.beta.-induced
neuronal perturbation in Alzheimer-type pathologies, and suggesting
that RAGE is a potential therapeutic target to ameliorate cellular
dysfunction.
[0032] The structure of RAGE has not been solved. Homology to other
proteins leads to a model where RAGE has several domains. These
domains are named in analogy to immunoglobulins: (i) V-like domain
at the N-terminus: this equivalent domain in immunoglobulins binds
antigen and represents the only binding region within these
proteins. In RAGE, this domain binds to some ligands like S100
(Ostendorp et al. EMBO J. 26,3875, 2007; Leclerc et al. JBC 282,
31317, 2007). A monoclonal antibody binding to the v-like domain in
RAGE competes with binding of different ligands S100b, HMGB1, and
amyloid .beta. (WO 2007/109749(A2)) implying that these ligands
would also bind to RAGE via this same domain. (ii) First C2-like
domain: Two domains within RAGE have homology to the C2 domains of
immunoglobulins; one of these domains has been called C1
(nomenclature also used by Ostendorp et al. Biochem Biophys Res
Comm 2006; 347: 4-11). Several ligands binding to this domain have
been described, for instance, S100A12 (also called ENRAGE or
Calgranulin C), which binds with an affinity of K.sub.d=90 nM
(Hofmann et al. Cell 97, 889, 1999; Xie et al. JBC, 282, 4218,
2007). A.beta. competes with S100A12 for this domain, suggesting
that A.beta. also binds to the C1-domain. (iii) Second C2-like
domain: this domain is called C2 (also used by Ostendorp et al.
Biochem Biophys Res Comm 2006; 347: 4-11). RAGE ligand S100A6 binds
to the C2 domain and an antibody generated against a peptide from
the C2 domain was shown to compete against S100A6 for this binding;
the antibody lead to reduced signal transduction in SH-SY5Y
(Leclerc et al. JBC 282, 31317, 2007).
[0033] A "RAGE domain" may be defined in line with different
definitions provided in the state of the art:
[0034] According to Xie et al. 2007, J. Biol. Chem., Vol.
282:4218-4231 the following definition of h RAGE domains applies:
[0035] the V domain (amino acids 24-129 of SEQ ID NO:60), [0036]
the C1 domain (amino acids 130-234 of SEQ ID NO:60), [0037] the C2
domain (amino acids 235-336 of SEQ ID NO:60),
[0038] According to a more recent definition by Hudson et al, The
FASEB Journal. 2008; 22:1572-1580, h RAGE (404 amino acid residues
according to SEQ ID NO:60) has an extracellular region (amino acids
1-342 of SEQ ID NO:60) composed of [0039] a signal peptide (amino
acids 1-22 of SEQ ID NO:60), followed by three immunoglobulin-like
domains, including [0040] an Ig-like V-type domain (amino acids
23-116 of SEQ ID NO:60) and [0041] two Ig-like C2-type 1/2 domains
(amino acids 124-221 of SEQ ID NO:60; also designated C1 domain;
and amino acids 227-317 of SEQ ID NO:60; also designated C2
domain); [0042] a single transmembrane domain (amino acids 343-363
of SEQ ID NO:60), and [0043] a short cytoplasmic tail (amino acids
364-404 of SEQ ID NO:60).
[0044] In view of the high degree of identity, in particular with
respect to the definition of domains V, C1 and C2, and unless
otherwise stated, each of the definitions may be applied in order
to define the binding characteristics of the binding proteins of
the present invention.
[0045] As indicated above, RAGE is capable of binding different
ligands via different domains. Results from competition experiments
with other ligands seem to indicate that A.beta. binds to the
C1-domain (Hofmann et al. Cell 97, 889, 1999 or Xie et al. JBC,
282, 4218, 2007). As non-limiting examples of RAGE ligands there
may be mentioned: [0046] Advanced glycation end products (AGEs),
(Baynes J. W., 1991, Diabetes. 1991, 40:405-412; Ahmed K. A., 2007,
J Clin Biochem Nutr. 41 (2):97-105); [0047] Members of the
S100/calgranulin family (e.g., calgranulin C (also known as ENRAGE
and S100A12), S100A1, S100A4, S100A11, S100A13, S100B, and S100P);
[0048] Amyloid-.beta.-peptide (A.beta.), as for example A.beta.
1-40 peptide [0049] Amyloid-.beta. globulomers; as for example
A.beta.1-42, A.beta.12-42, A.beta.20-42 globulomers (see Barghorn
et al., Globular amyloid .beta. peptide 1-42 oligomer--a homogenous
and stable neuropathological protein in Alzheimer's disease)
Journal of Neurochemistry. 95(3):834-847, November 2005; WO
2007/062852; WO 2008/150949; all incorporated by reference); [0050]
eukocyte integrins (e.g., Mac-1)
[0051] The term "RAGE" as used herein, particularly refers to human
RAGE, also designated "hRAGE" or "huRAGE". Unless otherwise stated
the term "RAGE" also encompasses RAGE molecules isolated or
obtained from other, different from human, species, as for example,
rodents, like mice or rats; or bovine RAGE molecules.
[0052] The term "sRAGE" refers to a soluble form of RAGE, derived
from the extra cellular domain of RAGE. For example, a sRAGE
molecule derived from human RAGE, also designated as husRAGE
comprises amino acid residues 1 to 331 (see SEQ ID NO: 61) of human
RAGE (see SEQ ID NO: 60).
[0053] "Biological activity" as used herein, refers to all inherent
biological properties of RAGE as defined herein.
[0054] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an "antigenic determinant" or "epitope" as defined
below) on the chemical species; for example, an antibody recognizes
and binds to a specific protein structure rather than to proteins
generally. If an antibody is specific for epitope "A", the presence
of a molecule containing epitope A (or free, unlabeled A), in a
reaction containing labeled "A" and the antibody, will reduce the
amount of labeled A bound to the antibody.
[0055] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art.
Nonlimiting embodiments of which are discussed below. An antibody
is said to be "capable of binding" a molecule if it is capable of
specifically reacting with the molecule to thereby bind the
molecule to the antibody.
[0056] A "monoclonal antibody" as used herein is intended to refer
to a preparation of antibody molecules, which share a common heavy
chain and common light chain amino acid sequence, in contrast with
"polyclonal" antibody preparations that contain a mixture of
different antibodies. Monoclonal antibodies can be generated by
several novel technologies like phage, bacteria, yeast or ribosomal
display, as well as classical methods exemplified by
hybridoma-derived antibodies (e.g., an antibody secreted by a
hybridoma prepared by hybridoma technology, such as the standard
Kohler and Milstein hybridoma methodology ((1975) Nature
256:495-497).
[0057] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG 1, IgG2, IgG 3, IgG 4, IgA1 and IgA2) or subclass.
[0058] The term "antigen-binding portion" or "antigen-binding
fragment" of an antibody (or simply "antibody portion" or "antibody
fragment"), as used herein, refers to one or more fragments of an
antibody that retain the ability to specifically bind to an antigen
(e.g., RAGE). It has been shown that the antigen-binding function
of an antibody can be performed by fragments of a full-length
antibody. Such antibody embodiments may also be bispecific, dual
specific, or multi-specific formats; specifically binding to two or
more different antigens. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include
(i) a Fab fragment, a monovalent fragment consisting of the VL, VH,
CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the VH and
CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains
of a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546, Winter et al., PCT publication WO
90/05144 A1 herein incorporated by reference), which comprises a
single variable domain; and (vi) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, VL and VH, are coded for by separate genes, they
can be joined, using recombinant methods, by a synthetic linker
that enables them to be made as a single protein chain in which the
VL and VH regions pair to form monovalent molecules (known as
single chain Fv (scFv); see e.g., Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see e.g., Holliger, P., et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et
al. (1994) Structure 2:1121-1123). Such antibody binding portions
are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN
3-540-41354-5).
[0059] The term "antibody construct" as used herein refers to a
polypeptide comprising one or more the antigen binding portions of
the invention linked to a linker polypeptide or an immunoglobulin
constant domain. Linker polypeptides comprise two or more amino
acid residues joined by peptide bonds and are used to link one or
more antigen binding portions. Such linker polypeptides are well
known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure
2:1121-1123). An immunoglobulin constant domain refers to a heavy
or light chain constant domain. Human IgG heavy chain and light
chain constant domain amino acid sequences are known in the art and
represented in Table 1.
TABLE-US-00001 TABLE 1 Sequence of human IgG heavy chain constant
domain and light chain constant domain ##STR00001##
[0060] Still further, an antibody or antigen-binding portion
thereof may be part of larger immunoadhesion molecules, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein.
[0061] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds human RAGE is substantially free
of antibodies that specifically bind antigens other than human
RAGE). An isolated antibody that specifically binds human RAGE may,
however, have cross-reactivity to other antigens, such as RAGE
molecules from other species. Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0062] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0063] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell (described further below), antibodies
isolated from a recombinant, combinatorial human antibody library
(Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and
Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V.,
and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H.,
and Chames P. (2000) Immunology Today 21:371-378), antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992)
Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L.
(2002) Current Opinion in Biotechnology 13:593-597; Little M. et al
(2000) Immunology Today 21:364-370) or antibodies prepared,
expressed, created or isolated by any other means that involves
splicing of human immunoglobulin gene sequences to other DNA
sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0064] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions. The chimeric antibody can be
produced through recombinant molecular biological techniques, or
may be physically conjugated together.
[0065] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0066] The terms "Kabat numbering", "Kabat definitions and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e. hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0067] As used herein, the terms "acceptor" and "acceptor antibody"
refer to the antibody or nucleic acid sequence providing or
encoding at least 50, 55, 60, 65, 70, 75 or 80%, at least 85%, at
least 90%, at least 95%, at least 98% or 100% of the amino acid
sequences of one or more of the framework regions. In some
embodiments, the term "acceptor" refers to the antibody amino acid
or nucleic acid sequence providing or encoding the constant
region(s). In yet another embodiment, the term "acceptor" refers to
the antibody amino acid or nucleic acid sequence providing or
encoding one or more of the framework regions and the constant
region(s). In a specific embodiment, the term "acceptor" refers to
a human antibody amino acid or nucleic acid sequence that provides
or encodes at least 50, 55, 60, 65, 70, 75 or 80%, particularly, at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%
or 100% of the amino acid sequences of one or more of the framework
regions. In accordance with this embodiment, an acceptor may
contain at least 1, at least 2, at least 3, least 4, at least 5, or
at least 10 amino acid residues that does (do) not occur at one or
more specific positions of a human antibody. An acceptor framework
region and/or acceptor constant region(s) may be, e.g., derived or
obtained from a germline antibody gene, a mature antibody gene, a
functional antibody (e.g., antibodies well-known in the art,
antibodies in development, or antibodies commercially
available).
[0068] As used herein, the term "CDR" refers to the complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the variable regions. The term "CDR set" as used herein refers
to a group of three CDRs that occur in a single variable region
capable of binding the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and
coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and
Chothia et al., Nature 342:877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as L1,
L2 and L3 or H1, H2 and H3 where the "L" and the "H" designates the
light chain and the heavy chains regions, respectively. These
regions may be referred to as Chothia CDRs, which have boundaries
that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan
(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45
(1996)). Still other CDR boundary definitions may not strictly
follow one of the above systems, but will nonetheless overlap with
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. The methods used herein may
utilize CDRs defined according to any of these systems, although
particular embodiments use Kabat or Chothia defined CDRs.
[0069] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al. (J. Mol. Biol.
196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992),
both are incorporated herein by reference). According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0070] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In a particular
embodiment, the donor antibody is an antibody from a species
different from the antibody from which the framework regions are
obtained or derived. In the context of a humanized antibody, the
term "donor antibody" refers to a non-human antibody providing one
or more CDRs.
[0071] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems, the meaning of a framework
sequence is subject to correspondingly different interpretations.
The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2,
and -H3 of heavy chain) also divide the framework regions on the
light chain and the heavy chain into four sub-regions (FR1, FR2,
FR3 and FR4) on each chain, in which CDR1 is positioned between FR1
and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
Without specifying the particular sub-regions as FR1, FR2, FR3 or
FR4, a framework region, as referred by others, represents the
combined FR's within the variable region of a single, naturally
occurring immunoglobulin chain. As used herein, a FR represents one
of the four sub-regions, and FRs represents two or more of the four
sub-regions constituting a framework region.
[0072] Human heavy chain and light chain acceptor sequences are
known in the art. In one embodiment of the invention the human
heavy chain and light chain acceptor sequences are selected from
the sequences described in Table 2 and Table 3. Different
combinations for human framework sequences FR1 to FR4 are stated in
said tables.
TABLE-US-00002 TABLE 2 Human heavy chain acceptor sequences
##STR00002##
TABLE-US-00003 TABLE 3 Human light chain acceptor sequences
##STR00003##
[0073] As used herein, the term "germline antibody gene" or "gene
fragment" refers to an immunoglobulin sequence encoded by
non-lymphoid cells that have not undergone the maturation process
that leads to genetic rearrangement and mutation for expression of
a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev.
Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med
Biol. 484:13-30 (2001)). One of the advantages provided by various
embodiments of the present invention stems from the recognition
that germline antibody genes are more likely than mature antibody
genes to conserve essential amino acid sequence structures
characteristic of individuals in the species, hence less likely to
be recognized as from a foreign source when used therapeutically in
that species.
[0074] As used herein, the term "key" residues refer to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with the
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0075] The term "humanized antibody" generally refers to antibodies
which comprise heavy and light chain variable region sequences from
a non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences.
[0076] In particular, the term "humanized antibody" as used herein,
is an antibody or a variant, derivative, analog or fragment thereof
which immunospecifically binds to an antigen of interest and which
comprises a framework (FR) region having substantially the amino
acid sequence of a human antibody and a complementary determining
region (CDR) having substantially the amino acid sequence of a
non-human antibody. As used herein, the term "substantially" in the
context of a CDR refers to a CDR having an amino acid sequence at
least 50, 55, 60, 65, 70, 75 or 80%, particularly at least 85%, at
least 90%, at least 95%, at least 98% or at least 99% identical to
the amino acid sequence of a non-human antibody CDR. A humanized
antibody comprises substantially all of at least one, and typically
two, variable domains (Fab, Fab', F(ab').sub.2, FabC, Fv) in which
all or substantially all of the CDR regions correspond to those of
a non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. Particularly, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. In
some embodiments, a humanized antibody contains both the light
chain as well as at least the variable domain of a heavy chain. The
antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions
of the heavy chain. In some embodiments, a humanized antibody only
contains a humanized light chain. In some embodiments, a humanized
antibody only contains a humanized heavy chain. In specific
embodiments, a humanized antibody only contains a humanized
variable domain of a light chain and/or humanized heavy chain.
[0077] The humanized antibody can be selected from any class of
immunoglobulins, including IgY, IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgA1, IgA2, IgG1, IgG2, IgG3
and IgG4. The humanized antibody may comprise sequences from more
than one class or isotype, and particular constant domains may be
selected to optimize desired effector functions using techniques
well-known in the art.
[0078] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In a particular embodiment, such mutations, however, will not be
extensive. Usually, at least 50, 55, 60, 65, 70, 75 or 80%,
particularly at least 85%, more particularly at least 90%, and in
particular at least 95% of the humanized antibody residues will
correspond to those of the parental FR and CDR sequences. As used
herein, the term "consensus framework" refers to the framework
region in the consensus immunoglobulin sequence. As used herein,
the term "consensus immunoglobulin sequence" refers to the sequence
formed from the most frequently occurring amino acids (or
nucleotides) in a family of related immunoglobulin sequences (See
e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft,
Weinheim, Germany 1987). In a family of immunoglobulins, each
position in the consensus sequence is occupied by the amino acid
occurring most frequently at that position in the family. If two
amino acids occur equally frequently, either can be included in the
consensus sequence.
[0079] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (1992, J. Mol.
Biol. 224:487-499, which is incorporated herein by reference).
Vernier zone residues form a layer underlying the CDRs and may
impact on the structure of CDRs and the affinity of the
antibody.
[0080] The term "inhibition of binding" of RAGE to one of his
ligands as used herein encompasses partial (as for example by about
1 to 10% or more, in particular about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 85%, 90%, or 95% or more) or complete reduction of said ligand
binding activity. Said "inhibition of binding" may be determined by
any suitable method available in the art, preferably by any method
as exemplified herein, as for example HTRF assays described
herein.
[0081] As used herein, the term "neutralizing" refers to
neutralization of biological activity of a target protein when a
binding protein specifically binds the target protein. Neutralizing
may be the result of different ways of binding of said binding
protein to the target. For example, neutralizing may be caused by
binding of the binding protein in a region of the target, which
does not affect receptor binding to the target molecule.
Alternatively binding of a binding protein may result in a blockade
of the receptor binding to the target, which blockade finally
neutralizes the target protein activity. Each of said different
mechanism may occur according to the invention.
[0082] Particularly a neutralizing binding protein is a
neutralizing antibody whose binding to RAGE results in
neutralization of a biological activity of RAGE. Particularly the
neutralizing binding protein binds RAGE and reduces a biologically
activity of RAGE by at least about 1 to 10%, at least about 20%,
30%, 40%, 50%, 60%, 70%, 80%, 85% or more. Neutralization of a
biological activity of RAGE by a neutralizing binding protein can
be assessed by measuring one or more indicators of RAGE biological
activity well known in the art.
[0083] An "inhibition of soluble A.beta.1-40 peptide-induced
reduction of cerebral blood volume" determined in vivo in an animal
model relates to a partial or complete inhibition, as for example
by at least about 1 to 10%, at least about 15%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 85% or more, if compared to a non-treated control,
or in particular, if compared to a negative, monoclonal or
polyclonal, immunoglobulin-isotype control.
[0084] An "improvement of the cerebral blood volume" as in vivo in
an animal model over-expressing human APP, relates to a
statistically significant improvement of CBV, if compared to a
non-treated control, or in particular, if compared to a negative,
monoclonal or polyclonal, immunoglobulin-isotype control.
[0085] A "reduction of amyloid plaque number and/or amyloid plaque
area" as measured in vivo in an animal model over-expressing human
APP relates to a partial or complete reduction, as for example by
at least about 1 to 10%, at least about 15%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 85% or more, if compared to a non-treated control,
or in particular, if compared to a negative, monoclonal or
polyclonal, immunoglobulin-isotype control.
[0086] An "inhibition of aggregated A.beta.1-40 peptide-induced
dynamin cleavage of hippocampal neurons in vitro; relates to a
partial or complete inhibition, as for example by at least about 1
to 10%, at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%
or more, if compared to a non-treated control, or in particular, if
compared to a negative, monoclonal or polyclonal,
immunoglobulin-isotype control.
[0087] A "reversal of A.beta.1-42 globulomer-induced reduction of
synaptic transmission" in vitro, relates to a partial or complete
reversial, as for example by at least about 1 to 10%, at least
about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85% or more, if
compared to a non-treated control, or in particular, if compared to
a negative, monoclonal or polyclonal, immunoglobulin-isotype
control.
[0088] A "neutralizing monoclonal antibody" as used herein is
intended to refer to a preparation of antibody molecules, which
upon binding to the specific antigen are able to compete and
inhibit the binding of the natural ligand for said antigen. In a
particular embodiment of the present application, the neutralizing
antibodies of the present invention are capable of competing with
RAGE for binding to at least one of its ligands, in particular a
ligand selected from A.beta.-peptides, A.beta.-globulomers, S100b
and Amphoterin, and to prevent RAGE biological activity or
function.
[0089] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-RAGE antibody that binds to an RAGE antigen and/or the
neutralizing potency of an antibody, for example, an anti-RAGE
antibody whose binding to RAGE neutralizes the biological activity
of RAGE.
[0090] The term "hyperalgesia" refers to an increased response to a
stimulus which is normally painful. Hyperalgesia reflects increased
pain on suprathreshold stimulation.
[0091] The term "allodynia" refers to pain due to a stimulus which
does not normally provoke pain.
[0092] The term "neuropathic pain" is pain initiated or caused by a
primary lesion or dysfunction in the nervous system.
[0093] The term "attenuation of established hyperalgesia" includes
reduction in behavioral responses to applied mechanical, thermal or
chemical stimuli to animals in vivo model of neuropathic or
inflammatory pain. It is important to note that the attenuation of
established hyperalgesia is distinct from prevention of development
of hyperalgesia.
[0094] The "biological function" or "activity" of RAGE may be
described as that of a signal transducing cell surface receptor for
A.beta. or a receptor that mediates transport of proteins into or
through the cell.
[0095] The term "epitope" or "antigenic determinant" includes any
polypeptide determinant capable of specific binding to an
immunoglobulin or T-cell receptor. In certain embodiments, epitope
determinants include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and/or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody. In certain embodiments, an antibody is said to
specifically bind an antigen when it preferentially recognizes its
target antigen in a complex mixture of proteins and/or
macromolecules.
[0096] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0097] The term "k.sub.on", as used herein, is intended to refer to
the on rate constant for association of an antibody to the antigen
to form the antibody/antigen complex as is known in the art.
[0098] The term "k.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex as is known in the art.
[0099] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction as is known in the art.
[0100] The term "labelled binding protein" as used herein, refers
to a protein with a label incorporated that provides for the
identification of the binding protein. Particularly, the label is a
detectable marker, e.g., incorporation of a radiolabelled amino
acid or attachment to a polypeptide of biotinyl moieties that can
be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Examples of labels for
polypeptides include, but are not limited to, the following:
radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I .sup.177Lu,
.sup.166Ho, or .sup.153Sm); fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), enzymatic labels (e.g.,
horseradish peroxidase, luciferase, alkaline phosphatase);
chemiluminescent markers; biotinyl groups; predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags); and magnetic
agents, such as gadolinium chelates.
[0101] The term "antibody conjugate" refers to a binding protein,
such as an antibody, chemically linked to a second chemical moiety,
such as a therapeutic or cytotoxic agent. The term "agent" is used
herein to denote a chemical compound, a mixture of chemical
compounds, a biological macromolecule, or an extract made from
biological materials. Particularly the therapeutic or cytotoxic
agents include, but are not limited to, pertussis toxin, taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0102] The terms "crystal", and "crystallized" as used herein,
refer to an antibody, or antigen binding portion thereof, that
exists in the form of a crystal. Crystals are one form of the solid
state of matter, which is distinct from other forms such as the
amorphous solid state or the liquid crystalline state. Crystals are
composed of regular, repeating, three-dimensional arrays of atoms,
ions, molecules (e.g., proteins such as antibodies), or molecular
assemblies (e.g., antigen/antibody complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships that are well-understood in the field.
The fundamental unit, or building block, that is repeated in a
crystal is called the asymmetric unit. Repetition of the asymmetric
unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the
crystal.
[0103] Repetition of the unit cell by regular translations in all
three dimensions provides the crystal. See Giege, R. and Ducruix,
A. Barrett, Crystallization of Nucleic Acids and Proteins, a
Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press,
New York, N.Y., (1999)."
[0104] The term "polynucleotide" as referred to herein shall mean a
polymeric form of two or more nucleotides, either ribonucleotides
or deoxynucleotides or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA but particularly is double-stranded DNA.
[0105] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, the
"isolated polynucleotide": is not associated with all or a portion
of a polynucleotide with which the "isolated polynucleotide" is
found in nature; is operably linked to a polynucleotide that it is
not linked to in nature; or does not occur in nature as part of a
larger sequence.
[0106] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0107] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include both expression control sequences that are contiguous with
the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. The term
"expression control sequence" as used herein refers to
polynucleotide sequences, which are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0108] "Transformation", as defined herein, refers to any process
by which exogenous DNA enters a host cell. Transformation may occur
under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for
the insertion of foreign nucleic acid sequences into a prokaryotic
or eukaryotic host cell. The method is selected based on the host
cell being transformed and may include, but is not limited to,
viral infection, electroporation, lipofection, and particle
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. They also include cells which transiently express the
inserted DNA or RNA for limited periods of time.
[0109] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which exogenous
DNA has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but,
to the progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. Particularly host
cells include prokaryotic and eukaryotic cells selected from any of
the Kingdoms of life. Particular eukaryotic cells include protist,
fungal, plant and animal cells. In particular host cells include
but are not limited to the prokaryotic cell line E. coli; mammalian
cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the
fungal cell Saccharomyces cerevisiae.
[0110] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be 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. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by
reference for any purpose.
[0111] "Transgenic organism", as known in the art and as used
herein, refers to an organism having cells that contain a
transgene, wherein the transgene introduced into the organism (or
an ancestor of the organism) expresses a polypeptide not naturally
expressed in the organism. A "transgene" is a DNA construct, which
is stably and operably integrated into the genome of a cell from
which a transgenic organism develops, directing the expression of
an encoded gene product in one or more cell types or tissues of the
transgenic organism.
[0112] The term "regulate" and "modulate" are used interchangeably,
and, as used herein, refers to a change or an alteration in the
activity of a molecule of interest (e.g., the biological activity
of RAGE). Modulation may be an increase or a decrease in the
magnitude of a certain activity or function of the molecule of
interest. Exemplary activities and functions of a molecule include,
but are not limited to, binding characteristics, enzymatic
activity, cell receptor activation, and signal transduction.
[0113] Correspondingly, the term "modulator," as used herein, is a
compound capable of changing or altering an activity or function of
a molecule of interest (e.g., the biological activity of RAGE). For
example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator.
[0114] The term "agonist", as used herein, refers to a modulator
that, when contacted with a molecule of interest, causes an
increase in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function
observed in the absence of the agonist. Particular agonists of
interest may include, but are not limited to, RAGE polypeptides or
polypeptides, nucleic acids, carbohydrates, or any other molecules
that bind to RAGE.
[0115] The term "antagonist", as used herein, refer to a modulator
that, when contacted with a molecule of interest causes a decrease
in the magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in
the absence of the antagonist. Exemplary antagonists include, but
are not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies are
described, e.g., in WO01/83525.
[0116] Particular antagonists of interest include those that block
or modulate the biological or immunological activity of RAGE.
Antagonists of RAGE may include, but are not limited to, proteins,
nucleic acids, carbohydrates, or any other molecules, which bind to
RAGE, particularly monoclonal antibodies that interact with the
RAGE molecule. It should be noted that the interaction with RAGE
may result in binding and neutralization of other ligands/cell
membrane components, and may be useful for additive or synergistic
functioning against multiple diseases.
[0117] As used herein, the term "effective amount" refers to the
amount of a therapy which is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof, prevent the advancement of a disorder, cause regression of
a disorder, prevent the recurrence, development, onset or
progression of one or more symptoms associated with a disorder,
detect a disorder, or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or
therapeutic agent).
[0118] The term "sample", as used herein, is used in its broadest
sense. A "biological sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
serum, urine, synovial fluid, cells, organs, tissues, bone marrow,
lymph nodes and spleen.
2. Uses of RAGE Antibodies
[0119] Ligands of RAGE include S100/calgranulins, HMGB1 and MAC-1,
among others. Activation of RAGE by its ligands triggers signaling
cascades that involve MAP kinases and nuclear translocation of
nuclear factor kappa B (NF-.kappa.B) (Ramasamy et al. Glycobiology,
2005; 15(7): 16R-28R). S100/calgranulins are expressed by
polymorphonuclear leukocytes, dendritic cells (DC), macrophages
(MP), and lymphocytes (Zimmer et al., Brain Res Bull. 1995; 37(4):
417-429; Schafer and Heinzmann, Trends Biochem Sci, 1996
21(4):134-40; Donato, Int J Biochem Cell Biol. 2001; 33(7):
637-68). Many of these proteins were shown to play a role in
diseases that have a clear inflammatory component. For example,
S100A12 was shown to be released by neutrophils at sites of
inflammation, and was shown to play a role in the Kawasaki disease
(Leclerc et al. Biochem Biophys Acta, 2009; 1793: 993-1007, Boussac
et al. Electrophoresis, 2000; 21(3):665-72; Foell et al. Lancet,
2003; 21(3):665-672). Another ligand of RAGE, MAC-1 (CD11b/CD18)
promotes leucocyte extravasation into inflamed tissue in a
RAGE-dependent manner (Chavakis et al. J Exp Med, 2003;
198(10):1507-15). Along the same lines, pro-inflammatory cytokine
HMGB1 is upregulated in synovial tissues of patients with
rheumathoid arthritis (Kokkola et al. Arthritis Rheum, 2002;
46(10):2598-603). On the other hand, S100B protein is increased in
the lumbar spinal cords of both rats and mice following 14 days
after the lumbar L5 spinal nerve transaction (Tanga et al.
Neuroscience, 2006; 140(3):1003-10). Overexpression of S100B in the
transgenic mice leads to a significantly decreased tactile
threshold responses. Conversely, in the S100B knock-out mice
tactile thresholds (reduced response to non-noxious stimuli) are
significantly increased implicating S100B protein in the
pathophysiology neuropathic pain (Tanga et al. Neuroscience, 2006;
140(3):1003-10). Well-documented role of some S100 proteins in
inflammatory processes and provides a rationale for therapeutic
interventions that target. RAGE. Two general outcomes of
RAGE-mediated cellular activation are induction of a
proinflammatory phenotype and upregulation of the receptor itself
by activated NF-.kappa.B (Ramasamy et al. Glycobiology, 2005;
15(7): 16R-28R).
[0120] Evidence of involvement of RAGE and AGEs in diabetic
neuropathy has been recently established. Clinical evidence
suggests that ligands of RAGE, RAGE itself, activated NF-.kappa.B,
and IL-6 are colocalized in sural nerve biopsies of patients with
diabetes mellitus, and those with impaired glucose
tolerance-related diabetic peripheral neuropathy, but not in
biopsies of control patients (Bierhaus et al, J. Clin. Invest.
2004; 114:1741-1751; Haslbeck et al, Exp Clin Endocrinol Diabetes.
2005 May; 113(5):288-91). CML (protein-bound
N.sup..epsilon.-(carboxymethyl)lysine, an AGE adduct) could be
detected in axons, Schwann cells, and endoneurial microvessels of
human peripheral nerves. The staining intensity was shown to be
significantly increased in diabetic patients and to correlate with
myelinated fiber loss (Sugimoto et al, Diabetologia. 1997 December;
40(12):1380-7). In addition, pronounced AGE immunoreactivity was
detected in the axons and myelin sheaths of 90% of type 2 diabetic
patients, but not in control subjects. The intensity of the axonal
AGE immunoreactivity was shown to correlate with the severity of
perineurial thickening, microvascular luminal narrowing, and axonal
loss characteristic of diabetic neuropathy (Misur et al, Acta
Diabetol. 2004 December; 41(4):158-66). The use of RAGE expression
as a biomarker for human pathological pain conditions has also been
reported. Expression of RAGE, a ligand of RAGE
(N.epsilon.-carboxymethyllysine), and a signaling molecule
associated with RAGE activation (NF-.kappa.B) were reported
increased in muscle tissue from patients with Fibromyalgia
Syndrome, as compared to healthy controls (Ruster et al., Scand J
Rheumatol 2005; 34:460-463; Hein & Franke, Rheumatology
(Oxford). 2002 October; 41(10):1163-7).
[0121] RAGE expression has been investigated in tissues from normal
mice, and from mice with streptozotocin (STZ)-induced diabetic
neuropathy. Expression of RAGE is mostly absent in sensory tissues
of healthy rodents. RAGE expression in mice is increased in tissues
involved in sensory processing following STZ-induced diabetic
neuropathy (Toth et al., Current Molecular Medicine 2007, 7,
766-776). RAGE expression was reported increased in skin
(subcutaneous tissues, blood vessels, and epidermal nerve fibers),
in sural nerve glial cells, and in neurons in dorsal root ganglia,
spinal cord and hippocampus (Toth et al., Current Molecular
Medicine 2007, 7, 766-776). The functional effects of RAGE
activation in models of chronic pain are much less understood.
[0122] In human patients, diabetic neuropathy often results in a
loss of sensation, which has been attributed to loss of sensory
nerve fiber innervations in the tissue(s) where sensation is lost.
In a mouse model of STZ-induced diabetic neuropathy, RAGE knockout
mice, like their wild-type counterparts, have a measurable loss of
nerve fiber innervations in the footpads of the hindpaw (Bierhaus
et al., J. Clin. Invest. 2004, 114:1741-1751). Nociceptive
threshold in the diabetic-RAGE mice was unaltered compared to
wild-type, suggesting that RAGE contributes to the functional
sensory deficits associated with diabetic neuropathy (Bierhaus et
al., J. Clin. Invest. 2004, 114:1741-1751). In a pharmacological
study, soluble RAGE attenuated thermal hyperalgesia in diabetic
(STZ) mice (Bierhaus et al., J. Clin. Invest. 2004, 114:1741-1751).
In addition to anti-nociceptive efficacy in neuropathic pain
models, it is possible that RAGE antibodies will contribute to
attenuation of pain resulting from inflammation. For example, the
chronic constriction injury model of pain (Bennett, G J & Xie,
Y K, Pain. 1988 April; 33(1):87-107), is neuropathic in origin, and
has a strong peripheral inflammatory component resulting from
chromic gut sutures loosely tied around the nerve (Kawakami, M., et
al., Spine (Phila Pa. 1976). 1994 Aug. 15; 19(16):1780-94; Hurty, C
A et al., Vet Rec. 2002 Sep. 14; 151(11):324-8).
[0123] Given their ability to bind to human RAGE, the neutralizing
antibodies of the present application, or portions thereof, can be
used to detect human RAGE (e.g., in a biological sample, such as
serum or plasma), using a conventional immunoassay, such as an
enzyme linked immunosorbent assays (ELISA), a radioimmunoassay
(RIA) or tissue immunohistochemistry. The present application
provides a method for detecting human RAGE in a biological sample
comprising contacting a biological sample with an antibody, or
antibody portion, of the invention and detecting either the
antibody (or antibody portion) bound to human RAGE or unbound
antibody (or antibody portion), to thereby detect human RAGE in the
biological sample. The antibody is directly or indirectly labeled
with a detectable substance to facilitate detection of the bound or
unbound antibody. Suitable detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, .sup.153Sm.
[0124] The antibodies and antibody portions of the present
application particularly are capable of neutralizing human RAGE
activity both in vitro and in vivo. Accordingly, such antibodies
and antibody portions of the invention can be used to inhibit RAGE
binding to its ligands and therefore neutralize the resulting
activity.
[0125] In another embodiment, the present application provides a
method for reducing RAGE activity in a subject, advantageously from
a subject suffering from a disease or disorder in which RAGE
resulting activity is detrimental. The present application provides
methods for reducing RAGE activity in a subject suffering from such
a disease or disorder, by preventing RAGE binding to at least one
of its ligands, through the use of the monoclonal antibodies of the
present application. The antibodies of the present invention, in
particular, the humanized antibodies disclosed herein, can be
administered to a human subject for therapeutic purposes.
[0126] As used herein, the term "a disorder in which RAGE activity
is detrimental" is intended to include diseases and other disorders
in which the presence of RAGE or its resulting activity in a
subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder. Accordingly, a disorder in which RAGE activity is
detrimental is a disorder in which reduction of RAGE activity is
expected to alleviate the symptoms and/or progression of the
disorder. Non-limiting examples of disorders that can be treated
with the antibodies of the invention include those disorders
discussed in the section below pertaining to pharmaceutical
compositions of the antibodies of the invention.
[0127] In addition to pain including chronic pain, diabetic
neuropathy and fibromyalgia syndrome, it is recognized that RAGE
plays an important role in the pathology associated with a variety
of diseases involving neurological diseases selected from the group
comprising amytropic lateral sclerosis, brachial plexus injury,
brain injury, including traumatic brain injury, cerebral palsy,
Friedrich's Ataxia, Guillain Bane, leukodystrophies, multiple
sclerosis, post polio, spina bifida, spinal cord injury, spinal
muscle atrophy, spinal tumors, stroke, transverse myelitits,
dementia, senile dementia, mild cognitive impairment, Alzheimer's
disease and related dementia, Huntington's chorea, tardive
dyskinesia, hyperkinesias, manias, Morbus Parkinson, steel-Richard
syndrome, Down's syndrome, myasthenia gravis, nerve trauma,
vascular amyloidosis, cerebral hemorrhage I with amyloidosis, brain
inflammation, Friedrich's ataxia, acute confusion disorder,
amyotrophic lateral sclerosis, glaucoma, Alzheimer's disease,
diabetic nephropathy, sepsis, rheumatoid arthritis and related
inflammatory diseases. Diabetes and resulting complications like
diabetic retinopathy, nephropathy, vascular complications;
atherosclerotic complications, pulmonary fibrosis,
cancer--especially melanomas, other amyloidoses. (See for example
the following references: Amyloidosis, cancer, arthritis, Crohn's
disease, chronic and acute inflammatory diseases: Schmidt A M et
al: J Clin Invest. 2001 October; 108(7):949-55; cardiovascular
diseases, diabetes, diabetic complications: Yan S D et al: Eur J
Clin Invest. 1997 March; 27(3):179-81; Prion-associated diseases:
Sasaki N et al: Neurosci Lett. 2002 Jun. 28; 326(2):117-20;
vascularitis, nephropathies, retinopathies and neuropathies:
Thornalley P J.: Int Rev Neurobiol. 2002; 50:37-57; Alzheimer
disease: Weldon D T et al: Geriatrics. 1997 September; 52 Suppl
2:S13-6; Yan S D et al: Biochim Biophys Acta. 2000 Jul. 26;
1502(1):145-57; rheumatoid arthritis, osteoarthritis: Drinda S et
al: Rheumatol Int. 2004 Mar. 26; bowel disease: Foell D et al:Gut.
2003 June; 52(6):847-53; multiple sclerosis: Yan S S et al: Nat
Med. 2003 March; 9(3):287-93; psoriasis: Foell D et al:
Rheumatology (Oxford). 2003 November; 42(11):1383-9: lupus: Tanji N
et. al.: J Am Soc Nephrol. 2000 September; 11(9):1656-66; general
autoimmune diseases, sepsis: Liliensiek B et al: J Clin Invest.
2004 June; 113(11):1641-50; arteriosclerosis and restenosis:
Schmidt A M et al: Circ Res. 1999 Mar. 19; 84(5):489-97; U.S.
patent application Ser. No. 12/437,715 and WO 2009/136382, which
are herein incorporated by reference).
[0128] Also, DVD immunoglobulins, or dual-specific antibodies
between any one of the partners described above may be of use. Such
antibody preparations as described above may be useful for the
treatment of such diseases.
[0129] The antibodies of the present application may also be
combined with peptides allowing the trans-membrane transfer to
include targeting of intracellular target proteins. Such peptide
sequences may include, but are not limited to, tat, antennapedia,
poly-args, some anti-microbial peptides. Such peptides may allow
transfer through membranes, including cellular plasma membranes,
but also epithelia and endothelial membranes, including the
blood-brain-barrier, gut mucosa, meninges, and others.
[0130] An antibody, or antibody portion, of the present application
also can be administered with one or more additional small molecule
therapeutic agents useful in the treatment of disorders in which
RAGE activity is involved as discussed in the foregoing paragraphs.
It should be understood that the antibodies of the present
application or antigen binding portion thereof can be used alone or
in combination with an additional agent, e.g., a therapeutic agent,
said additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent that imparts a
beneficial attribute to the therapeutic composition e.g., an agent
that affects the viscosity of the composition.
[0131] The present invention encompasses a number of embodiments, a
non-exhaustive list of which are included below. Other embodiments
not listed below shall be apparent upon further reading of this
description. However, by way of example, such specific embodiments
include:
1. An isolated binding protein that dissociates from human RAGE
with a K.sub.D of 1.times.10.sup.-7 M or less and a k.sub.off rate
constant of 1.times.10.sup.-2 S.sup.-1 or less, both determined by
surface plasmon resonance. 1A. i) The binding protein of embodiment
1, that binds to human RAGE and modulates, in particular inhibits,
the ability of RAGE to bind to at least one of its ligands, as
determined in a standard in vitro assay, as for example, a HTRF
assay. ii) The binding protein of embodiment 1, which is capable of
inhibiting a RAGE-mediated biological activity. iii) A binding
protein, in particular, according to one of the preceding
embodiments, having at least one of the following biological
activities: [0132] a. inhibition of soluble A.beta.1-40
peptide-induced reduction of cerebral blood volume (CBV) in vivo in
an animal model, like C57BL/6 female mice, as for example described
in more detail in the experimental part, in particular example 7
and references cited therein; [0133] b. improvement of the cerebral
blood volume in vivo in an animal model over-expressing human APP,
like the transgenic Tg2576 mouse model; [0134] c. reduction of
amyloid plaque number and/or amyloid plaque area in vivo in an
animal model over-expressing human APP, like the transgenic Tg2576
mouse model; [0135] d. inhibition of aggregated A.beta.1-40
peptide-induced dynamin cleavage of hippocampal neurons in vitro,
like hippocampal neurons as obtained from embryonic rats; and
[0136] e. reversal of A.beta.1-42 globulomer-induced reduction of
synaptic transmission in vitro, determined in hippocampal slice
cultures. 2. The binding protein of embodiment 1, wherein the
ligand is selected from A.beta. peptides, A.beta.-globulomers,
S100b and Amphoterin. 3. The binding protein of one of the
preceding embodiments, which is a neutralizing binding protein. 4.
The binding protein of one of the preceding embodiments, which is
capable of blocking, in particular inhibiting, the binding of
A.beta. globulomer to human RAGE. 5. The binding protein of one of
the preceding embodiments wherein said wherein said A.beta.
globulomer is A.beta.1-42. 6. The binding protein of one of the
preceding embodiments, wherein said binding protein interacts with
at least one, as for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
amino acid residues of the C1- and/or C2-domain of human RAGE. 7.
The binding protein according to one of the preceding embodiments,
which is a humanized antibody. 8. The binding protein according to
one of the preceding embodiments, comprising an antigen binding
domain, said binding protein capable of binding an epitope of a
human RAGE molecule, said antigen binding domain comprising at
least one CDR comprising an amino acid sequence selected from the
CDR-H3 group of amino acid sequences selected from the group
consisting of SEQ ID NOs: 12 and 20; and modified CDR amino acid
sequences having a sequence identity of at least 50%, as for
example at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identity, to
one of said sequences; the CDR-L3 group of amino acid sequences
selected from the group consisting of SEQ ID NOs: 8, 16 and 24; and
modified CDR amino acid sequences having a sequence identity of at
least 50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90,
95% identity, to one of said sequences. 9. A binding protein
comprising an antigen binding domain, said binding protein capable
of binding an epitope of a human RAGE molecule, said antigen
binding domain comprising at least one CDR comprising an amino acid
sequence selected from the group consisting of: the CDR-H3 group of
amino acid sequences consisting of SEQ ID NOs: 12 and 20; and
modified CDR amino acid sequences having a sequence identity of at
least 50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90,
95% identity, to one of said sequences; the CDR-L3 group of amino
acid sequences selected from the group consisting of SEQ ID NOs: 8,
16 and 24; and modified CDR amino acid sequences having a sequence
identity of at least 50%, as for example at least 55, 60, 65, 70,
75, 80, 85, 90, 95% identity, to one of said sequences. 10. The
binding protein according to one of the preceding embodiments,
further comprising at least one CDR comprising an amino acid
sequence selected from the CDR-H1 group consisting of SEQ ID NOs:
10 and 18; or selected from the CDR-H2 group consisting of SEQ ID
NOs: 11 and 19; or selected from the CDR-L1 group consisting of SEQ
ID NOs: 14, and 22; or selected from the CDR-L2 group consisting of
SEQ ID NOs: 15 and 23; and modified CDR amino acid sequences having
a sequence identity of at least 50%, as for example at least 55,
60, 65, 70, 75, 80, 85, 90, 95 or 99% identity, to one of said
sequences. 11. The binding protein according to any one of the
preceding embodiments, wherein said at least one CDR comprises an
amino acid sequence selected from the group consisting of:
TABLE-US-00004 [0136] SEQ ID NO: 10 Residues 31-35 of SEQ ID NO: 9
SEQ ID NO: 11 Residues 50-66 of SEQ ID NO: 9 SEQ ID NO: 12 Residues
97-109 of SEQ ID NO: 9 SEQ ID NO: 14 Residues 24-34 of SEQ ID NO:
13 SEQ ID NO: 15 Residues 50-56 of SEQ ID NO: 13 SEQ ID NO: 16
Residues 89-97 of SEQ ID NO: 13 SEQ ID NO: 18 Residues 31-35 of SEQ
ID NO: 17 SEQ ID NO: 19 Residues 50-66 of SEQ ID NO: 17 SEQ ID NO:
20 Residues 99-109 of SEQ ID NO: 17 SEQ ID NO: 22 Residues 24-34 of
SEQ ID NO: 21 SEQ ID NO: 23 Residues 50-56 of SEQ ID NO: 21 SEQ ID
NO: 24 Residues 89-97 of SEQ ID NO: 21
12. The binding protein according to embodiment 11, comprising at
least 3 CDRs which are selected from a variable domain CDR set
comprising:
TABLE-US-00005 VH 11E6 set VH 11E6 CDR-H1 Residues 31-35 of SEQ ID
NO: 9 SEQ ID NO: 10 VH 11E6 CDR-H2 Residues 50-66 of SEQ ID NO: 9
SEQ ID NO: 11 VH 11E6 CDR-H3 Residues 99-109 of SEQ ID NO: 9 SEQ ID
NO: 12 VL 11E6 set VL 11E6 CDR-L1 Residues 24-34 of SEQ ID NO: 13
SEQ ID NO: 14 VL 11E6 CDR-L2 Residues 50-56 of SEQ ID NO: 13 SEQ ID
NO: 15 VL 11E6 CDR-L3 Residues 89-97 of SEQ ID NO: 13 SEQ ID NO: 16
VH 4E5 set VH 4E5 CDR-H1 Residues 31-35 of SEQ ID NO: 17 SEQ ID NO:
18 VH 4E5 CDR-H2 Residues 50-66 of SEQ ID NO: 17 SEQ ID NO: 19 VH
4E5 CDR-H3 Residues 99-109 of SEQ ID NO: 17 SEQ ID NO: 20 VL 4E5
set VL 4E5 CDR-L1 Residues 24-34 of SEQ ID NO: 21 SEQ ID NO: 22 VL
4E5 CDR-L2 Residues 50-56 of SEQ ID NO: 21 SEQ ID NO: 23 VL 4E5
CDR-L3 Residues 89-97 of SEQ ID NO: 21 SEQ ID NO: 24
or a variable domain set wherein at least one of said 3 CDRs is a
modified CDR amino acid sequence having a sequence identity of at
least 50%, as for example at least 55, 60, 65, 70, 75, 80, 85, 90,
95% identity, to the parent sequence. 13. The binding protein
according to embodiment 12, comprising at least two variable domain
CDR sets. 14. The binding protein according to embodiment 13,
wherein said at least two variable domain CDR sets are from at
least one of the below sets: [0137] (a) VH 4E5 set and VL 4E5 set;
or [0138] (b) VH 11E6 set and VL 11E6 set. 15. The binding protein
according to one of the preceding embodiments, further comprising a
human acceptor framework. 16. The binding protein according to
embodiment 15, wherein said human acceptor framework comprises at
least one amino acid sequence selected from the group consisting of
SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 54 and 55.
17. The binding protein of any one of the preceding embodiments
comprising at least one heavy chain variable domain selected from
the group consisting of: SEQ ID NOs: 56 and 57; and/or at least one
light chain variable domain selected from the group consisting of:
SEQ ID NOs: 58 and 59. 18. The binding protein of embodiment 17,
wherein said binding protein comprises two variable domains,
wherein the two variable domains have amino acid sequences of: SEQ
ID NOs: 56 and 58; 56 and 59; 57 and 58; or 57 and 59. 19. The
binding protein according to any one of the embodiment 16 to 18,
wherein said human acceptor framework comprises at least one
framework region amino acid substitution at a key residue, said key
residue selected from the group consisting of a residue adjacent to
a CDR; a glycosylation site residue; a rare residue; a residue
capable of interacting with a RAGE epitope; a residue capable of
interacting with a CDR; a canonical residue; a contact residue
between heavy chain variable region and light chain variable
region; a residue within a Vernier zone; an N-terminal residue
capable of para-glutamate formation; and a residue in a region that
overlaps between a Chothia-defined variable heavy chain CDR1 and a
Kabat-defined first heavy chain framework. 20. The binding protein
according to embodiment 19, wherein said key residue comprises (a)
at least one heavy chain sequence position selected from the group
consisting of: 1, 2, 68, 70, 72, 76, 85, 89 and 95; (b) at least
one light chain sequence position selected from the group
consisting of: 11, 13, 43, 49, 58, 70 and 87; or (c) combinations
of (a) and (b). 21. The binding protein of any one of the preceding
embodiments, wherein the binding protein is a consensus human
variable domain. 22. The binding protein of any one of the
embodiments 16 to 21, wherein said human acceptor framework
comprises at least one framework region amino acid substitution, as
for example 1 to 20, 1 to 15, 1 to 10, or 2, 3, 4, 5, 6, 7, 8 or 9
substitutions, wherein the amino acid sequence of the framework is
at least 65% identical to the sequence of said human acceptor
framework and comprises at least 70 amino acid residues identical
to said human acceptor framework. 23. The binding protein of any
one of the preceding embodiments, wherein said binding protein
comprises (a) at least one framework mutation at a position
selected from the group consisting of: 62, 67, 68 and 69; (b) at
least one light chain framework mutation at a position selected
from the group consisting of: 63, 64, 65 and 66; or (c)
combinations of (a) and (b). 24. The binding protein of embodiment
23, wherein said binding protein comprises two variable domains,
wherein said two variable domains have amino acid sequences from
one of the below groups of amino acid sequences: [0139] (a) SEQ ID
NOs: 62 and 63; 62 and 64; 62 and 65; or 62 and 66; [0140] (b) SEQ
ID NOs: 67 and 63; 67 and 64; 67 and 65; or 67 and 66; or [0141]
(c) SEQ ID NOs: 68 and 63; 68 and 64; 68 and 65; or 68 and 66. 25.
The binding protein of any one of the preceding embodiments,
wherein said binding protein is capable of binding a target,
selected from RAGE molecules. 26. The binding protein of any one of
the preceding embodiments capable of binding to human RAGE. 27. The
binding protein of embodiment 26, having at least one of the
following additional functional characteristics: binding to mouse
and rat RAGE. 28. The binding protein of any one of the preceding
embodiments, wherein the binding protein is capable of modulating,
in particular neutralizing, a biological function of a target,
selected from RAGE molecules. 29. The binding protein of embodiment
28, wherein said binding protein modulates, in particular inhibits,
the ability of RAGE to bind to at least one of its ligands. 30. The
binding protein of embodiment 29, wherein said binding protein
modulates, in particular inhibits, at least one of the following
interactions: binding of human RAGE to A.beta. peptides,
A.beta.-globulomers, S100b and amphoterin. 31. The binding protein
of any one of the preceding embodiments, wherein said binding
protein is capable of neutralizing a RAGE biological activity, as
for example, AP-induced cleavage of dynamin in primary neurons,
Globulomer induced synaptic deficits in hippocampal slices, or
AP-induced decrease in CBV. 32. The binding protein of embodiment
31, wherein the RAGE molecule is RAGE or a RAGE fragment, such as
sRAGE. 33. The binding protein of embodiment 32, wherein the RAGE
is selected from the group consisting of: human, rat and mouse. 34.
The binding protein of any one of the preceding embodiments,
wherein said binding protein has an on rate constant (k.sub.on) to
said target selected from the group consisting of: at least about
10.sup.2M.sup.-1s.sup.-1; at least about 10.sup.3M.sup.-1s.sup.-1;
at least about 10.sup.4M.sup.-1s.sup.-1; at least about
10.sup.5M.sup.-1s.sup.-1; at least about 10.sup.6M.sup.-1s.sup.-1,
and at least about 10.sup.7M.sup.-1s.sup.-1 as measured by surface
plasmon resonance. 35. The binding protein of any one of the
preceding embodiments, wherein said binding protein has an off rate
constant (k.sub.off) to said target selected from the group
consisting of: at most about 10.sup.-2s.sup.-1; at most about
10.sup.-3s.sup.-1; at most about 10.sup.-4s.sup.-1; at most about
10.sup.-5s.sup.-1; and at most about 10.sup.-6s.sup.-1, as measured
by surface plasmon resonance. 36. The binding protein of any one of
the preceding embodiments, wherein said binding protein has a
dissociation constant (K.sub.D) to said target selected from the
group consisting of: at most about 10.sup.-7 M; at most about
10.sup.-8 M; at most about 10.sup.-9 M; at most about 10.sup.-10 M;
at most about 10.sup.-11 M; at most about 10.sup.-12 M; and at most
10.sup.-13M. 37. An antibody construct comprising a binding protein
described in any one of the preceding embodiments, said antibody
construct further comprising a linker polypeptide or an
immunoglobulin constant domain. 38. The antibody construct
according to embodiment 37, wherein said binding protein is
selected from the group consisting of an immunoglobulin molecule, a
monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a
humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide
linked Fv, a scFv, a single domain antibody, a diabody, a
multispecific antibody, a dual specific antibody, a dual variable
domain immunoglobulin, and a bispecific antibody. 39. The antibody
construct according to any on of the embodiments 37 and 38, wherein
said binding protein comprises a heavy chain immunoglobulin
constant domain selected from the group consisting of a human IgM
constant domain, a human IgG1 constant domain, a human IgG2
constant domain, a human IgG3 constant domain, a human IgG4
constant domain, a human IgE constant domain, a human IgD constant
domain, a human IgA1 constant domain, a human IgA2 constant domain,
a human IgY constant domain and corresponding mutated domains. 40.
The antibody construct according to any on of the embodiments 37 to
39, comprising an immunoglobulin constant domain having an amino
acid sequence selected from the group consisting of: SEQ ID NOs:
25, 41, 42, and 26. 41. An antibody conjugate comprising an
antibody construct described in any one of embodiments 37 to 40,
said antibody conjugate further comprising an agent selected from
the group consisting of: an immunoadhension molecule, an imaging
agent, a therapeutic agent, and a cytotoxic agent. 42. The antibody
conjugate according to embodiment 41, wherein said agent is an
imaging agent selected from the group consisting of a radiolabel,
an enzyme, a fluorescent label, a luminescent label, a
bioluminescent label, a magnetic label, and biotin. 43. The
antibody conjugate according to embodiment 42, wherein said imaging
agent is a radiolabel selected from the group consisting of:
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm. 44.
The antibody conjugate according to embodiment 41, wherein said
agent is a therapeutic or cytotoxic agent selected from the group
consisting of: an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent. 45. The antibody construct according to any on of the
embodiments 37 to 40, wherein said binding protein possesses a
human glycosylation pattern. 46. The antibody conjugate according
to any on of the embodiments 41 to 45, wherein said binding protein
possesses a human glycosylation pattern. 47. The binding protein
according to any one of the embodiments 1 to 36, wherein said
binding protein exists as a crystal. 48. The antibody construct
according to any one of the embodiments 37 to 40, wherein said
antibody construct exists as a crystal. 49. The antibody conjugate
according to any one of the embodiments 41 to 44, wherein said
antibody construct exists as a crystal. 50. The binding protein
according to embodiment 47, wherein said crystal is a carrier-free
pharmaceutical controlled release crystal. 51. The antibody
construct according to embodiment 50, wherein said crystal is a
carrier-free pharmaceutical controlled release crystal. 52. The
antibody conjugate according to embodiment 51, wherein said crystal
is a carrier-free pharmaceutical controlled release crystal. 53.
The binding protein according to embodiment 50, wherein said
binding protein has a greater half life in vivo than the soluble
counterpart of said binding protein. 54. The antibody construct
according to embodiment 51, wherein said antibody construct has a
greater half life in vivo than the soluble counterpart of said
antibody construct. 55. The antibody conjugate according to
embodiment 52, wherein said antibody conjugate has a greater half
life in vivo than the soluble counterpart of said antibody
conjugate. 56. The binding protein according to embodiment 53,
wherein said binding protein retains biological activity. 57. The
antibody construct according to embodiment 54, wherein said
antibody construct retains biological activity. 58. The antibody
conjugate according to embodiment 55, wherein said antibody
conjugate retains biological activity. 59. An isolated nucleic acid
encoding a binding protein amino acid sequence of any one of
embodiments 1-36. 60. An isolated nucleic acid encoding an antibody
construct amino acid sequence of any one of embodiments 37-40. 61.
An isolated nucleic acid encoding an antibody conjugate amino acid
sequence of any one of embodiments 41-44. 62. A vector comprising
an isolated nucleic acid according to any one of embodiments 59 to
61. 63. The vector of embodiment 62 wherein said vector is selected
from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV,
pHybE, and pBJ. 64. A host cell comprising a vector according to
any one of embodiments 62 and 63. 65. The host cell according to
embodiment 64, wherein said host cell is a prokaryotic cell. 66.
The host cell according to embodiment 65, wherein said host cell is
E. coli. 67. The host cell according to embodiment 66, wherein said
host cell is a eukaryotic cell. 68. The host cell according to
embodiment 67, wherein said eukaryotic cell is selected from the
group consisting of protist cell, animal cell, plant cell and
fungal cell. 69. The host cell according to embodiment 68, wherein
said eukaryotic cell is an animal cell selected from the group
consisting of: a mammalian cell, an avian cell, and an insect cell.
70. The host cell according to embodiment 69, wherein said host
cell is selected from the group consisting of: HEK Cells, CHO cells
COS cells and yeast cells. 71. The host cell according to
embodiment 70, wherein said yeast cell is Saccharomyces cerevisiae.
72. The host cell according to embodiment 70, wherein said host
cell is an insect Sf9 cell. 73. A method of producing a protein
capable of binding RAGE, comprising culturing a host cell of any
one of embodiments 64 to 72 in culture medium under conditions
sufficient to produce a binding protein capable of binding RAGE.
74. A protein produced according to the method of embodiment 73.
75. A composition for the release of a binding protein said
composition comprising [0142] (a) a formulation, wherein said
formulation comprises a crystallized product protein according to
any one of embodiments 47 to 52, and an ingredient; and [0143] (b)
at least one polymeric carrier. 76. The composition according to
embodiment 75, wherein said polymeric carrier is a polymer selected
from one or more of the group consisting of: poly(acrylic acid),
poly(cyanoacrylates), poly(amino acids), poly(anhydrides),
poly(depsipeptide), poly(esters), poly(lactic acid),
poly(lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate),
poly(caprolactone), poly(dioxanone); poly(ethylene glycol),
poly((hydroxypropyl) methacrylamide, poly[(organo)phosphazene],
poly(ortho esters), poly(vinyl alcohol), poly(vinylpyrrolidone),
maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives, collagen,
fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polyeaccharides, blends and copolymers
thereof. 77. The composition according to embodiment 75, wherein
said ingredient is selected from the group consisting of albumin,
sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol. 78. A method for treating a mammal comprising
the step of administering to the mammal an effective amount of the
composition according to any one of the embodiments 75 and 76. 79.
A pharmaceutical composition comprising the product of any one of
embodiments 1 to 61, and a pharmaceutically acceptable carrier. 80.
The pharmaceutical composition of embodiment 79, wherein said
pharmaceutically acceptable carrier functions as adjuvant useful to
increase the absorption, or dispersion of said binding protein.
81. The pharmaceutical composition of embodiment 80, wherein said
adjuvant is hyaluronidase. 82. The pharmaceutical composition of
embodiment 81 further comprising at least one additional
therapeutic agent for treating a disorder in which RAGE activity is
detrimental. 83. The pharmaceutical composition of embodiment 82,
wherein said additional agent is selected from the group consisting
of: therapeutic agent, imaging agent, cytotoxic agent, angiogenesis
inhibitors; kinase inhibitors; co-stimulation molecule blockers;
adhesion molecule blockers; anti-cytokine antibody or functional
fragment thereof; methotrexate; cyclosporin; rapamycin; FK506;
detectable label or reporter; a TNF antagonist; an antirheumatic; a
muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug
(NSAID), an analgesic, an anesthetic, a sedative, a local
anesthetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a corticosteriod, an anabolic steroid, an
erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog, a cytokine, and a cytokine
antagonist. Further examples are: Dimebon, anti-A.beta.-antibodies,
beta-secretase inhibitors, tau-modulators, cognition enhancers like
e.g. 5-HT6 antagonists, cholesterinase inhibitor (e.g., tactrine,
donepezil, rivastigmine or galantamine), a partial NMDA receptor
blocker (e.g., memantine), a glycosaminoglycan mimetic (e g,
Alzhemed), an inhibitor or allosteric modulator of gamma secretase
(e.g., R-flurbiprofen), a luteinizing hormone blockade gonadotropin
releasing hormone agonist (e.g., leuprorelin), a serotinin 5-HT1A
receptor antagonist, a chelatin agent, a neuronal selective L-type
calcium channel blocker, an immunomodulator, an amyloid
fibrillogenesis inhibitor or amyloid protein deposition inhibitor
(e.g., M266), another antibody (e.g., bapineuzumab), a 5-HT1a
receptor antagonist, a PDE4 inhibitor, a histamine agonist, a
receptor protein for advanced glycation end products, a PARP
stimulator, a serotonin 6 receptor antagonist, a 5-HT4 receptor
agonist, a human steroid, a glucose uptake stimulant which enhanced
neuronal metabolism, a selective CB1 antagonist, a partial agonist
at benzodiazepine receptors, an amyloid beta production antagonist
or inhibitor, an amyloid beta deposition inhibitor, a NNR alpha-7
partial antagonist, a therapeutic targeting PDE4, a RNA translation
inhibitor, a muscarinic agonist, a nerve growth factor receptor
agonist, a NGF receptor agonist and a gene therapy modulator. 84. A
method for reducing human RAGE activity comprising contacting human
RAGE with the product of any one of embodiments 1 to 61 such that
human RAGE activity is reduced. 85. A method for decreasing hRAGE
binding to at least one ligand selected from A.beta. peptides,
globulomers, S100b and Amphoterin in a subject in need thereof,
comprising the step of administering to the subject a product of
any one of embodiments 1 to 61. 86. A method of treating a subject
for a disorder associated with RAGE activity comprising the step of
administering alone or in combination with other therapeutic agents
a product of any one of embodiments 1 to 61. 87. A method of
embodiment 86 where an RAGE therapeutic antibody is delivered
systemically with effects in the peripheral nervous system. 88. A
method for reducing RAGE activity in a subject suffering from a
disorder in which RAGE activity is detrimental, comprising
administering to the subject a product of any one of embodiments 1
to 61, alone or in combination with other therapeutic agents. 89.
The method of embodiment 88, wherein the disorder comprises
neurological diseases selected from the group comprising Amytropic
Lateral Sclerosis, Brachial Plexus Injury, Brain Injury, including
traumatic brain injury, Cerebral Palsy, Friedrich's Ataxia,
Guillain Bane, Leukodystrophies, Multiple Sclerosis, Post Polio,
Spina Bifida, Spinal Cord Injury, Spinal Muscle Atrophy, Spinal
Tumors, Stroke, Transverse Myelitits, dementia, senile dementia,
mild cognitive impairment, Alzheimer-related dementia, Huntington's
chorea, tardive dyskinesia, hyperkinesias, manias, Morbus
Parkinson, steel-Richard syndrome, Down's syndrome, myasthenia
gravis, nerve trauma, vascular amyloidosis, cerebral hemorrhage I
with amyloidosis, brain inflammation, Friedrich's ataxia, acute
confusion disorder, amyotrophic lateral sclerosis, glaucoma,
Alzheimer's disease, diabetic nephropathy, sepsis, rheumatoid
arthritis and related inflammatory diseases; Diabetes and resulting
complications like diabetic retinopathy, nephropathy, neuropathy,
vascular complications; atherosclerotic complications, pulmonary
fibrosis, Cancer especially melanomas, other amyloidoses, and
chronic pain. 90. An isolated CDR of a binding protein as defined
in any one of the embodiments 1 to 36. 91. An isolated binding
protein that specifically interacts to at least one epitope of a
Receptor of Advanced Glycation Endproducts (RAGE) protein. 92. The
isolated binding protein of embodiment 91, wherein the isolated
protein is a monoclonal antibody or antigen binding fragment
thereof. 93. The monoclonal antibody or antigen binding fragment
according to embodiment 92, which comprises a VH and a VL domain.
94. The monoclonal antibody according to embodiment 92 wherein said
monoclonal antibody diminishes the ability of RAGE to bind to its
ligands. 95. The ligands according to embodiment 94, wherein the
ligands comprise A.beta. peptides, globulomers, S100b and
amphoterin. 96. The monoclonal antibody according to embodiment 92
wherein said antibody is capable of blocking the binding of A.beta.
globulomer to RAGE. 97. The monoclonal antibody or antigen binding
fragment thereof according to embodiment 92 wherein the antibody or
antigen binding fragment comprises: [0144] a heavy chain variable
region having an amino acid sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 17; [0145]
a light chain variable region having an amino acid sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 13,
and SEQ ID NO: 21; [0146] a human Immunoglobulin gamma 1 heavy
chain constant region with amino acid sequence SEQ ID NO: 25; and
[0147] a human Immunoglobulin kappa light chain constant region
with amino acid sequence SEQ ID NO: 26. 98. The monoclonal antibody
of embodiment 93, wherein the antigen binding domain comprises at
least one complementarity determining region (CDR) comprising an
amino acid sequence with at least 90% homology with the sequence
selected from the group consisting of SEQ ID NOs: 2, 3, 4, 6, 7, 8,
10, 11, 12, 14, 15, 16, 18, 19, and 20. 99. The monoclonal antibody
according to embodiment 93 wherein said VH domain comprises a heavy
chain variable region having an amino acid sequence that has at
least 90% homology with the sequence selected from the group
consisting of SEQ ID NO 1, SEQ ID NO 9, and SEQ ID NO 17. 100. The
monoclonal antibody according to embodiment 99 wherein said VH
domain comprises at least one CDR region comprising an amino acid
sequence with at least 90% homology with the sequence selected from
the group consisting of SEQ ID NOs: 2, 3, 4, 10, 11, 12, 18, 19,
and 20. 101. The monoclonal antibody according to embodiment 100
wherein said VH domain comprises at least three CDR regions
selected from the set of SEQ ID NOs: 2, 3, and 4; SEQ ID NOs: 10,
11, and 12; or SEQ ID NOs: 18, 19, and 20. 102. The monoclonal
antibody according to embodiment 93 wherein said VL domain
comprises a light chain variable region having an amino acid
sequence that has at least 90% homology with the sequence selected
from the group consisting of SEQ ID NO: 5, SEQ ID NO: 13, and SEQ
ID NO: 21. 103. The monoclonal antibody according to embodiment 102
wherein said VL domain comprises at least one CDR region comprising
an amino acid sequence with at least 90% homology with the sequence
selected from the group consisting of SEQ ID NOs: 6, 7, 8, 14, 15,
16, 22, 23, and 24. 104. The monoclonal antibody according to
embodiment 103 wherein said VL domain comprises at least three CDR
regions selected from at least one set of SEQ ID NOs: 6, 7, and 8;
SEQ ID NOs: 14, 15 and 16; or SEQ ID NOs: 22, 23, and 24. 105. The
antibody or antigen-binding fragment of embodiment 92, wherein said
antibody or antigen-binding fragment is a mouse antibody, a
humanized antibody, a fully human, a chimeric antibody, an
antigen-binding fragment of a humanized antibody, or an
antigen-binding fragment of a chimeric antibody. 106. The antibody
or antigen-binding fragment of embodiment 92, wherein said antibody
or antigen-binding fragment is an antigen-binding fragment selected
from the group consisting of a Fab fragment, a Fab' fragment, a
F(ab').sub.2 fragment and a Fv fragment. 107. A hybridoma cell line
that produces a monoclonal antibody or antigen-binding fragment
thereof according to embodiment 96. 108. The hybridoma cell line of
embodiment 107, wherein the hybridoma is selected from the group
consisting of mouse, human, rat, sheep, pig, cattle, goat, and
horse hybridoma. 109. A hybridoma cell line that produces a
monoclonal antibody, which specifically binds to at least one
epitope of a RAGE protein. 110. The hybridoma cell line of
embodiment 107, wherein the hybridoma is selected from the group
consisting of mouse and human hybridoma. 111. The hybridoma cell
line of embodiment 107, wherein the hybridoma is selected from the
group consisting of rat, sheep, pig, cattle, goat, and horse
hybridoma. 112. A vector comprising the isolated nucleic acid
comprising the isolated nucleic acid that encodes any of the amino
acid sequences of embodiment 97, wherein said vector is selected
from the group consisting of pcDNA; pTT; pTT3; pEFBOS; pBV; pJV;
and pBJ. 113. A host cell transformed with the vector according to
embodiment 112, wherein the host cell is selected from the group
consisting of protist cell, animal cell, plant cell and fungal
cell. 114. The host cell of embodiment 113 wherein the animal cell
is a mammalian cell selected from the group comprising HEK293, CHO
and COS. 115. A method of producing the isolated binding protein
according to embodiment 91, comprising culturing a host cell in a
culture medium under conditions sufficient to produce the binding
protein, collecting the culture media, and purifying the produced
isolated binding protein. 116. A pharmaceutical composition
comprising the monoclonal antibody or antigen-binding portion
according to any of embodiments 99 or 102 and a pharmaceutically
acceptable carrier. 117. A method of treating a disease or disorder
comprising administering the monoclonal antibodies of embodiments
99 or 102 that bind to the C2-domain in RAGE. 118. The method of
embodiment 117 wherein the disorder comprises neurological diseases
selected from the group consisting of: Amytropic Lateral Sclerosis,
Brachial Plexus Injury, Brain Injury, including traumatic brain
injury, Cerebral Palsy, Friedrich's Ataxia, Guillain Bane,
Leukodystrophies, Multiple Sclerosis, Post Polio, Spina Bifida,
Spinal Cord Injury, Spinal Muscle Atrophy, Spinal Tumors, Stroke,
Transverse Myelitits, dementia, senile dementia, mild cognitive
impairment, Alzheimer-related dementia, Huntington's chorea,
tardive dyskinesia, hyperkinesias, manias, Morbus Parkinson,
steel-Richard syndrome, Down's syndrome, myasthenia gravis, nerve
trauma, vascular amyloidosis, cerebral hemorrhage I with
amyloidosis, brain inflammation, Friedrich's ataxia, acute
confusion disorder, amyotrophic lateral sclerosis, glaucoma,
Alzheimer's disease, diabetic nephropathy, sepsis, rheumatoid
arthritis and related inflammatory diseases. 119. The antibody of
embodiment 105, comprising at least one VH region comprising an
amino acid sequence selected from the group consisting of: SEQ ID
NOs: 56 and 57. 120. The antibody of embodiment 105, comprising at
least one VL region comprising an amino acid sequence selected from
the group consisting of: SEQ ID NO: 58 and 59. 121. The antibody of
embodiment 119 or 120, additionally modified by 1 to 10 mutations
in an VH or VL sequence. 122. The antibody of embodiment 121,
wherein the mutations are selected from the group consisting of:
framework back mutations and mutations of Vernier and VH/VL
interfacing residues. 123. An antibody or binding protein of one of
the preceding embodiments which inibits the binding of RAGE to an
HMGB1-CpG DNA complex; or an antibody or binding protein of one of
the preceding embodiments which does not inhibit the binding of
RAGE to an HMGB1-CpG DNA complex. 124. A method for treating pain,
comprising the step of administering to a mammal in need thereof an
effective amount of a composition comprising an isolated binding
protein, antibody construct, antibody conjugate or antibody as
defined in any one of the above embodiments 1 to 58, 91 to 94, 96
to 106 or 119 to 123. 125. A method of embodiment 124, comprising
the step of administering to a mammal in need thereof an effective
amount of a composition comprising a monoclonal antibody comprising
an antigen binding domain, said antibody capable of binding an
epitope of a human RAGE molecule, said antigen binding domain
comprising at least one CDR comprising an amino acid sequence
selected from: a) the CDR-H3 group of amino acid sequences selected
from the group consisting of SEQ ID NOs: 4, 12 and 20; modified CDR
amino acid sequences having a sequence identity of at least 50% to
one of said sequences; and/or b) the CDR-L3 group of amino acid
sequences selected from the group consisting of SEQ ID NOs: 8, 16
and 24; and modified CDR amino acid sequences having a sequence
identity of at least 50% to one of said sequences. 126. The method
according to embodiment 125, wherein the antibody construct,
antibody conjugate or antibody further comprises at least one CDR
comprising an amino acid sequence selected from the CDR-H1 group
consisting of SEQ ID NOs: 2, 10 and 18; or selected from the CDR-H2
group consisting of SEQ ID NOs: 3, 11 and 19; or selected from the
CDR-L1 group consisting of SEQ ID NOs: 6, 14, 22; or selected from
the CDR-L2 group consisting of SEQ ID NOs: 7, 15, and 23; and
modified CDR amino acid sequences having a sequence identity of at
least 50% to one of said sequences. 127. The method according to
embodiment 126, wherein the antibody construct, antibody conjugate
or antibody further comprises at least 3 CDRs which are selected
from a variable domain CDR set comprising:
TABLE-US-00006 [0147] VH 11E6 set VH 11E6 CDR-H1 Residues 31-35 of
SEQ ID NO: 9 SEQ ID NO: 10 VH 11E6 CDR-H2 Residues 50-66 of SEQ ID
NO: 9 SEQ ID NO: 11 VH 11E6 CDR-H3 Residues 99-109 of SEQ ID NO: 9
SEQ ID NO: 12 VL 11E6 set VL 11E6 CDR-L1 Residues 24-34 of SEQ ID
NO: 13 SEQ ID NO: 14 VL 11E6 CDR-L2 Residues 50-56 of SEQ ID NO: 13
SEQ ID NO: 15 VL 11E6 CDR-L3 Residues 89-97 of SEQ ID NO: 13 SEQ ID
NO: 16 VH 4E5 set VH 4E5 CDR-H1 Residues 31-35 of SEQ ID NO: 17 SEQ
ID NO: 18 VH 4E5 CDR-H2 Residues 50-66 of SEQ ID NO: 17 SEQ ID NO:
19 VH 4E5 CDR-H3 Residues 99-109 of SEQ ID NO: 17 SEQ ID NO: 20 VL
4E5 set VL 4E5 CDR-L1 Residues 24-34 of SEQ ID NO: 21 SEQ ID NO: 22
VL 4E5 CDR-L2 Residues 50-56 of SEQ ID NO: 21 SEQ ID NO: 23 VL 4E5
CDR-L3 Residues 89-97 of SEQ ID NO: 21 SEQ ID NO: 24
or a variable domain set wherein at least one of said 3 CDRs is a
modified CDR amino acid sequence having a sequence identity of at
least 50% to the parent sequence. 128. The method according to
embodiment 127, wherein said at least two variable domain CDR sets
are from at least one of the below sets: [0148] (a) VH 4E5 set and
VL 4E5 set; or [0149] (b) VH 11E6 set and VL 11E6 set. 129. The
method according to embodiment 125, wherein the antibody construct,
antibody conjugate or antibody further comprises a human acceptor
framework. 130. The method of embodiment 125, wherein the antibody
construct, antibody conjugate or antibody comprises at least one
heavy chain variable domain selected from the group consisting of:
SEQ ID NOs: 56 and 57; and/or at least one light chain variable
domain selected from the group consisting of: SEQ ID NOs: 58 and
59. 131. The method according to embodiment 130, wherein said
binding protein comprises (a) at least one heavy chain sequence
mutation selected from the group consisting of: 1, 2, 68, 70, 72,
76, 85, 89 and 95; (b) at least one light chain sequence mutation
selected from the group consisting of: 11, 13, 43, 49, 58, 70 and
87; or (c) combinations of (a) and (b). 132. The method of
embodiment 125, wherein the antibody construct, antibody conjugate
or antibody comprises (a) at least one framework mutation at a
position selected from the group consisting of: 62, 67, 68 and 69;
(b) at least one light chain framework mutation at a position
selected from the group consisting of: 63, 64, 65 and 66; or (c)
combinations of (a) and (b). 133. The method of embodiment 125,
wherein the antibody construct, antibody conjugate or antibody is
selected from the group consisting of antibodies 11E6 and 4E5. 134.
The method of embodiment 125, wherein the composition comprises a
formulation, wherein said formulation comprises the antibody of
embodiment 1 in crystallized form as an ingredient; and at least
one polymeric carrier. 135. The method of embodiment 134, wherein
the composition comprises 11E6. 136. The method of one of the
embodiments 124 to 135, wherein said pain is selected from the
group consisting of chronic and acute pain. 137. The method of
embodiment 136, wherein said pain is selected from the group
consisting of peripheral or central neuropathic pain, nociceptive
pain (like superficial somatic pain, deep somatic pain and visceral
pain); and psychogenic pain. 138. The method of embodiment 125,
wherein said pain is inflammatory pain. 139. A binding protein,
antibody construct, antibody conjugate or antibody as defined in
any one of the above embodiments 1 to 58, 91 to 94, 96 to 106 or
119 to 123 for use in treating pain. 140. The binding protein,
antibody construct, antibody conjugate or antibody for the use of
embodiment 139, wherein said pain is selected from the group
consisting of chronic or acute pain. 141. The binding protein m
antibody construct, antibody conjugate or antibody for the use of
embodiment 140, wherein said pain is selected from the group
consisting of peripheral or central neuropathic pain, nociceptive
pain (like superficial somatic pain, deep somatic pain and visceral
pain); and psychogenic pain. 142. The binding protein, antibody
construct, antibody conjugate or antibody for the use of embodiment
141, wherein said pain is inflammatory pain. 143. The use of a
binding protein, antibody construct, antibody conjugate or antibody
as defined in any one of the above embodiments 1 to 58, 91 to 94,
96 to 106 or 119 to 123 for preparing a pharmaceutical composition
for treating pain. 144. The use of embodiment 143, wherein said
pain is selected from the group consisting of chronic and acute
pain. 145. The use of embodiment 144, wherein said pain is selected
from the group consisting of peripheral or central neuropathic
pain, nociceptive pain (like superficial somatic pain, deep somatic
pain and visceral pain); and psychogenic pain. 146. The binding
protein, antibody construct, antibody conjugate or antibody of
embodiment 125, wherein said pain is inflammatory pain. 147. The
method as in embodiments 124-135, wherein said pain is neuropathic
pain. 148. The method as in embodiment 147, wherein said
neuropathic pain is peripheral or central neuropathic pain. 149.
The method as in embodiments 143, wherein said pain is neuropathic
pain. 150. The method as in embodiment 149, wherein said
neuropathic pain is peripheral or central neuropathic pain.
3. Generation of Anti-RAGE Antibodies
3.1. General
[0150] Antibodies of the application can be generated by
immunization of a suitable host (e.g., vertebrates, including
humans, mice, rats, sheep, goats, pigs, cattle, horses, reptiles,
fishes, amphibians, and in eggs of birds, reptiles and fish). To
generate the antibodies of the present application, the host is
immunized with an immunogenic RAGE polypeptide or fragment thereof
of the invention. The term "immunization" refers herein to the
process of presenting an antigen to an immune repertoire whether
that repertoire exists in a natural genetically unaltered organism,
or a transgenic organism, including those modified to display an
artificial human immune repertoire. Similarly, an "immunogenic
preparation" is a formulation of antigen that contains adjuvants or
other additives that would enhance the immunogenicity of the
antigen.
[0151] Immunization of animals may be done by any method known in
the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1990. Methods for
immunizing non-human animals such as mice, rats, sheep, goats,
pigs, cattle and horses are well known in the art. See, e.g.,
Harlow and Lane (supra) and U.S. Pat. No. 5,994,619. In a
particular embodiment, the RAGE antigen is administered with an
adjuvant to stimulate the immune response. Such adjuvants include
complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides)
or ISCOM (immunostimulating complexes). Such adjuvants may protect
the polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Particularly, if a polypeptide is
being administered, the immunization schedule will involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0152] It is contemplated that the animal host is immunized with
the antigen associated with the cell membrane of an intact or
disrupted cell and antibodies of the present application are
identified by binding to an immunogenic polypeptide of the
invention. After immunization of the animal host with the antigen,
antibodies may be obtained from the animal. The antibody-containing
serum is obtained from the animal by bleeding or sacrificing the
animal. The serum may be used as it is obtained from the animal, an
immunoglobulin fraction may be obtained from the serum, or the
antibodies may be purified from the serum. Serum or immunoglobulins
obtained in this manner are polyclonal, thus having a heterogeneous
array of properties.
3.2 Anti-RAGE Monoclonal Antibodies Using Hybridoma Technology
[0153] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0154] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In one embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, particularly, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention. Briefly, mice can be immunized with an RAGE antigen. In
a particular embodiment, the RAGE antigen is administered with an
adjuvant to stimulate the immune response. Such adjuvants include
complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides)
or ISCOM (immunostimulating complexes). Such adjuvants may protect
the polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Particularly, if a polypeptide is
being administered, the immunization schedule will involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0155] Once an immune response is detected, e.g., antibodies
specific for the antigen RAGE are detected in the mouse serum, the
mouse spleen is harvested and splenocytes isolated. The splenocytes
are then fused by well-known techniques to any suitable myeloma
cells, for example cells from cell line SP20 available from the
ATCC. Hybridomas are selected and cloned by limited dilution. The
hybridoma clones are then assayed by methods known in the art for
cells that secrete antibodies capable of binding RAGE. Ascites
fluid, which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0156] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
See, e.g., Harlow and Lane, supra. In a particular embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using RAGE or a portion thereof, or a
cell expressing RAGE. In a particular embodiment, the initial
screening is performed using an enzyme-linked immunoassay (ELISA)
or a radioimmunoassay (RIA), particularly an ELISA. An example of
ELISA screening is provided in WO 00/37504, herein incorporated by
reference.
[0157] Anti-RAGE antibody-producing hybridomas are selected, cloned
and further screened for desirable characteristics, including
robust hybridoma growth, high antibody production and desirable
antibody characteristics, as discussed further below. Hybridomas
may be cultured and expanded in vivo in syngeneic animals, in
animals that lack an immune system, e.g., nude mice, or in cell
culture in vitro. Methods of selecting, cloning and expanding
hybridomas are well known to those of ordinary skill in the
art.
[0158] In a particular embodiment, the hybridomas are mouse
hybridomas, as described above. In another particular embodiment,
the hybridomas are produced in a non-human, non-mouse species such
as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human
non-secretory myeloma is fused with a human cell expressing an
anti-RAGE antibody.
[0159] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments). F(ab').sub.2 fragments contain the variable region, the
light chain constant region and the CHI domain of the heavy
chain.
3.3 Anti-RAGE Monoclonal Antibodies Using SLAM
[0160] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052, WO 92/02551 and
Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA
93:7843-7848. In this method, single cells secreting antibodies of
interest, e.g., lymphocytes derived from any one of the immunized
animals described above, are screened using an antigen-specific
hemolytic plaque assay, wherein the antigen RAGE, a subunit of
RAGE, or a fragment thereof, is coupled to sheep red blood cells
using a linker, such as biotin, and used to identify single cells
that secrete antibodies with specificity for RAGE. Following
identification of antibody-secreting cells of interest, heavy- and
light-chain variable region cDNAs are rescued from the cells by
reverse transcriptase-PCR and these variable regions can then be
expressed, in the context of appropriate immunoglobulin constant
regions (e.g., human constant regions), in mammalian host cells,
such as COS or CHO cells. The host cells transfected with the
amplified immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example by panning the transfected cells to isolate
cells expressing antibodies to RAGE. The amplified immunoglobulin
sequences further can be manipulated in vitro, such as by in vitro
affinity maturation methods such as those described in WO 97/29131
and PCT Publication WO 00/56772.
3.4 Anti-RAGE Monoclonal Antibodies Using Transgenic Animals
[0161] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with an RAGE antigen. In a
particular embodiment, the non-human animal is a XENOMOUSE
transgenic mouse, an engineered mouse strain that comprises large
fragments of the human immunoglobulin loci and is deficient in
mouse antibody production. See, e.g., Green et al. Nature Genetics
7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615,
5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also
WO 91/10741, published Jul. 25, 1991, WO 94/02602, published Feb.
3, 1994, WO 96/34096 and WO 96/33735, both published Oct. 31, 1996,
WO 98/16654, published Apr. 23, 1998, WO 98/24893, published Jun.
11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031,
published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO
00/09560, published Feb. 24, 2000 and WO 00/037504, published Jun.
29, 2000. The XENOMOUSE transgenic mouse produces an adult-like
human repertoire of fully human antibodies, and generates
antigen-specific human Mabs. The XENOMOUSE transgenic mouse
contains approximately 80% of the human antibody repertoire through
introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and x light chain loci. See
Mendez et al., Nature Genetics 15:146-156 (1997), Green and
Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of
which are hereby incorporated by reference.
3.5 Anti-RAGE Monoclonal Antibodies Using Recombinant Antibody
Libraries
[0162] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, Ladner et
al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO
92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et
al. PCT Publication No. WO 92/20791; Markland et al. PCT
Publication No. WO 92/15679; Breitling et al. PCT Publication No.
WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;
Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.
(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol
226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.
(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology
9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137;
and Barbas et al. (1991) PNAS 88:7978-7982, US patent application
publication 2003/0186374, and PCT Publication No. WO 97/29131, the
contents of each of which are incorporated herein by reference.
[0163] The recombinant antibody library may be from a subject
immunized with RAGE, or a portion of RAGE. Alternatively, the
recombinant antibody library may be from a naive subject, i.e., one
who has not been immunized with RAGE, such as a human antibody
library from a human subject who has not been immunized with human
RAGE. Antibodies of the invention are selected by screening the
recombinant antibody library with the peptide comprising human RAGE
to thereby select those antibodies that recognize RAGE. Methods for
conducting such screening and selection are well known in the art,
such as described in the references in the preceding paragraph. To
select antibodies of the invention having particular binding
affinities for hRAGE, such as those that dissociate from human RAGE
with a particular k.sub.off rate constant, the art-known method of
surface plasmon resonance can be used to select antibodies having
the desired K.sub.off rate constant. To select antibodies of the
invention having a particular neutralizing activity for hRAGE, such
as those with a particular an IC.sub.50, standard methods known in
the art for assessing the inhibition of hRAGE activity may be
used.
[0164] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds human
RAGE. Particularly, the antibody is a neutralizing antibody. In
various embodiments, the antibody is a recombinant antibody or a
monoclonal antibody.
[0165] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles, which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0166] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication WO 92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et
al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043
(1988) (said references incorporated by reference in their
entireties). Examples of techniques, which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988).
[0167] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0168] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed Wittrup, et al. U.S.
Pat. No. 6,699,658 incorporated herein by reference.
4. Production of Particular Recombinant RAGE Antibodies of the
Invention
[0169] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is preferable, and most preferable in mammalian
host cells, because such eukaryotic cells (and in particular
mammalian cells) are more likely than prokaryotic cells to assemble
and secrete a properly folded and immunologically active
antibody.
[0170] Particular mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, in particular, secretion of the antibody into
the culture medium in which the host cells are grown. Antibodies
can be recovered from the culture medium using standard protein
purification methods.
[0171] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention and the other heavy and light chain are specific for an
antigen other than the antigens of interest by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0172] In a particular system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr- CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
4.1 Anti RAGE Antibodies
[0173] Table 4 is a list of amino acid sequences of VH and VL
regions of particular anti-hRAGE antibodies of the invention.
TABLE-US-00007 TABLE 4A List of Amino Acid Sequences of VH and VL
regions of anti-huRAGE antibodies Seq. ID Protein Sequence No.
Region 1234567890123456789012345678901 9 VH 11E6
QIQLVQSGPELKKPGETVKISCKASGYTFTN FGMNWVKQAPGKGLKWMGYINTNTGESIYSE
EFKGRFAFSLETSASTAYLQINNLKNEDTAT YFCARSRMVTAYGMDYWGQGTSVTVSS VH 11E6
Residues NFGMN CDR-H1 31-35 of SEQ ID NO: 9 VH 11E6 Residues
YINTNTGESIYSEEFKG CDR-H2 50-66 of SEQ ID NO: 9 VH 11E6 Residues
SRMVTAYGMDY CDR-H3 99-109 of SEQ ID NO: 9 13 VL
DIVMTQSQKFMSTSVGDRVSITCKASQNVGT 11E6
AVAWYQQRPGQSPKLLIFSASNRYTGVPDRF TGSGSGTDFTLTLSNMQPEDLADYFCQQYSS
YPLTFGVGTKLELKR VL 11E6 Residues KASQNVGTAVA CDR-L1 24-34 of SEQ ID
NO: 13 VL 11E6 Residues SASNRYT CDR-L2 50-56 of SEQ ID NO: 13 VL
11E6 Residues QQYSSYPLT CDR-L3 89-97 of SEQ ID NO: 13 17 VH 4E5
QVQLQQSGAELVRPGTSVKVSCKASGYAFNN YLIEWIKQRPGQGLEWIGVINPGSGGTNHNE
KFKVKATLTADKSSSTAYIQLSSLTSDDSAV YFCARSAGTARARFAYWGQGTLVTVSA VH 4E5
Residues NYLIE CDR-H1 31-35 of SEQ ID NO: 17 VH 4E5 Residues
VINPGSGGTNHNEKFKV CDR-H2 50-66 of SEQ ID NO: 17 VH 4E5 Residues
CDR-H3 99-109 of SAGTARARFAY SEQ ID NO: 17 21 VL 4E5
DIQMTQSPSSLSASLGERVSLTCRASQDIGS SLNWLQQEPDGTIKRLIYATSSLDSGVPKRF
SGSRSGSDYSLTISSLESEDFVDYYCLQYAS FPFTFGSGTKLEIKR VL 4E5 Residues
RASQDIGSSLN CDR-L1 24-34 of SEQ ID NO: 21 VL 4E5 Residues ATSSLDS
CDR-L2 50-56 of SEQ ID NO: 21 VL 4E5 Residues LQYASFPFT CDR-L3
89-97 of SEQ ID NO: 21
[0174] The foregoing isolated anti-RAGE antibody CDR sequences
establish a novel family of RAGE binding proteins, isolated in
accordance with this invention. To generate and to select CDR's of
the invention having particular RAGE binding and/or neutralizing
activity with respect to hRAGE, standard methods known in the art
for generating binding proteins of the present invention and
assessing the RAGE binding and/or neutralizing characteristics of
those binding protein may be used, including but not limited to
those specifically described herein.
[0175] The below humanized antibody 7F9 could also be useful as an
anti-RAGE antibody.
TABLE-US-00008 TABLE 4B Sequences for antibody 7F9 Seq. ID Protein
Sequence No. Region 1234567890123456789012345678901 1 VH 7F9*
EEKLEESGGGLVQLGGSMKISCVASGFTLSN YWMDWVRQSPEKGLEWIAEIRLKSNYYSTHY
AESVKGRFSISRDDSKGSVSLQMDNLTAEDT GIYFCARNAYWYFDVWGTGTTVTVSS VH 7F9
Residues NYWMD CDR-H1 31-35 of SEQ ID NO: 1 VH 7F9 Residues
EIRLKSNYYSTHYAESVKG CDR-H2 50-68 of SEQ ID NO: 1 VH 7F9 Residues
NAYWYFDV CDR-H3 101-108 of SEQ ID NO: 1 5 VL 7F9
DIVMTQSHKFMSTSVGDRVSATCKASQDVGT SVAWYQQKLGQSPKLLIYWTSTRHTGVPDRF
TGSGSGTDFTLTISNVQSEDLADYFCQQYNN YPLTFGDGTKLELKR VL 7F9 Residues
KASQDVGTSVA CDR-L1 24-34 of SEQ ID NO: 5 VL 7F9 Residues WTSTRHT
CDR-L2 50-56 of SEQ ID NO: 5 VL 7F9 Residues QQYNNYPLT CDR-L3 89-97
of SEQ ID NO: 5
4.2 Anti RAGE Chimeric Antibodies
[0176] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art. See
e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214 (1986); Gillies et al., (1989) J. Immunol. Methods
125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,
which are incorporated herein by reference in their entireties. In
addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.
81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454 which are incorporated herein by
reference in their entireties) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used.
[0177] In one embodiment, the chimeric antibodies of the invention
are produced by replacing the heavy chain constant region of the
murine monoclonal anti human RAGE antibodies described herein with
a human IgG1 constant region.
4.3 Anti RAGE CDR Grafted Antibodies
[0178] CDR-grafted antibodies of the invention comprise heavy and
light chain variable region sequences from a human antibody wherein
one or more of the CDR regions of V.sub.H and/or V.sub.L are
replaced with CDR sequences of non-human, as for example, murine
antibodies of the invention. A framework sequence from any human
antibody may serve as the template for CDR grafting. However,
straight chain replacement onto such a framework often leads to
some loss of binding affinity to the antigen. The more homologous a
human antibody is to the original murine antibody, the less likely
the possibility that combining the murine CDRs with the human
framework will introduce distortions in the CDRs that could reduce
affinity. Therefore, it is preferable that the human variable
framework that is chosen to replace the murine variable framework
apart from the CDRs have at least a 65% sequence identity with the
murine antibody variable region framework. It is more preferable
that the human and murine variable regions apart from the CDRs have
at least 70% sequence identify. It is even more preferable that the
human and murine variable regions apart from the CDRs have at least
75% sequence identity. It is most preferable that the human and
murine variable regions apart from the CDRs have at least 80%
sequence identity. Methods for producing CDR-grafted antibodies are
known in the art (Jones et al., Nature 321:522-525 (1986); U.S.
Pat. No. 5,225,539).
[0179] In a specific embodiment the invention provides CDR grafted
antibodies with V.sub.H and/or V.sub.L chains as described in Table
5.
TABLE-US-00009 TABLE 5 CDR Grafted antibodies SEQ ID Sequence NO
Protein region 123456789012345678901234567890 56 VH 11E6.1-GL
QVQLVQSGSELKKPGASVKVSCKASGYTFT (43) (VH7-4.1/
NFGMNWVRQAPGQGLEWMGYINTNTGESIY JH6 FR1) (44) (VH7-4.1/
SEEFKGRFVFSLDTSVSTAYLQICSLKAED JH6 FR2) (45) (VH7-4.1/
TAVYYCARSRMVTAYGMDYWGQGTTVTVSS JH6 FR3) (46) (VH7-4.1/ JH6 FR4) 57
VH 11E6.2-GL QVQLVQSGAEVKKPGASVKVSCKASGYTFT (47) (VH1-2/
NFGMNWVRQAPGQGLEWMGYINTNTGESIY JH6 FR1) (44) (VH1-2/
SEEFKGRVTMTRDTSISTAYMELSRLRSDD JH6 FR2) (48) (VH1-2/
TAVYYCARSRMVTAYGMDYWGQGTTVTVSS JH6 FR3) (46) (VH1-2/) JH6 FR4 58 VL
11E6.1-GL DIQMTQSPSSVSASVGDRVTITCKASQNVG (49) (1-12/L5/
TAVAWYQQKPGKAPKLLIYSASNRYTGVPS JK2 FR1) (50) (1-12/L5/
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ JK2 FR2) (51) (1-12/L5/
YSSYPLTFGQGTKLEIKR JK2 FR3) (52) (1-12/L5/ JK2 FR4) 59 VL 11E6.2-GL
EIVMTQSPATLSVSPGERATLSCKASQNVG (53) (3-15/L2/
TAVAWYQQKPGQAPRLLIYSASNRYTGIPA JK2 FR1) (54) (3-15/L2/
RFSGSGSGTEFTLTISSLQSEDFAVYYCQQ JK2 FR2) (55) (3-15/L2/)
YSSYPLTFGQGTKLEIKR JK2 FR3 (52) (3-15/L2/ JK2 FR4)
CDR sequences derived from mAb 11E6 are stated in bold letters.
Reference is also made to the specific framework sequences (FR1 to
FR4) by stating the corresponding SEQ ID NOs: (see also Tables 2
and 3).
4.4 Anti RAGE Humanized Antibodies
[0180] Humanized antibodies are antibody molecules from non-human
species antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Known human Ig sequences are disclosed, e.g., www.ncbi.nlm
nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;
www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uni-heidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH-05/kuby05.htm;
www.library.thinkques t.org/12429/Immune/Antibody.html; www.hhmi
org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.-html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html-;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/lin-ks.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;
baserv.uci.kun.nl/about.jraats/linksl.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;
www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.unizh ch/.about.honegger/AHOsem-inar/Slide01.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr
mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;
www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983), each entirely incorporated herein by reference. Such
imported sequences can be used to reduce immunogenicity or reduce,
enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic, as
known in the art.
[0181] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, particularly improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties.)
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as but not limited to those described in Jones et
al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534
(1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and
Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl.
Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);
Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO
91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939,
US94/01234, GB89/01334, GB91/01134, GB92/01755; WO 90/14443, WO
90/14424, WO 90/14430, EP 229246, EP 592,106; EP 519,596, EP
239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514,
5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352,
6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;
4,816,567, each entirely incorporated herein by reference, included
references cited therein.
5. Further Embodiments of Antibodies of the Invention
5.1 Fusion Antibodies and Immunoadhesins
[0182] The present application also describes a fusion antibody or
immunoadhesin that may be made which comprises all or a portion of
a RAGE antibody of the present application linked to another
polypeptide. In some embodiments, only the variable region of the
RAGE antibody is linked to the polypeptide. In other embodiments,
the VH domain of a RAGE antibody of this application is linked to a
first polypeptide, while the VL domain of the antibody is linked to
a second polypeptide that associates with the first polypeptide in
a manner that permits the VH and VL domains to interact with one
another to form an antibody binding site. In other embodiments, the
VH domain is separated from the VL domain by a linker that permits
the VH and VL domains to interact with one another (see below under
Single Chain Antibodies). The VH-linker-VL antibody is then linked
to a polypeptide of interest. The fusion antibody is useful to
directing a polypeptide to a cell or tissue that expresses a RAGE.
The polypeptide of interest may be a therapeutic agent, such as a
toxin, or may be a diagnostic agent, such as an enzyme; that may be
easily visualized, such as horseradish peroxidase. In addition,
fusion antibodies can be created in which two (or more)
single-chain antibodies are linked to one another. This is useful
if one wants to create a divalent or polyvalent antibody on a
single polypeptide chain, or if one wants to create a bispecific
antibody.
[0183] One embodiment provides a labelled binding protein wherein
an antibody or antibody portion of the present application is
derivatized or linked to another functional molecule (e.g., another
peptide or protein). For example, a labelled binding protein of the
present application can be derived by functionally linking an
antibody or antibody portion of the present application (by
chemical coupling, genetic fusion, noncovalent association or
otherwise) to one or more other molecular entities, such as a
nucleic acid, another antibody (e.g., a bispecific antibody or a
diabody), a detectable agent, a cytotoxic agent, a pharmaceutical
agent, and/or a protein or peptide that can mediate association of
the antibody or antibody portion with another molecule (such as a
streptavidin core region or a polyhistidine tag).
[0184] Useful detectable agents with which an antibody or antibody
portion of the present application may be derivatized include
fluorescent compounds. Exemplary fluorescent detectable agents
include fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with a nucleic acid, biotin, and
detected through indirect measurement of avidin or streptavidin
binding.
5.2 Single Chain Antibodies
[0185] The present application includes a single chain antibody
(scFv) that binds an immunogenic RAGE of the invention. To produce
the scFv, VH- and V-encoding DNA is operatively linked to DNA
encoding a flexible linker, e.g., encoding the amino acid sequence
(Gly4-Ser), such that the VH and VL sequences can be expressed as a
contiguous single-chain protein, with the VL and VH regions joined
by the flexible linker (see e.g., Bird et al. (1988) Science
242:423-42 6; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:
5879-5883; McCafferty et al., 30 Nature (1990) 34 8: 552-554). The
single chain antibody may be monovalent, if only a single VH and VL
are used, bivalent, if two VH and VL are used, or polyvalent, if
more than two VH and VL are used. Two of said scFv fragments
coupled via a linker are called "diabody" which form is also
encompassed by the invention.
5.3 Bispecific Antibodies
[0186] The present application further includes a bispecific
antibody or antigen-binding fragment thereof in which one
specificity is for an immunogenic RAGE polypeptide of the present
application. For example, a bispecific antibody can be generated
that specifically binds to an immunogenic RAGE polypeptide of the
invention through one binding domain and to a second molecule
through a second binding domain In addition, a single chain
antibody containing more than one VH and VL may be generated that
binds specifically to an immunogenic polypeptide of the invention
and to another molecule that is associated with attenuating myelin
mediated growth cone collapse and inhibition of neurite outgrowth
and sprouting. Such bispecific antibodies can be generated using
techniques that are well known for example, Fanger et al. Immunol
Methods 4: 72-81 (1994).
[0187] In some embodiments, the bispecific antibodies are prepared
using one or more of the variable regions from an antibody of the
invention. In another embodiment, the bispecific antibody is
prepared using one or more CDR regions from said antibody.
5.4 Derivatized and Labeled Antibodies
[0188] An antibody or an antigen-binding fragment of the present
application can be derivatized or linked to another molecule (e.g.,
another peptide or protein). In general, the antibody or
antigen-binding fragment is derivatized such that binding to an
immunogenic polypeptide of the invention is not affected adversely
by the derivatization or labeling.
[0189] For example, an antibody or antibody portion of the present
application can be functionally linked (by chemical coupling,
genetic fusion, noncovalent association or otherwise) to one or
more other molecular entities, such as another antibody (e.g., a
bispecific antibody or a diabody), a detection reagent, a cytotoxic
agent, a pharmaceutical agent, and/or a protein or peptide that can
mediate association of the antibody or antigen-binding fragment
with another molecule (such as a streptavidin core region or a
polyhistidine tag). Still further, an antibody or antigen-binding
portion thereof may be part of a larger immunoadhesion molecule,
formed by covalent or non-covalent association of the antibody or
antibody portion with one or more other or different proteins or
peptides. Examples of such immunoadhesion molecules include use of
the streptavidin core region to make a tetrameric scFv molecule
(Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101)
and use of a cysteine residue, a marker peptide and a C-terminal
polyhistidine tag to make bivalent and biotinylated scFv molecules
(Kipriyanov et al. (1994) Molecular Immunology 31:1047-1058).
Antibody portions, such as Fab and F(ab').sub.2 fragments, can be
prepared from whole antibodies using conventional techniques, such
as papain or pepsin digestion, respectively, of whole antibodies.
Moreover, antibodies, antibody portions and immunoadhesion
molecules can be obtained using standard recombinant DNA
techniques.
[0190] A derivatized antibody may be produced by crosslinking two
or more antibodies (of the same type or of different types, e.g.,
to create bispecific antibodies). Suitable crosslinkers include
those that are heterobifunctional, having two distinctly reactive
groups separated by an appropriate spacer (e.g.
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional
(e.g., disuccinimidyl suberate). Such linkers are available from
Pierce Chemical Company, Rockford, Ill.
[0191] A derivatized antibody may also be a labeled antibody. For
instance, detection agents with which an antibody or antibody
portion of the invention may be derivatized are fluorescent
compounds, including fluorescein, fluorescein isothiocyanate,
rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride,
phycoerythrin, lanthanide phosphors and the like. An antibody also
may be labeled with enzymes that are useful for detection, such as
horseradish peroxidase, galactosidase, luciferase, alkaline
phosphatase, glucoseoxidase and the like. In embodiments that are
labeled with a detectable enzyme, the antibody is detected by
adding additional reagents that the enzyme uses to produce a
detectable reaction product. For example, horseradish peroxidase
with hydrogen peroxide and diaminobenzidine. An antibody also may
be labeled with biotin, and detected through indirect measurement
of avidin or streptavidin binding. An antibody may also be labeled
with a predetermined polypeptide epitope recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope: tags). An
RAGE antibody or an antigen fragment thereof also may be labeled
with a radio-labeled amino acid. The radiolabel may be used for
both diagnostic and therapeutic purposes. The radio-labeled RAGE
antibody may be used diagnostically, for example, for determining
RAGE receptor levels in a subject. Further, the radio-labeled RAGE
antibody may be used therapeutically for treating spinal cord
injury.
[0192] Examples of labels for polypeptides include, but are not
limited to, the following radioisotopes or radionucleotides
.sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I,
.sup.131I, .sup.177Lu, .sup.166Ho, .sup.153Sm. A RAGE antibody or
an antigen fragment thereof may also be derivatized with a chemical
group such as polyethylene glycol (PEG), a methyl or ethyl group,
or a carbohydrate group. These groups may be useful to improve the
biological characteristics of the antibody, e.g., to increase serum
half-life or to increase tissue binding. Also, a label for
polypeptides can include a nucleic acid, for example DNA for
detection by PCR, or enhancing gene expression, or siRNA to
suppress gene expression in RAGE-bearing cells or tissues.
[0193] The class and subclass of RAGE antibodies may be determined
by any method known in the art. In general, the class and subclass
of an antibody may be determined using antibodies that are specific
for a particular class and subclass of antibody. Such antibodies
are available commercially. The class and subclass can be
determined by ELISA, Western Blot as well as other techniques.
Alternatively, the class and subclass may be determined by
sequencing all or a portion of the constant domains of the heavy
and/or light chains of the antibodies, comparing their amino acid
sequences to the known amino acid sequences of various classes and
subclasses of immunoglobulins, and determining the class and
subclass of the antibodies.
5.5 Dual Variable Domain Immunoglobulins
[0194] Dual variable domain (DVD) binding proteins or
immunoglobulins as used herein, are binding proteins that comprise
two or more antigen binding sites and are tetravalent or
multivalent binding proteins, as for example divalent and
tetravalent. The term "multivalent binding protein" is used in this
specification to denote a binding protein comprising two or more
antigen binding sites. The multivalent binding protein is
particularly engineered to have the two or more antigen binding
sites, and is generally not a naturally occurring antibody. The
term "multispecific binding protein" refers to a binding protein
capable of binding two or more related or unrelated targets. Such
DVDs may be monospecific, i.e capable of binding one antigen or
multispecific, i.e. capable of binding two or more antigens. DVD
binding proteins comprising two heavy chain DVD polypeptides and
two light chain DVD polypeptides are referred to a DVD Ig. Each
half of a DVD Ig comprises a heavy chain DVD polypeptide, and a
light chain DVD polypeptide, and two antigen binding sites. Each
binding site comprises a heavy chain variable domain and a light
chain variable domain with a total of 6 CDRs involved in antigen
binding per antigen binding site. DVD binding proteins and methods
of making DVD binding proteins are disclosed in U.S. patent
application Ser. No. 11/507,050 and incorporated herein by
reference. It is intended that the present invention comprises a
DVD binding protein comprising binding proteins capable of binding
RAGE. Particularly the DVD binding protein is capable of binding
RAGE and a second target. The second target is selected from the
group consisting of anti inflammatory MAB activities (IL-1, IL-6,
IL-8, IL-11, IL-12, IL-17, IL-18, IL-23,TNF alpha/beta, IFN-beta,
gamma, LIF, OSM, CNTF, PF-4, Platelet basic protein (PBP), NAP-2,
beta-TG, MIP-1, MCP2/3, RANTES, lymphotactin), of
transport-mediating proteins (insulin receptor, transferrin
receptor, thrombin receptor, leptin receptor, LDL receptor), of
other neuroregenerative MABs (NgR, Lingo, p75, CSPG (e.g. NG-2,
neurocan, brevican, versican, aggrecan) hyaluronic acid, mAG,
tenascin, NI-35, NI-250, IMP, perlecan, neurocan, phosphacan,
nogo-A, OMGP, Sema4D, Sema 3A, ephrin B3, ephrin A2, ephrin A5,
MAG, EphA4, plexin B1, TROY, wnts, ryk rec., BMP-2, BMP-4, BMP-7),
of neuroprotective MAB activities (EGF, EGFR, Sema 3), of
anti-amyloid beta MABs (e.g. m266, 3D6 (bapineuzumab),
anti-globulomer MABs 7C6), of CNS located receptors and
transporters (serotonin receptors, dopamine receptors, DAT, Asc-1,
GlyT1).
5.6 Dual-Specific Antibodies
[0195] The present application also describes "dual-specific
antibody" technology. Dual-specific antibodies may serve as
agonists, antagonists, or both in different combinations.
Dual-specific antibodies are antibodies in which the VH chain binds
to a first antigen and the VL chain binds to another antigen as
exemplified in WO 2008/082651.
5.7 Crystallized Antibodies
[0196] Another embodiment of the present application provides a
crystallized binding protein. The term "crystallized" as used
herein, refer to an antibody, or antigen binding portion thereof,
that exists in the form of a crystal. Crystals are one form of the
solid state of matter, which is distinct from other forms such as
the amorphous solid state or the liquid crystalline state. Crystals
are composed of regular, repeating, three-dimensional arrays of
atoms, ions, molecules (e.g., proteins such as antibodies), or
molecular assemblies (e.g., antigen/antibody complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships that are well understood in the field.
The fundamental unit, or building block, that is repeated in a
crystal is called the asymmetric unit. Repetition of the asymmetric
unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the crystal.
Repetition of the unit cell by regular translations in all three
dimensions provides the crystal. See Giege, R. and Ducruix, A.
Barrett, Crystallization of Nucleic Acids and Proteins, a Practical
Approach, 2.sup.nd ed., pp. 20 1-16, Oxford University Press, New
York, N.Y., (1999).
[0197] Particularly the present application describes crystals of
whole RAGE antibodies and fragments thereof as disclosed herein,
and formulations and compositions comprising such crystals. In one
embodiment the crystallized binding protein has a greater half-life
in vivo than the soluble counterpart of the binding protein. In
another embodiment the binding protein retains biological activity
after crystallization.
[0198] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in WO
02/072636, incorporated herein by reference.
5.8 Glycosylated Antibodies
[0199] Another embodiment of the invention provides a glycosylated
binding protein wherein the antibody or antigen-binding portion
thereof comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Antibodies are glycoproteins with one or more
carbohydrate residues in the Fc domain, as well as the variable
domain. Carbohydrate residues in the Fc domain have important
effect on the effector function of the Fc domain, with minimal
effect on antigen binding or half-life of the antibody (R.
Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast,
glycosylation of the variable domain may have an effect on the
antigen binding activity of the antibody. Glycosylation in the
variable domain may have a negative effect on antibody binding
affinity, likely due to steric hindrance (Co, M. S., et al., Mol.
Immunol. (1993) 30:1361-1367), or result in increased affinity for
the antigen (Wallick, S. C., et al., Exp. Med. (1988)
168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717
2723).
[0200] One aspect of the present invention is directed to
generating glycosylation site mutants in which the O- or N-linked
glycosylation site of the binding protein has been mutated. One
skilled in the art can generate such mutants using standard
well-known technologies. Glycosylation site mutants that retain the
biological activity but have increased or decreased binding
activity are another object of the present invention.
[0201] In still another embodiment, the glycosylation of the
antibody or antigen-binding portion of the invention is modified.
For example, an aglycoslated antibody can be made (i.e., the
antibody lacks glycosylation). Glycosylation can be altered to, for
example, increase the affinity of the antibody for antigen. Such
carbohydrate modifications can be accomplished by, for example,
altering one or more sites of glycosylation within the antibody
sequence. For example, one or more amino acid substitutions can be
made that result in elimination of one or more variable region
glycosylation sites to thereby eliminate glycosylation at that
site. Such aglycosylation may increase the affinity of the antibody
for antigen. Such an approach is described in further detail in PCT
Publication WO 2003/016466A2, and U.S. Pat. Nos. 5,714,350 and
6,350,861, each of which is incorporated herein by reference in its
entirety.
[0202] Additionally or alternatively, a modified antibody of the
invention can be made that has an altered type of glycosylation,
such as a hypofucosylated antibody having reduced amounts of
fucosyl residues or an antibody having increased bisecting GlcNAc
structures. Such altered glycosylation patterns have been
demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example,
expressing the antibody in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibodies of the invention to thereby produce
an antibody with altered glycosylation. See, for example, Shields,
R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.
(1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP
1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of
which is incorporated herein by reference in its entirety.
[0203] Protein glycosylation depends on the amino acid sequence of
the protein of interest, as well as the host cell in which the
protein is expressed. Different organisms may produce different
glycosylation enzymes (eg., glycosyltransferases and glycosidases),
and have different substrates (nucleotide sugars) available. Due to
such factors, protein glycosylation pattern, and composition of
glycosyl residues, may differ depending on the host system in which
the particular protein is expressed. Glycosyl residues useful in
the invention may include, but are not limited to, glucose,
galactose, mannose, fucose, n-acetylglucosamine and sialic acid.
Particularly the glycosylated binding protein comprises glycosyl
residues such that the glycosylation pattern is human.
[0204] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0205] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (U.S. patent applications
20040018590 and 20020137134 and PCT publication WO 2005/100584
A2).
[0206] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. Particularly, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
5.9 Anti-Idiotypic Antibodies
[0207] In addition to the binding proteins, the present invention
is also directed to an anti-idiotypic (anti-Id) antibody specific
for such binding proteins of the invention. An anti-Id antibody is
an antibody, which recognizes unique determinants generally
associated with the antigen-binding region of another antibody. The
anti-Id can be prepared by immunizing an animal with the binding
protein or a CDR containing region thereof. The immunized animal
will recognize, and respond to the idiotypic determinants of the
immunizing antibody and produce an anti-Id antibody. The anti-Id
antibody may also be used as an "immunogen" to induce an immune
response in yet another animal, producing a so-called anti-anti-Id
antibody.
6. Pharmaceutical Compositions
[0208] The invention also provides pharmaceutical compositions
comprising an antibody, or antigen-binding portion thereof, of the
invention and a pharmaceutically acceptable carrier. The
pharmaceutical compositions comprising antibodies of the invention
are for use in, but not limited to, diagnosing, detecting, or
monitoring a disorder, in preventing, treating, managing, or
ameliorating of a disorder or one or more symptoms thereof, and/or
in research. In a specific embodiment, a composition comprises one
or more antibodies of the invention. In another embodiment, the
pharmaceutical composition comprises one or more antibodies of the
invention and one or more prophylactic or therapeutic agents other
than antibodies of the invention for treating a disorder in which
RAGE activity is detrimental. Particularly, the prophylactic or
therapeutic agents known to be useful for or having been or
currently being used in the prevention, treatment, management, or
amelioration of a disorder or one or more symptoms thereof. In
accordance with these embodiments, the composition may further
comprise of a carrier, diluent or excipient.
[0209] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0210] Various delivery systems are known and can be used to
administer one or more antibodies of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods
of administering a prophylactic or therapeutic agent of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidurala administration,
intratumoral administration, and mucosal adminsitration (e.g.,
intranasal and oral routes). In addition, pulmonary administration
can be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913,
5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO
97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which
is incorporated herein by reference their entireties. In one
embodiment, an antibody of the invention, combination therapy, or a
composition of the invention is administered using Alkermes
AIR.RTM. pulmonary drug delivery technology (Alkermes, Inc.,
Cambridge, Mass.). In a specific embodiment, prophylactic or
therapeutic agents of the invention are administered
intramuscularly, intravenously, intratumorally, orally,
intranasally, pulmonary, or subcutaneously. The prophylactic or
therapeutic agents may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0211] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion, by
injection, or by means of an implant, said implant being of a
porous or non-porous material, including membranes and matrices,
such as sialastic membranes, polymers, fibrous matrices (e.g.,
Tissel.RTM.), or collagen matrices. In one embodiment, an effective
amount of one or more antibodies of the invention antagonists is
administered locally to the affected area to a subject to prevent,
treat, manage, and/or ameliorate a disorder or a symptom thereof.
In another embodiment, an effective amount of one or more
antibodies of the invention is administered locally to the affected
area in combination with an effective amount of one or more
therapies (e.g., one or more prophylactic or therapeutic agents)
other than an antibody of the invention of a subject to prevent,
treat, manage, and/or ameliorate a disorder or one or more symptoms
thereof.
[0212] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release (see Langer, supra; Sefton, 1987, CRC Crit.
Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507;
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used to achieve controlled
or sustained release of the therapies of the invention (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253. Examples of polymers
used in sustained release formulations include, but are not limited
to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a
preferred embodiment, the polymer used in a sustained release
formulation is inert, free of leachable impurities, stable on
storage, sterile, and biodegradable. In yet another embodiment, a
controlled or sustained release system can be placed in proximity
of the prophylactic or therapeutic target, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984)).
[0213] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO
91/05548, PCT publication WO 96/20698, Ning et al., 1996,
"Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy & Oncology
39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Intl. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in their entireties.
[0214] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.
USA 88:1864-1868). Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression by homologous recombination.
[0215] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0216] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack
Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage
forms, viscous to semi-solid or solid forms comprising a carrier or
one or more excipients compatible with topical application and
having a dynamic viscosity particularly greater than water are
typically employed. Suitable formulations include, without
limitation, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, and the like, which are, if desired,
sterilized or mixed with auxiliary agents (e.g., preservatives,
stabilizers, wetting agents, buffers, or salts) for influencing
various properties, such as, for example, osmotic pressure. Other
suitable topical dosage forms include sprayable aerosol
preparations wherein the active ingredient, particularly in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon) or in a squeeze bottle. Moisturizers or humectants
can also be added to pharmaceutical compositions and dosage forms
if desired. Examples of such additional ingredients are well known
in the art.
[0217] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0218] If the method of the invention comprises oral
administration, compositions can be formulated orally in the form
of tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0219] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968, 5,985, 320, 5,985,309, 5,934,272, 5,874,064,
5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,
each of which is incorporated herein by reference their entireties.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0220] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use. The methods of the invention may additionally
comprise of administration of compositions formulated as depot
preparations. Such long acting formulations may be administered by
implantation (e.g., subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the compositions may be
formulated with suitable polymeric or hydrophobic materials (e.g.,
as an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0221] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0222] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0223] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. Particularly, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more particularly at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized
prophylactic or therapeutic agents or pharmaceutical compositions
of the invention should be stored at between 2.degree. C. and
8.degree. C. in its original container and the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
should be administered within 1 week, particularly within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied in liquid form in a
hermetically sealed container indicating the quantity and
concentration of the agent. Particularly, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, more particularly at least 0.5
mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at
least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25
mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml.
The liquid form should be stored at between 2.degree. C. and
8.degree. C. in its original container.
[0224] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Particularly, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ surfactants.
The pharmaceutical composition comprising the antibodies and
antibody-portions of the invention prepared as an injectable
solution for parenteral administration, can further comprise an
agent useful as an adjuvant, such as those used to increase the
absorption, or dispersion of a therapeutic protein (e.g.,
antibody). A particularly useful adjuvant is hyaluronidase, such as
Hylenex.RTM. (recombinant human hyaluronidase). Addition of
hyaluronidase in the injectable solution improves human
bioavailability following parenteral administration, particularly
subcutaneous administration. It also allows for greater injection
site volumes (i.e. greater than 1 ml) with less pain and
discomfort, and minimum incidence of injection site reactions. (see
WO 2004/078140, US2006104968 incorporated herein by reference).
[0225] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The particular form depends
on the intended mode of administration and therapeutic application.
Typical particular compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. A particular
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a particular
embodiment, the antibody is administered by intravenous infusion or
injection. In another particular embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0226] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, particular methods of preparation are vacuum
drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including, in the composition, an agent that delays absorption, for
example, monostearate salts and gelatin.
[0227] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, a particular
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0228] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0229] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which RAGE activity is detrimental. For
example, an anti-RAGE antibody or antibody portion of the invention
may be coformulated and/or coadministered with one or more
additional antibodies that bind other targets (e.g., antibodies
that bind cytokines or that bind cell surface molecules).
Furthermore, one or more antibodies of the invention may be used in
combination with two or more of the foregoing therapeutic agents.
Such combination therapies may advantageously utilize lower dosages
of the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies.
[0230] In certain embodiments, an antibody to RAGE or fragment
thereof is linked to a half-life extending vehicle known in the
art. Such vehicles include, but are not limited to, the Fc domain,
polyethylene glycol, and dextran. Such vehicles are described,
e.g., in U.S. application Ser. No. 09/428,082 and published PCT
Application No. WO 99/25044, which are hereby incorporated by
reference for any purpose.
[0231] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding an antibody of the invention or
another prophylactic or therapeutic agent of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded antibody or
prophylactic or therapeutic agent of the invention that mediates a
prophylactic or therapeutic effect.
[0232] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., 1993, Clinical
Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,
1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11(5):155-215. Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley
&Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY (1990). Detailed description
of various methods of gene therapy are disclosed in US20050042664
A1 which is incorporated herein by reference.
[0233] RAGE plays a critical role in the pathology associated with
a variety of diseases as defined herein above. Infusion of amyloid
A.beta.-peptides into animals leads to responses like inflammatory
responses in arteriolose, decrease in cerebral blood flow. These
effects could be prevented by antibodies against RAGE (Rhodin, J.
et al. World Congress for Microcirculation, submitted Papers, 7th,
Sydney, Australia, Aug. 19-22, 2001, 543-547; Deane et al. Nature
Med. 2003). RAGE is upregulated in the microvasculature of AD
patients and in transgenice mice where the human APP gene has been
overexpressed (Deane et al. Nature Med. 2003). Using
double-transgenic mice where the human APP gene is expressed and
RAGE is overexpressed it was shown that overexpression of the
normal RAGE gene leads to impairment in learning, increase in
plaques whereas overexpression of a dominant-negative signalling
defective RAGE variant leads to improvement in learning and lower
plaque levels (Arancio et al. 2004 EMBO J. 2004). Experimentation
in animal models of both Type 1 and 2 diabetes reveals that
antagonism of the ligand-RAGE axis suppresses the development and
progression of vascular and inflammatory cell perturbation in the
diabetic milieu, e.g. RAGE knock-out mice and Anti-RAGE antibodies
have been used to show an improvement in animal models for e.g.
diabetic nephropathy (Ravichandran R. et al. Canadian Journal of
Diabetes 2006; 30(4):422, Myint Khin et al. Diabetes (2006), 55(9),
2510; De-Vriese et al. Journal of the American Society of
Nephrology 2003, 14/8, 2109, Jensen et al), and diabetic neuropathy
(Bierhaus, A. J Clin Invest. 2004 December; 114(12):1741-51). Renal
effects of a neutralising RAGE-antibody in long-term
streptozotocin-diabetic mice. The Journal of Endocrinology, 2006,
188, 493). Positive long-term renal effects of a neutralizing RAGE
antibody in obese type 2 diabetic mice were shown by Flyvbj erg et
al (Diabetes, 2004, 53, 1, p. 166-72). RAGE knock-out mice were
used to show an involvement of RAGE in sepsis (Birgit Liliensiek et
al. J Clin Invest. 2004 June 1; 113(11): 1641-1650; Receptor for
advanced glycation end products (RAGE) regulates sepsis but not the
adaptive immune response). Blocking F(ab).sub.2 fragments derived
from anti-RAGE IgG reduces the inflammatory response in MOG or
MBP-induced EAE (Yan, S. S., et al. 2003. Nat. Med. 9:287-293.)
Involvement of RAGE in cancer was shown (Abe-R et al. Journal of
Investigative Dermatology, 2004, 122/2 (461-467). In tumor-bearing
mice, survival rates were prolonged, and spontaneous pulmonary
metastases were inhibited by treatment using anti-RAGE neutralizing
antibodies.
[0234] The antibodies, and antibody portions of the invention can
be used to treat humans suffering from such a diseases.
[0235] It should be understood that the antibodies of the invention
or antigen binding portion thereof can be used alone or in
combination with an additional agent, e.g., a therapeutic agent,
said additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent that imparts a
beneficial attribute to the therapeutic composition e.g., an agent,
which affects the viscosity of the composition.
[0236] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed
composition can perform its intended function.
[0237] Non-limiting examples of therapeutic agents for multiple
sclerosis with which an antibody, or antibody portion, of the
invention can be combined include the following: corticosteroids;
prednisolone; methylprednisolone; azathioprine; cyclophosphamide;
cyclosporine; methotrexate; 4-aminopyridine; tizanidine;
interferon-.beta.1a (AVONEX; Biogen); interferon-.beta.1b
(BETASERON; Chiron/Berlex); interferon .alpha.-n3) (Interferon
Sciences/Fujimoto), interferon-.alpha. (Alfa Wassermann/J&J),
interferon .beta.1A-IF (Serono/Inhale Therapeutics), Peginterferon
.alpha.2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE;
Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;
intravenous immunoglobulin; clabribine; antibodies to or
antagonists of other human cytokines or growth factors and their
receptors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,
IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF.
Antibodies of the invention, or antigen binding portions thereof,
can be combined with antibodies to cell surface molecules such as
CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. The antibodies of the invention,
or antigen binding portions thereof, may also be combined with
agents, such as methotrexate, cyclosporine, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNFalpha or IL-1 (e.g. IRAK, NIK,
IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting enzyme
inhibitors, TACE inhibitors, T-cell signaling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R)
and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-13 and
TGFbeta).
[0238] Particular examples of therapeutic agents for multiple
sclerosis in which the antibody or antigen binding portion thereof
can be combined to include interferon-beta, for example,
IFN.beta.1a and IFN.beta.1b; copaxone, corticosteroids, caspase
inhibitors, for example inhibitors of caspase-1, IL-1 inhibitors,
TNF inhibitors, and antibodies to CD40 ligand and CD80.
[0239] Particularly, the binding proteins and antibodies of the
present invention may be utilized to treat an amyloidosis, for
example, Alzheimer's disease and Down's syndrome. It should be
understood that the binding proteins and antibodies of the
invention can be used alone or in combination with at least one
additional agents suitable for treating one of the above diseases.
Said at least one additional agent may be selected by the skilled
artisan for its intended purpose. For example, the additional agent
can be a therapeutic agent such as a cholesterinase inhibitor
(e.g., tactrine, donepezil, rivastigmine or galantamine), a partial
NMDA receptor blocker (e.g., memantine), a glycosaminoglycan
mimetic (e g, Alzhemed), an inhibitor or allosteric modulator of
gamma secretase (e.g., R-flurbiprofen), a luteinizing hormone
blockade gonadotropin releasing hormone agonist (e.g.,
leuprorelin), a serotinin 5-HT1A receptor antagonist, a chelatin
agent, a neuronal selective L-type calcium channel blocker, an
immunomodulator, an amyloid fibrillogenesis inhibitor or amyloid
protein deposition inhibitor (e.g., M266), another antibody (e.g.,
bapineuzumab), a 5-HTla receptor antagonist, a PDE4 inhibitor, a
histamine agonist, a receptor protein for advanced glycation end
products, a PARP stimulator, a serotonin 6 receptor antagonist, a
5-HT4 receptor agonist, a human steroid, a glucose uptake stimulant
which enhanced neuronal metabolism, a selective CB1 antagonist, a
partial agonist at benzodiazepine receptors, an amyloid beta
production antagonist or inhibitor, an amyloid beta deposition
inhibitor, a NNR alpha-7 partial antagonist, a therapeutic
targeting PDE4, a RNA translation inhibitor, a muscarinic agonist,
a nerve growth factor receptor agonist, a NGF receptor agonist and
a gene therapy modulator (i.e., those agents currently recognized,
or in the future being recognized, as useful to treat the disease
or condition being treated by the antibody or binding protein of
the present invention). The additional agent also can be an agent
that imparts a beneficial attribute to the therapeutic composition
e.g., an agent that affects the viscosity of the composition.
[0240] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as alemtuzumab,
dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden
hydrochloride, fampridine, glatiramer acetate, natalizumab,
sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokine
receptor antagonists, BBR-2778, calagualine, CPI-1189, LEM
(liposome encapsulated mitoxantrone), THC.CBD (cannabinoid agonist)
MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor
antibody, neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1,
talampanel, teriflunomide, TGF-beta2, tiplimotide, VLA-4
antagonists (for example, TR-14035, VLA4 Ultrahaler,
Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4
agonists.
[0241] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may be determined by a person
skilled in the art and may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the antibody or antibody portion to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody,
or antibody portion, are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0242] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0243] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.1-20 mg/kg, more particularly 1-10
mg/kg. It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0244] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the
invention or the embodiments disclosed herein. Having now described
the present invention in detail, the same will be more clearly
understood by reference to the following examples, which are
included for purposes of illustration only and are not intended to
be limiting of the invention.
Example 1
Anti-huRAGE Antibodies
[0245] 1.1 Production of Hybridomas and Antibodies.
[0246] Balb/c and A/J mice, 4-6 weeks of age, were immunized and
boosted subcutaneously with human RAGE. Animals were injected every
three weeks, beginning with a primary injection of 30 .mu.g in
complete Freund's adjuvant and injection boosts of 30 .mu.g in
Incomplete Freund's Adjuvant. Mice selected for fusion were
injected intravenously with 10 .mu.g hRAGE in saline, four days
prior to fusion. Spleens from immunized animals were removed and
single cell suspensions were prepared. SP2/0 myeloma cells were
harvested from culture and washed. Spleen cells and tumor cells
were mixed at a ratio of 5:1 and fused using 50% PEG 3000 using
standard techniques (Kohler and Milstein, 1975). Fused cells were
seeded in 96 well plates in selective media, at a density of
2.5.times.10.sup.5 spleen cells per well. Fusions were incubated at
37.degree. C. for 7-10 days. When macroscopic colonies were
observed, supernatants were removed and tested in the hRAGE
ELISA.
[0247] Hybridomas that were producing mAbs with desired
characteristics were subcloned by the limiting dilution method.
Supernatant containing subclones were assayed for binding to hRAGE
by ELISA. Heavy and light chain subclasses of the mAbs were
determined using the Zymed EIA Isotyping kit.
[0248] 1.2. Determination of the Amino Acid Sequence of the
Variable Region for Each Murine Anti-Human RAGE mAb.
[0249] For each amino acid sequence determination, approximately
10.times.10.sup.6 hybridoma cells were isolated by centrifugation
and processed to isolate total RNA with Trizol (Gibco
BRL/Invitrogen, CA.) following manufacturer's instructions. Total
RNA was subjected to first strand DNA synthesis using the
SuperScript First-Strand Synthesis System (Invitrogen, CA) per the
manufacturers instructions. Oligo(dT) was used to prime
first-strand synthesis to select for poly(A).sup.+ RNA. The
first-strand cDNA product was then amplified by PCR with primers
designed for amplification of murine immunoglobulin variable
regions (Ig-Primer Sets, Novagen, Wis.). PCR products were resolved
on an agarose gel, excised, purified, and then subcloned with the
TOPO Cloning kit into pCR2.1-TOPO vector (Invitrogen, CA) and
transformed into TOP10 chemically competent E. coli (Invitrogen,
CA). Colony PCR was performed on the transformants to identify
clones containing insert. Plasmid DNA was isolated from clones
containing insert using a QIAprep Miniprep kit (Qiagen, Valencia,
Calif.). Inserts in the plasmids were sequenced on both strands to
determine the variable heavy or variable light chain DNA sequences
using M13 forward and M13 reverse primers (Fermentas Life Sciences,
Hanover Md.). Variable heavy and variable light chain sequences of
the 3 monoclonal antibodies 7F9, 11E6 and 4E5 and their three
variable heavy chain CDRs and three variable light chain CDRs are
listed in Table 4, above.
[0250] 1.3. Construction and Expression of Recombinant Anti Human
RAGE Antibodies
[0251] The DNA encoding the heavy chain constant region of murine
anti-human RAGE monoclonal antibodies 7F9, 11E6 and 4E5 was
replaced by a cDNA fragment encoding the human IgG1 constant region
by homologous recombination in bacteria. The light chain constant
region of each of these antibodies was replaced by a human kappa
constant region (Table 1, above). Full-length chimeric antibodies
were transiently expressed in COS cells or 293 cells by
co-transfection of chimeric heavy and light chain cDNAs ligated
into the pBOS or pTT3 expression plasmid (Mizushima and Nagata,
Nucleic Acids Research 1990, Vol 18, pg 5322). Cell supernatants
containing recombinant chimeric antibody were purified by Protein A
Sepharose chromatography and bound antibody was eluted by addition
of acid buffer. Antibodies were neutralized and dialyzed into
PBS.
[0252] 1.4. ELISA Binding of Recombinant Anti-Human RAGE mAbs and
of Hybridoma-Derived Anti-Human RAGE mAbs.
[0253] The purified chimeric anti-human RAGE monoclonal antibodies
were tested for their ability to bind human RAGE in a competition
ELISA. Recombinant chimeric anti-human RAGE monoclonal antibodies
or hybridoma-derived anti-human RAGE monoclonal antibodies were
diluted in PBST+10% Superblock (Pierce Biotech, Rockford, Ill.) and
made up as a 2.times. stock at various concentrations ranging from
320 .mu.g/mL to 0.0156 .mu.g/mL (7F9 and 11E6) and from 160
.mu.g/mL to 0.0078 .mu.g/mL (4E5). Biotinylated hybridoma-derived
anti-human RAGE monoclonal antibodies (7F9-biotin, 11E6-biotin and
4E5-biotin) were prepared at 8 .mu.g/mL in PBST+10% Superblock.
Equal volumes (50 .mu.L) of each recombinant chimeric anti-human
RAGE monoclonal antibodies or hybridoma-derived anti-human RAGE
monoclonal antibodies and each corresponding biotinylated
hybridoma-derived anti-RAGE mAbs were mixed. 50 .mu.L, of this
mixture was then added to ELISA plates pre-coated with recombinant
human RAGE at 2 .mu.g/mL and incubated for 1.5 hours at room
temperature. Wells were washed three times with PBS+0.05% Tween-20.
Streptavidin HRP (1 mg/mL) was diluted 1:16000 in PBST+10%
Superblock; 50 L/well was added and the plates incubated for 1 hour
at room temperature. Plates were washed 3 times with PBS+0.05%
Tween-20. 50 .mu.L of TMB solution (Sigma, St Louis, Mo.) was added
to each well and incubated for 10 minutes at room temperature. The
reaction was stopped by addition of 1N sulphuric acid. Plates were
read spectrophotometrically at a wavelength of 450 nm Results are
shown in FIGS. 1A, 1B, and 1C.
Example 2
Generation of Recombinant Human sRAGE (husRAGE)
[0254] Recombinant husRAGE protein 293/6.1 sRAGE His 6 was
expressed and purified in HEK293 cell (ATCC CRL-1573). The
expression vector used for the generation of the stable expression
was "pcDNA3 (-) 6.1 C HIS A".
[0255] Molecular biological standard techniques were used according
to Sambrook and Russel (Molecular Cloning: A Laboratory Manual, 3rd
edition, Cold Spring Harbor Laboratory Press. 2001). Total RNA from
human lymphocytes (PBL) was reverse transcribed into cDNA using
Superscript RT-PCR system (Invitrogen, Carlsbad USA). Using the
oligonucleodtide primers RAGE-SE: CCG AAT TCC GGA AGC AGG ATG GCA
GCC G (SEQ ID NO: 81) and RAGE-AS: CCC TCG AGC CCC TCA AGG CCC TCA
GTA CTA CT (SEQ ID NO: 82), the RAGE cDNA was amplified from the
cDNA (obtained above) yielding the RAGE cDNA as described in
reference sequence NM-001136. The PCR-fragment was run on an
agarose gel, purified and extracted with QIAquick Gelextraction Kit
(Qiagen GmbH, Germany). Afterwards the cDNA was cut with
restriction endonucleases EcoR1 and XhoI. The resulting fragment
was gel purified and ligated into vector pcDNA 3 (Invitrogen, USA)
which had been precut with XhoI/EcoR1. After transformation into E.
coli XL-1 blue cells (Invitrogen, USA) a positive recombinant clone
was identified. The sequence of this clone was verified and the
pcDNA3/RAGE 2.6 plasmid DNA isolated using plasmid mini-Kit
(Qiagen, Germany). The coding region for the extracellular part of
RAGE, husRAGE, was amplified from pcDNA3/RAGE 2.6 using PCR and
primers N-SE A: AGT AAC GGC CGC CAG TGT GCT GGA ATT CGG A (SEQ ID
NO: 83) and C-SE B: CCG GTA CCA CCT GCA GTT GGC CCC TCC TCG CC (SEQ
ID NO: 84). The resulting PCR product was cut with restriction
endonucleases EcoR1 and Kpn1, gel purified as described above and
ligated into "pcDNA3.1(-) Myc HIS" (Invitrogen, USA) which had been
precut with EcoR1/Kpn1. The resulting plasmid "pcDNA 3 (-) 6.1 C
HIS A" was transfected into HEK293 cells using Superfect (Qiagen,
Germany) according to manufacturers instructions. Selection of
resistant cells was done using 800 .mu.g/ml G418 in MEM Medium
(#M4528, Sigma, Germany)+10% FCS, 2 mM L-Glutamin, 100 U/ml
Pennicillin/Streptavidin (Invitrogen, USA). Cloning of single cells
through serial dilutions of cell suspensions lead to the
identification of a clone "293/6.1 sRAGE His 6" that secreted
husRAGE into the cell culture medium as confirmed by Western Blot
using a RAGE specific antibody (Santa Cruz; # sc5563). For
expression and purification of sufficient amounts of husRAGE
protein this clone was grown in serum containing cell culture
medium (see above) in cell factories (Nunc, Germany). Cells were
then switched to serum-free medium Pro293a-CDM (#12-764Q,
BioWhittaker, Belgiun) and incubated for 3 days at 37.degree. C. 80
liters of cell free medium was harvested and concentrated using
Hemoflow F-Series High-Flux columns (Fresenisus Medical Care AG,
Germany) to a volume of 1400 ml.
[0256] Protein purification was done using immobilized metal ion
affinity chromatography (IMAC) by Diarect AG (Freiburg, Germany)
and sepharose FF for chelation (Amersham-Bioscience, Sweden).
Equilibration of the column and binding of the hexa-His containing
protein from cell supernatants to the matrix were done according to
instructions by the manufacturer. Elution of the protein was done
using step gradients with increasing concentrations of imidazole.
Eluted fractions were analyzed for protein containing hexa-His
using Western Blots and anti HIS antibodies. Purified husRAGE
eluted specifically at 250-500 mM imidazole. Positive fractions
were combined, concentrated and dialyzed 3 times against PBS
(2.times.4 h, 1.times.16 h).
[0257] A N-terminal shortened version of husRAGE
(102-331-sRAGE-HIS) missing the first 101 amino acids of human RAGE
was generated by standard techniques in molecular biology as
described above for the husRAGE protein. This protein was generated
by the same basic procedure used for husRAGE (1-331). Using the
plasmid described above (pcDNA3/RAGE 2.6) and two primers (CGA AGC
TTG ATG AAC AGG AAT GGA AAG GAG ACC AAG (SEQ ID NO: 85) and TCC TCG
AGC ACC TGC AGT TGG CCC CTC CTC GCC T (SEQ ID NO: 86)) the shorter
version of the DNA for husRAGE was amplified by PCR. After agarose
gel and elution of the fragment the resulting pure fragment was
cleaved with restriction endonucleases HindIII and XhoI and
purified again using agarose gel and elution. The fragment was
ligated into psecTAG 2A (Invitrogen, USA) that was precut with
restriction endonucleases HindIII and XhoI. After transformation
into E. coli "TOP10 One Shot" cells (Invitrogen, USA) a positive
clone was picked and the plasmid DNA isolated. The DNA in the
expression vector was transfected into HEK293 F cells using the
Freestyle expression system (Invitrogen, USA). After 96 hours of
expression the cell free supernatant was used for the purification
using Ni-NTA Superflow beads (Quiagen, Germany). Equilibration and
binding were done according to instruction by the manufacturer.
Bound protein was eluted in buffer (PBS, 160 mM NaCl, 150 mM
Imidazole, pH8.0). Fractions containing protein were combined and
dialyzed over night at 4.degree. C. against TBS (Tris-buffered
saline; pH 7.4). Purified husRAGE (102-331) concentrations were
determined spectrophotometrically.
[0258] A C-terminal shortened version of husRAGE-fusion protein
(1-130-sRAGE-Fc) missing the amino acids following amino acid 130
of human RAGE was generated by standard techniques in molecular
biology as described above for the husRAGE protein. Using the
plasmid described above (pcDNA3/RAGE 2.6) and two primers
(GCACCATGGCAGCCGGAACAGCAGTTG (SEQ ID NO: 87) and
GAGTCTCGAGGCAGAATCTACAATTTCTG (SEQ ID NO: 88)) the shorter version
of the DNA for husRAGE was amplified by PCR. After agarose gel and
elution of the fragment the resulting pure fragment was cleaved
with restriction endonucleases NcoI and XhoI and purified again
using agarose gel and elution. The fragment was then ligated into
the plasmid pENTR4 that was precut with restriction endonucleases
NcoI and XhoI. The ligation mixtures were transformed into E. coli
"TOP10 One Shot" cells (Invitrogen, USA) to generate pENTR4-RAGE
1-130. A positive clone was picked and the plasmid DNA isolated.
Using site specific recombination and the gateway cloning system
(Invitrogen, Carlsbad, USA; attL.times.attR) with DNA of the clone
pENTR4 hRAGE 1-130 and DNA of the vector pcDNA3.1(+)Zeo hIgG lambda
hc 257-Stop a plasmid was constructed (see below) that after
transformation into "TOP10 One Shot" cells (Invitrogen, USA) and
purification encoded for husRAGE-1-130-Fc (plasmid called: pEXP
hRAGE 1-130/hIgG lambda hc 257-Stop). Expression of this plasmid
using the Freestyle Expression system and 293F cells (Example 2.1)
and purification of the resulting protein from the cell supernatant
using Protein G-beads (Example 2.2) resulted in a protein with
>95% purity.
Example 3
Construction of pcDNA3.1(+)Zeo hIgG Lambda hc 257-Stop
[0259] 2 oligonucleotide primers
(gtacgatatcgagggacgaatggatccaccgtgcccagcacc (SEQ ID NO: 91);
ctagtctagatcatttacccggagacagggag (SEQ ID NO: 92)) were used to
amplify the DNA sequence for hIgG lambda heavy chain from a human
placenta cDNA Library (Clontech # HL5014a) using EasyA Polymerase
in a PCR, polymerase chain reaction. The resulting DNA was gel
purified (as described above), cloned into pcDNA3.1 V5-His TOPO
Vektor (pcDNA3.1/V5/His TOPO TA Expression Kit Invitrogen
#K4800-01) using instruction from the manufacturer and transformed
into E. coli TOP10 cells as described above. Positive clones were
identified and the resulting plasmid DNA purified (named:
pcDNA3.1(V5His) FC/hIgG lambda hc Nr.2/7) using PCR and
olgionucleotide primers (gtacgatatcgagggacgaatggatccaccgtgcccagcacc
(SEQ ID NO: 93); ctagtctagatcatttacccggagacagggag (SEQ ID NO: 94)).
The hIgG lambda hc part of the DNA was amplified, cut with
EcoRV/XbaI, ligated to EcoRV/XbaI precut pcDNA3.1(+)Zeo vector DNA
and transformed into E. coli TOP10 cells. The resulting plasmid was
named: pcDNA3.1(+)Zeo hIgG lambda hc 257-Stop and used for further
work to express proteins N-terminally fused in frame to a
C-terminal part of immunoglobulin IgG heavy chains.
[0260] 3.1. Transfection and Expression of Proteins in HEK293F
Cells
[0261] HEK 293F cells that had been grown in culture for 2-3 days
in Free Style 293 Expression Medium were centrifuged at 400 g and
the supernatant discarded. The cell pellet was resuspended in
medium and adjusted to 3.times.107 cells in 28 ml fresh medium,
transferred to a 125 ml Erlenmeyer and incubated in an incubator at
37.degree. C., 8% CO2 on an orbital shaker at 150 rpm until the
transfection mixture was set up.
[0262] Transfection mixtures with 293fectin-DNA complex were set up
as follows:
[0263] (i) 30 .mu.g of DNA were diluted with Opti-MEM I to a total
volume of 1000 .mu.l (control 1000 .mu.l Opti-MEM I) and mixed.
[0264] (ii) 35 .mu.l of 293fectin (Invitrogen #12347-019; 1 ml)
were diluted with Opti-MEM I to a total volume of 1000 .mu.l, mixed
and incubated for 5 min at room temperature.
[0265] DNA mixture and 239fectin-solution from (i) and (ii) were
transferred to a new tube, mixed slightly and after incubation for
25 minutes at room temperature were added to the cells in the
Erlenmeyer.
[0266] Cells were incubated with this transfection mixture for the
indicated time in an incubator at 37.degree. C., 8% CO.sub.2 on an
orbital shaker at 150 rpm. Cell supernatants were harvested by
centrifugation at 400 g for 10 minutes.
[0267] 3.2. Purification of RAGE-Fc Fusion Proteins Using Protein
G-Sepharose
[0268] To couple the protein from cell supernatants to beads, beads
(protein G-sepharose 4 Fast Flow (Amersham Bioscience) were washed
3 times in PBS by suspending the beads in PBS and centrifugation at
13,500 rpm, discarding the supernatant. Beads were incubated with
the respective cell supernatants (300 ml cell supernatants per ml
beads) to be coupled for 1-2 hours on a rotator at room
temperature. The beads were washed 3 times with PBS and incubated
with the cell supernatants for 12 hours or overnight at 4.degree.
C. After incubation the beads were washed 3 times with PBS as
above. Bound protein was eluted by adding 200 .mu.l 140 mM
NaCl+0.1M glycine to the bead pellet and incubating for 30 minutes
on a rotator. After centrifugation the supernatant was immediately
neutralized by adding 2 M Tris to adjust the pH to 7.1-pH 7.4. The
bead pellet was discarded. Obtained Probes were dialyzed against
PBS and stored frozen in aliquots at -20.degree. C. RAGE-Fc fusion
protein containing the full extracellular ectodomain of RAGE was
obtained from R&D systems (no. 1145-RG; Recombinant Human
RAGE/Fc Chimera).
[0269] 3.3. Dot Blot Binding of Antibodies to Peptides or Fragments
of RAGE in a Non Denatured Form.
[0270] Dot blots were used to evaluate the binding of antibodies to
peptides or fragments of RAGE in a non-denatured form. Proteins
used were either sRAGE-Protein (1-331 sRAGE-HIS) or a N-terminal
shortened version (102-331-sRAGE-HIS). Peptides were ordered and
synthesized by Biotrend according to standard methods (solid phases
peptide synthesis on AMS 222 synthesizer using Fmoc/tBu-chemistry)
containing a free carboxyl terminus Peptides were HPLC-purified and
analysis of every peptide for purity was done using RP-HPLC. All
peptides had a purity of >80%. The identities of peptides were
verified by mass spectrometry.
[0271] Peptides used were 30mers spanning the extracellular region
of the human RAGE protein. Net charge of most peptides was
similar.
TABLE-US-00010 NtermR31: (SEQ ID NO: 70)
QNITARIGEPLVLKCKGAPKKPPQRLEWKLN Net charge: +7 Peptide 1: (SEQ ID
NO: 71) KLNTGRTEAWKVLSPQGGGPWDSVARVLPN Net charge: +2 Peptide 2:
(SEQ ID NO: 72) LPNGSLFLPAVGIQDEGIFRCQAMNRNGKE Net charge: 0
Peptide 3: (SEQ ID NO: 73) GKETKSNYRVRVYQIPGKPEIVDSASELTA Net
charge: +1 Peptide 4: (SEQ ID NO: 74)
LTAGVPNKVGTCVSEGSYPAGTLSWKLDGK Net charge: +1 Peptide 5: (SEQ ID
NO: 75) DGKPLVPNEKGVSVKEQTRRHPETGLFTLQ Net charge: +2 Peptide 6:
(SEQ ID NO: 76) TLQSELMVTPARGGDPRPTFSCSFSPGLPR Net charge: +1
Peptide 7: (SEQ ID NO: 77) LPRHRALRTAPIQPRVWEPVPLEEVQLVVE Net
charge: +2 Peptide 8: (SEQ ID NO: 78)
VVEPEGGAVAPGGTVTLTCEVPAQPSPQIH Net charge: -2 Peptide 9: (SEQ ID
NO: 79) QIHWMKDGVPLPLPPSPVLILPEIGPQDQG Net charge: +0 Peptide 10:
(SEQ ID NO: 80) DQGTYSCVATHSSHGPQESRAVSISIIEPG Net charge: -1
[0272] Dots consisting of different amount of protein/peptide (30
ng, 10 ng, 3 ng, 1 ng, 0.3 ng, 0.1 ng, 0.03 ng, and 0.01 ng) in a
volume of 1 .mu.l in 1.times.PBS were spotted onto a Hybond-ECL
Nitrocellulose Membrane (Amersham, RPN68D) in duplicates. Membranes
were dried and unspecific binding was blocked by shaking the
membranes for 1 hour at room temperature with Western Blocking
reagent (Roche, no. 1921673). Blocking reagent was discarded and
the membranes were incubated with antibodies in a concentration of
7.14 nM (shaking, 1 hour at room temperature). Monoclonal
antibodies were ML37-7F9, ML37-11E6, ML37-4E5, commercially
available antibodies from R&D systems (e.g. AF1145). Blots were
washed 4 times (each time 5 minute incubation with shaking at room
temperature) with 1.times.PBS. Blots were then incubated with goat
anti mouse IgG AP secondary antibody (Sigma no. A-7434) diluted
1:2000 in Western Blocking Reagent (Roche, no. 192173). Incubation
for 1 hour was as before (shaking, room temperature). The filters
were washed 4 times (5 minutes each) in 1.times.PBS. Development of
signals was according to manufacturers instructions with NBT/BCIP
substrate solution (Roche, no. 1697471). Color development was
stopped after 10 minutes with bi-distilled water. See FIG. 2.
[0273] Although husRAGE was detected in dot blots by all three
monoclonal antibodies of the present invention and by polyclonal
antibody AF1145 (from a commercial source, R&D) none of the
peptides were detected by the monoclonal antibodies of the present
invention. However the polyclonal antibody detected several
peptides. Peptide 9, which did include the amino acid sequence used
to generate polyclonal antibodies as described by Ostendorp et al.
(EMBO J. 26, 3875, 2007), was clearly detected by the commercially
available polyclonal antibody. These results indicate that the
monoclonal antibodies of the present invention clearly recognize a
different epitope than currently available antibodies.
[0274] Further characterization of the binding was done by analysis
of RAGE mutants of human sRAGE expressed in E. coli. Monoclonal
antibodies 11E6 and 4E5 bind to a region around the C2 domain,
since binding is lost in deletion mutants lacking amino acids
235-336 and binding, is apparent in mutant RAGE protein consisting
of the amino acids 235-336.
Example 4
Construction, Expression and Purification of the E. coli RAGE
Fragments
4.1. Preparation of Constructs
[0275] The E. coli RAGE constructs listed in Table 6 were generated
as follows. Construct 1 was created by PCR amplification from the
template plasmid pcDNA 3 (-) 6.1 C HIS A using the forward primer
(atgctacatatgaaaaagacagctatcgcgatt
gcagtggcactggctggtttcgctaccgtagcgcaggccgctcaaaacatcacagcc (SEQ ID
NO: 89)) and reverse primer (atgctactcgagtcagtggtggtgg
tggtggtgagttcccagccctgatcctcccacagagcctgcagttggcccctcc (SEQ ID NO:
90)) which introduced Nde I and Xho I restriction sites which were
utilized for subcloning into the analogous sites of pET29. The
remaining constructs (#2-#7, Table 6) were generated using
Construct 1 as a template. Sequences encoding RAGE amino acid
residues 24-129, 24-234, 24-336, 130-234, 130-336, 235-336 were PCR
amplified from Construct 1. The resulting DNA fragments were run on
a 1.0% agarose gel, and the DNA purified using the QIAquick Gel
Extraction Kit from Qiagen. The DNA fragments were digested with
NdeI and XhoI, and ligated into similarly digested pET28a. The
ligation mix was transformed into Max Efficiency DH5.alpha.
competent cells and plated onto LB agar plates containing 50 mg/L
kanamycin. After overnight incubation at 37.degree. C., three
colonies for each clone were inoculated into 3 ml of LB broth
containing 50 mg/L kanamycin and shaken overnight at 37.degree. C.
The DNA was isolated using the QIAprep Spin Miniprep Kit from
Qiagen, and the insert sequenced using T7 promoter and T7
terminator specific primers. The DNA sequence of the plasmids
encoding constructs #1-#7 are listed as SEQ ID NOs: 27-33, and the
corresponding translated regions are listed as SEQ ID NOs:
34-40.
TABLE-US-00011 TABLE 6 RAGE constructs Protein Plasmid Construct #
Protein Form Name SEQ ID SEQ ID 1 OmpA-[RAGE (23-340)]-6His 34 27 2
6His-(Thr)-[RAGE (24-129)] 35 28 3 6His-(Thr)-[RAGE (24-234)] 36 29
4 6His-(Thr)-[RAGE (24-336)] 37 30 5 6His-(Thr)-[RAGE (130-234)] 38
31 6 6His-(Thr)-[RAGE (130-336)] 39 32 7 6His-(Thr)-[RAGE
(235-336)] 40 33
[0276] E. coli strain BL21(DE3) was transformed with Construct #1
plasmid DNA, plated on LB plates containing kanamycin (50 mg/L),
and incubated at 37.degree. C. overnight. The next day 14 fernbach
flasks, each containing 1 L of Terrific Broth with kanamycin (50
mg/L), were inoculated with a CFU and placed shaking (180 rpm) in
an incubator at 37.degree. C. When the cultures reached an
OD.sub.600nm of 0.47, the flasks were transferred to a 30.degree.
C. incubator (still shaking at 180 rpm) and expression was induced
by addition of 0.4 mM IPTG. Cells were harvested 4 hours after
induction by centrifugation (15,900 g, 8 minutes, 4.degree. C.),
and the cell paste then frozen at -80.degree. C. until
purification.
[0277] Purification of RAGE Construct 1 proceeded by first thawing
and resuspending an .about.20 g cell pellet in 180 ml of lysis
buffer [50 mM Tris pH 7.6, 300 mM NaCl, 10% glycerol, 0.1% Triton
X-100, 0.5 mM MgCl.sub.2, 20 mM imidazole, 1.times. Roche EDTA-free
protease inhibitors, 20 U/ml DNase I]. Cells were lysed by passing
the suspension three consecutive times through an Avestin
Emulsiflex microfluidizer at 3.degree. C. Clarified lysate was then
loaded onto a 5 ml HiTrap IMAC-column (GE Healthcare, 17-5255-01)
at 2 ml/min. The column was then washed with 10 CV of wash buffer
[50 mM Tris pH 7.6, 300 mM NaCl, 10% glycerol, 20 mM imidazole].
Following the wash step, RAGE was gradient eluted using elution
buffer [50 mM Tris pH 7.6, 300 mM NaCl, 10% glycerol, 500 mM
imidazole]. Fractions containing RAGE were pooled and then dialyzed
against 50 mM Tris pH 7.6, 20 mM NaCl, 10% glycerol. Mass-spec
analysis of the purified material confirmed the OmpA-leader had
been processed off and that the purified material began with
residue 23 of RAGE (i.e. the sequence A-Q-N- . . . ) as
expected.
[0278] Plasmids encoding Constructs #2-#7 were separately
transformed into E. coli strain BL21(DE3), plated onto LB plates
containing kanamycin (50 mg/L), and incubated at 37.degree. C.
overnight. The next day 1 L of Overnight Express Instant TB Medium
(Novagen) was inoculated with a colony and shaken for 19 hours at
30.degree. C. The cells were pelleted by centrifugation
(15,900.times.g, 10 minutes, 4.degree. C.) and then frozen at
-80.degree. C. The pellets (5-6 grams each) were thawed and
resuspended in 50 ml of lysis buffer [50 mM Tris, pH 8, 300 mM
NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mg/ml lysozyme, 1 ml of
protease inhibitor cocktail set III (Calbiochem), 20 U/ml
benzonase, 5 mM B-mercaptoethanol]. The lysates were sonicated on a
Vibra Cell Sonicator for 2 minutes, followed by centrifugation at
20K.times.g for 30 minutes. Econo-Pac 10 columns from Bio-Rad were
filled with a 2 ml bed volume of ProBond Nickel Resin and
equilibrated with lysis buffer. The clarified lysates were passed
through the columns 3 consecutive times, followed by washing with
3.times.10 column volumes (60 ml total) of wash buffer
[2.times.PBS, 20 mM imidazole, 10% glycerol, 5 mM
B-mercaptoethanol]. The proteins were eluted off the columns with
5.times.1 column volume (10 ml total) of elution buffer
[2.times.PBS, 500 mM imidazole, 10% glycerol, 5 mM
B-mercaptoethanol]. The eluted material was transferred into PBS,
10% glycerol, and 1 mM DTT using Bio-Rad Econo-Pac 10DG
Columns.
[0279] 4.2. Expression of Anti-RAGE Monoclonal Antibodies 11E6, 4E5
and 7F9
[0280] The media used for hybridoma cell expansion consisted of BD
Cell MAb Quantum Yield Medium (Becton Dickenson--catalog #220511)
containing 10% ultra low IgG fetal bovine serum
(Invitrogen--catalog #16250-078). Briefly, multiple 300 ml seed
cultures of the murine hybridoma cell line expressing RAGE
monoclonal antibody 11E6 were expanded in a 2 L roller bottle
shaking in an incubator (65 rpm, 8% CO.sub.2, 37.degree. C.) until
reaching a density of 1.0.times.10.sup.6 cell/ml. Cells were then
seeded into 20 L of media at a density 0.06.times.10.sup.6 cells/mL
in a 25 L Wave BioReactor with operational settings of 14
rocks/minute, a rock angle of 6.degree., temperature of 37.degree.
C., and an 8% CO.sub.2 sparge-rate of 0.15 Lpm. After two days, the
culture was further expanded by addition of media to a final volume
of 24 L, resulting in a new cell density of 0.43.times.10.sup.6
cells/mL. The culture was harvested 12 days after being expanded to
full volume. Cells were removed by continuous centrifugation (Carr
ViaFuge, 6000 rpm, 1.7 Lpm). After addition of 5 mM NaN.sub.3 (from
a 1 M NaN.sub.3 stock--Hampton Research) to the clarified media,
the material immediately was utilized in the purification
process.
[0281] The media used for hybridoma cell expansion consisted of BD
Cell MAb Quantum Yield Medium (Becton Dickenson--catalog #220511)
containing 10% ultra low IgG fetal bovine serum
(Invitrogen--catalog #16250-078). Briefly, multiple 300 ml seed
cultures of the murine hybridoma cell line expressing RAGE
monoclonal antibody 4E5 were expanded in a 2 L roller bottle
shaking in an incubator (65 rpm, 8% CO.sub.2, 37.degree. C.) until
reaching a density of 1.0.times.10.sup.6 cell/ml. Cells were then
seeded into 5 L of media at a density 0.12.times.10.sup.6 cells/mL
in a 25 L Wave BioReactor with operational settings of 12
rocks/minute, a rock angle of 6.degree., temperature of 37.degree.
C., and an 8% CO.sub.2 sparge-rate of 0.15 Lpm. After four days,
the culture was further expanded by addition of media to a final
volume of 24 L, resulting in a new cell density of
0.24.times.10.sup.6 cells/mL. The rock-rate was increased to 14
rocks/minute. The culture was harvested 12 days after being
expanded to full volume. Cells were removed by continuous
centrifugation (Carr ViaFuge, 6000 rpm, 1.7 Lpm). After addition of
5 mM NaN.sub.3 (from a 1 M NaN.sub.3 stock--Hampton Research) to
the clarified media, the material immediately was utilized in the
purification process.
[0282] The media used for hybridoma cell expansion consisted of BD
Cell MAb Quantum Yield Medium (Becton Dickenson--catalog #220511)
containing 10% ultra low IgG fetal bovine serum
(Invitrogen--catalog #16250-078). Briefly, multiple 300 ml seed
cultures of the murine hybridoma cell line expressing RAGE
monoclonal antibody 7F9 were expanded in a 2 L roller bottle
shaking in an incubator (65 rpm, 8% CO.sub.2, 37.degree. C.) until
reaching a density of 1.0.times.10.sup.6 cell/ml. Cells were then
seeded into 10 L of media at a density 0.05.times.10.sup.6 cells/mL
in a 25 L Wave BioReactor with operational settings of 12
rocks/minute, a rock angle of 6.degree., temperature of 37.degree.
C., and an 8% CO.sub.2 sparge-rate of 0.15 Lpm. After four days,
the culture was further expanded by addition of media to a final
volume of 25 L, resulting in a new cell density of
0.25.times.10.sup.6 cells/mL. The rock-rate was increased to 14
rocks/minute. The culture was harvested 10 days after being
expanded to full volume. Cells were removed by continuous
centrifugation (Carr ViaFuge, 6000 rpm, 1.7 Lpm). After addition of
5 mM NaN.sub.3 (from a 1 M NaN.sub.3 stock--Hampton Research) to
the clarified media, the material immediately was utilized in the
purification process.
[0283] 4.3. Purification of Anti RAGE Monoclonal Antibodies 11E6,
4E5, 7F9
[0284] To the clarified hybridoma culture media, glycine and NaCl
were added to final concentrations of 3M and 1.5M, respectively.
The pH was adjusted to 8.0 with NaOH. Material is filtered using a
5 .mu.m Pall Capsule #120 membrane filter and loaded onto a 200 mL
BioSepra Protein A column Protein solution was washed with 11 CV of
wash buffer (20 mM sodium phosphate pH 8.0, 1 mM sodium azide) and
eluted using a step gradient of 50 mM glycine (pH 3.0). Fractions
of 100 mL size were collected and protein concentration determined
by measuring the A280 nm. All column-processing steps were run at
4.degree. C. Pooled material was dialyzed against 20 L of 10 mM
Tris pH 8.0 overnight at 4.degree. C. Further chromatographic
polishing was achieved using a SartoBind Q strong basic anion
exchanger Singlesep Mini cartridge (Sartorius) in flow through mode
at 10 mL/min. The antibody does not bind the column and is
collected as one pool. Following this step, the material was
concentrated to 5 mg/ml using an Amicon stirred pressure cell (5000
MWCO membrane, 75 psi, 4.degree. C.). Finally, the antibody was
dialyzed two times against 20 L of PBS buffer (10 mM phophate,
0.138 M NaCl, 0.0027 M KCl, pH 7.4), each cycle for 24 h at
4.degree. C.
[0285] 4.4. ELISA Binding Experiments:
[0286] Antigens (purified construct #1-7 proteins) were diluted
into Coating Buffer [100 mM NaHCO.sub.3, pH 8.2] to 1 .mu.g/ml, and
100 .mu.l of the resulting solution was then aliquoted into a
96-well Nunc Immuno Plate (Maxi-Sorb Surface, flat bottom, catalog
#439454). The plate was sealed with sealing film and incubated at
4.degree. C. overnight. The next day, the plate wells were each
washed 3 times with 150 .mu.L PBST buffer [Sigma PBS+0.05% Tween
20]. Then 300 ul of blocking solution (3% NFDM in PBST) was added
to each well. The plate was incubated for 2 hours at room
temperature and shaking at 100 rpm. After the incubation step, each
well was again washed three times with 150 uL PBST. 100 ul of the
corresponding antibody to be tested (i.e. 7F9, 11E6, and 4E5) was
added at various dilutions made in PBST/0.5% BSA. The plate was
then sealed with sealing film and incubated for 2 hours at room
temperature and shaking at 100 rpm. Next the antibody solution was
drained out of the wells, and each well then washed three times
with 200 ul PBST. To each well then 200 ul of a 1:5000 dilution (in
PBST/1% NFDM) of conjugated secondary antibody [Donkey anti-mouse
HRPO conjugate, Jackson Immuno Research, Catalog #715-035-150] was
added. The plate was covered and allowed to incubate for 1 hour at
room temperature while shaking at 100 rpm. Following this
incubation, the solution was removed from the wells and each well
was washed three times with 200 ul PBST. To each well 100 .mu.L of
HRPO substrate [3,3',5',5'-Tetramethylbenzidine Liquid Substrate
(TMB), Supersensitive for ELISA, Sigma Catalog #T4444]solution was
added and the plate incubated at room temperature for 10 minutes.
Finally, 50 .mu.L of 2M H.sub.2SO.sub.4 was added to each well to
stop the reaction and the A.sub.540nm of each well was measured
using a microtiter plate reader.
[0287] Results of these binding experiments are shown in FIGS. 3 A,
3B and 3C, and FIGS. 4A, 4B, and 4C. FIGS. 3A, 3B, and 3C shows
that RAGE residues 24-234 were not involved in binding of RAGE
monoclonal antibodies 11E6, 4E5, or 7F9. Conversely, as shown in
FIGS. 4A, 4B, and 4C, RAGE residues 235-336 were sufficient for
binding of RAGE monoclonal antibodies 11E6 and 4E5. RAGE mAb 7F9
did not show any binding to any of these (E. coli expressed) RAGE
fragments.
Example 5
Epitope Mapping
[0288] 5.1. Immobilization of the 11E6, 4E5 and 7F9 Antibodies
[0289] Approximately 20 mg of CNBr-activated Sepharose fast flow
resin was weighed into three compact reaction columns fitted with
35 .mu.m frits. The resins were permitted to swell in 200 .mu.L of
1 mM HCl before washing 3 times with 200 .mu.L of 1 mM HCl. The
resins were subsequently rinsed 3 times with 200 .mu.L of 100 mM
sodium bicarbonate buffer (pH 8.3) containing 500 mM sodium
chloride (buffer A). Once completed, the solutions were flushed
from the columns so that only a thin layer of buffer remained on
the surface of each resin. Approximately 5.5 nmoles of the
antibodies were immobilized to the resins, which required the
addition of 235 .mu.L of 7F9 (3.4 mg/mL), 500 .mu.L of 11E6 (1.6
mg/mL), and 200 .mu.L of 4E5 (3.75 mg/mL). For the 7F9 and 4E5
antibody immobilizations, 200 .mu.L of buffer A was also included.
The compact reaction columns were sealed and permitted to mix
through inversion at room temperature for 4 hours. Once completed,
the compact reaction columns were unsealed and flushed with three
additions of 200 .mu.L of buffer A to remove unbound antibody.
After the flush, 200 .mu.L of a buffer containing 100 mM Tris-HCl
(pH 8) and 500 mM sodium chloride (buffer B) was added to each
column. The columns were resealed and allowed to mix through
inversion at room temperature to block unbound but activated sites
on the resins. After 2 hours, the columns were unsealed and flushed
first with 200 .mu.L of 100 mM sodium acetate buffer (pH 4)
containing 500 mM sodium chloride (buffer C) followed by 200 .mu.L
of buffer B. This process was repeated an additional two times to
ensure complete removal of unbound antibody and to fully block the
remaining sites of attachment on the resins. The resins were then
washed four times with 200 .mu.L of 100 mM sodium bicarbonate
buffer (pH 8.3) containing 100 mM sodium chloride (buffer D) prior
to coupling the antigen.
[0290] 5.2. Proteolytic Excision of the E. coli and
BacMam-Expressed sRAGE Antigens
[0291] The sRAGE antigens were permitted to bind to the antibody
columns by adding 75 .mu.L of the E. coli-expressed antigen to the
11E6 and 4E5 resins and adding 125 .mu.L of the BacMam-expressed
antigen to the 11E6, 4E5 and 7E9 resins. The columns were sealed
and the samples allowed to mix by inversion at room temperature for
2 hours. After this time, the columns were unsealed and flushed
with 4 additions of 200 .mu.L of buffer D. After flushing through
the rinses, the resins were resuspended in 200 .mu.L of buffer D as
well as with the proteases, generated as 0.1 mg/mL solutions of
either Trypsin, endoproteinase Glu-C or Chymotrypsin. The amounts
of the proteases varied between the experiments to attenuate the
digestions, but ranged from 200-400 fold excess of the antigen over
the protease by weight. Proteolysis was permitted to occur at room
temperature with mixing by inversion for 12 hours. After this time,
the columns were unsealed and the proteolytic solution was flushed
through and saved for further analysis. For samples subjected to
dual digestion, the resins were resuspended in 200 .mu.L of buffer
D and a second protease before the next steps. For those samples
not treated in a second protease, the columns were subjected to two
individual 200 .mu.L washes in buffer D followed by a 200 .mu.L
wash in buffer A then a final 200 .mu.L wash in buffer D. Each wash
was retained separately for later analysis. The epitope containing
peptides were eluted from the column with three individual 200
.mu.L washes in 2% formic acid. Each elution sample was retained
separately for later analysis.
[0292] 5.3. Mass Spectrometry Analysis of the Epitope-Containing
Peptides
[0293] The samples were analyzed using both a Bruker Apex QE 7T
Fourier-Transform Ion Cyclotron Resonance (FT-ICR) mass
spectrometer as well as an Applied Biosystems Q-STAR Pulsar I mass
spectrometer. For the FT-ICR mass spectrometric analysis, 8 .mu.L
of the epitope excision samples were injected onto a Jupiter C4
reversed phase column (0.5.times.150 mm, 5.mu. particle size, 300
.ANG. pore size) by an Agilent series 1100 capillary HPLC. The
samples were washed for 5 minutes in 90% water with 0.1% formic
acid (solvent A)/10% acetonitrile with 0.1% formic acid (solvent B)
at a 5 .mu.L/min flow rate to desalt. The peptides were eluted into
the mass spectrometer by changing the mobile phase composition to
5% solvent A/95% solvent B. Samples requiring direct infusion for
tandem mass spectrometry were injected onto a protein Microtrap
(Michrom) equilibrated in 98% water, 1% acetonitrile and 1% formic
acid. The samples were washed with 1 mL of the equilibration
solvent before being eluted in 300 .mu.L of 60% acetonitrile, 40%
water and 0.1% formic acid. The eluent was directly infused into
the FT-ICR mass spectrometer at 2 .mu.L/min. For the Q-STAR Pulsar
mass spectrometer, between 5-30 .mu.L of sample was injected onto a
protein Microtrap (Michrom) by an Agilent series 1100 HPLC. The
samples were washed in 95% solvent A/5% solvent B for 1 minute
prior to elution of the bound peptides into the mass spectrometer
in 5% solvent A/95% solvent B.
[0294] Proteolytic excision of the E. coli-expressed sRAGE bound to
the 11E6 antibody and elution of epitope-containing peptides
revealed the presence of a peptide with a mass of 12,204.5 Da. Mass
selection and collisionally-activated dissociation of the 10.sup.+
charge state confirmed the identity of this peptide, which
corresponded to the residues Val.sup.229-His.sup.346 (this His
residue is due to adding the Hexa-His-tag to the sRAGE
protein).Further epitope mapping using Trypsin followed by
Chymotrypsin proteolysis revealed cleavage of the C-terminal
hexahistidine tag, thus refining the epitope to residues
Val.sup.229-His.sup.341 (this His residue is due to adding the
Hexa-His-tag to the sRAGE protein). No further refinement of this
epitope could be obtained using proteolysis. Similarly executed
proteolytic excision of the E. coli-expressed sRAGE bound to the
4E5 antibody revealed the same peptide of 12,204.5 Da that was
observed for the 11E6 antibody epitope. Correspondingly, excision
of the BacMam-expressed sRAGE bound to either the 11E6 or 4E5
antibody revealed two major epitope containing peptides of masses
10,671.9 Da and 10,614.0 Da. These peptides match to the C-terminal
of the BacMam-expressed construct, spanning residues
Val.sup.229-Gly.sup.331 and Val.sup.229-Ala.sup.330,
respectively.
[0295] Proteolytic excision of the BacMam-expressed sRAGE bound to
the 7F9 antibody and elution of epitope-containing peptides
revealed multiple species representing several overlapping
peptides. Deconvolution of the mass spectrum revealed peptides of
masses 12,079.6 Da, 12,372.9 Da, 13,477.3 Da and 24,132.3 Da. These
masses match to residues Asn.sup.105-Arg.sup.216,
Asn.sup.105-Arg.sup.218, Asn.sup.105-Arg.sup.228 and
Asn.sup.105-Gly.sup.331, respectively, suggesting a minimal epitope
spanning residues Asn.sup.105-Arg.sup.216.
[0296] These results indicate that antibodies 11E6 and 4E5 possess
epitopes on the C-termini of both E. coli and BacMam-expressed
sRAGE, and that antibody 7F9 recognizes an epitope on the center
domain of BacMam-expressed sRAGE.
Example 6
Biacore and Surface Plasmon Resonance Measurements
[0297] The affinity of the three monoclonal antibodies of the
present invention, i.e. 7F9, 11E6 and 4E5, was measured using
Biacore and surface plasmon resonance measurements.
[0298] 6.1. Materials and Methods for Anti-RAGE Binding Kinetic
Determinations:
[0299] Biacore 2000 instrument was used to measure mouse anti-RAGE
mAb binding kinetics. The assay format for mAb affinity analysis
was Fc-based capture via immobilized anti-Fc antibodies. A standard
amine coupling protocol was employed to immobilize Fc-specific IgG
via primary amines to the carboxy-methyl (CM) dextran surface of
CM5 sensorchips (Biacore). For the study of mouse anti-RAGE mAbs,
anti-mouse Fc (Biacore, anti-mouse, BR-1005-14) was used as the
immobilized capture reagent. An automated protocol, available on
the Biacore 2000, was used to immobilize 8000-10000RU of capture
reagent in all 4 flowcells of the sensorchip. Briefly, the
CM-dextran surfaces were activated by freshly prepared 1:1 50 mM
N-hydroxysuccinimide (NHS):200 mM 3-(N,N-dimethylamino)
propyl-N-ethylcarbodiimide (EDC). Then the anti-Fc IgG capture
reagent (20 ug/ml in 10 mM sodium acetate, pH4.5) was applied to
the surface followed by deactivation of the surface and blocking of
the residual reactive sites with 1M ethanolamine (pH 8.5).
[0300] The running buffer employed was HBS-EP+[10 mM HEPES, pH 7.4,
150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant (Biacore)] for the
mouse anti-RAGE mAbs. Each experimental cycle consisted of the
following steps: 1) anti-RAGE mAbs were captured in flowcells 2, 3
or 4 to a level of 50-200RU (depending on the antigen). All
measurements were referenced against flowcell 1 which had no
captured anti-RAGE mAb. 2) Antigen was injected through all 4
flowcells, 180 ul at 60 ul/min. After the antigen injection,
dissociation was monitored for 600 seconds at 60 ul/min 3) The
anti-Fc capture surface was regenerated with low pH glycine. For
kinetic determinations, RAGE injections were a 2 fold dilution
series from 20 nM-0.31 nM and buffer only in randomized
duplicates.
[0301] 6.2. Evaluation and Results
[0302] Data were processed using Biacore evaluation software.
Briefly, the data were double referenced by first, subtracting the
signal from the reference cell and second, by subtracting the
signal from buffer-only injections. The double referenced data from
the RAGE injection series were then fit globally to a 1:1
(Langmuir) binding model, which included a mass transfer term, to
determine the binding kinetic rate constants, ka and kd, and the
affinity, K.sub.D.
[0303] Table 7 shows that 4E5 did not bind to mouse RAGE (Mu-RAGE).
11E6 and 4E5 cross-competed with each other for binding to RAGE.
7F9 does not bind to RAGE produced in E. coli lacking
glycosylation.
[0304] Table 7 also shows the specific epitopes to which the three
antibodies of the present invention bound to human RAGE, via
epitope mapping using protection of human sRAGE from proteolytic
digestion and identification of the protected peptides with mass
spectrometry. MAb 7F9 bound to C1 (Asn.sup.105-Pro.sup.215); mAb
4E5 bound to C2 (Val.sup.229-Thr.sup.340 in E. coli RAGE;
Val.sup.229-Gly.sup.331 in husRAGE produced in mammalian cells),
and 11E6 bound to C2 (Val.sup.238-Arg.sup.314 in E. coli RAGE;
Val.sup.229-Gly.sup.331 in husRAGE produced in mammalian
cells).
TABLE-US-00012 TABLE 7 epitopes to which the antibodies are binding
Mab Affinity (nM) Epitope Mouse-RAGE 7F9 0.11 C1; glycosylation
sensitive + 11E6 0.29 C2; overlap with 4E5 + 4E5 2.2 C2; overlap
with 11E6 -
Example 7
Construction of CDR-Grafted Antibodies
[0305] By applying standard methods well known in the art, the CDR
sequences of VH and VL chains of monoclonal antibody 11E6 (see
Table 4 above) are grafted into different different human heavy and
light chain acceptor sequences. Based on sequence VH and VL
alignments with the VH and VL sequences of monoclonal antibody 11E6
of the present invention the following known human sequences are
selected: [0306] a) VH7-4.1 and VH1-2 as well as the joining
sequences hJH6 for constructing heavy chain acceptor sequences
(according to Table 2 above); [0307] b) 1-12/L5 and 3-15/L2 as well
as hJK2 for constructing light chain acceptor sequences (according
to Table 3 above).
[0308] By grafting the corresponding VH and VL CDRs of 11E6 into
said acceptor sequences the following CDR grafted, humanized,
modified VH and VL sequences were prepared (see also Table 5,
above): VH 11E6.1-GL, VH 11E6.2-GL, VL 11E6.1-GL and VL
11E6.2-GL.
Example 8
Construction of Framework Back Mutations in CDR-Grafted
Antibodies
[0309] To generate humanized antibody framework mutations,
mutations are introduced into the CDR-grafted antibodies using de
novo synthesis of variable domains and/or mutagenic primers and
PCR, using methods well known in the art. Different combinations of
back mutations and other mutations are constructed for each of the
CDR-grafts as follows.
[0310] For heavy chain VH 11E6.1-GL one or more of the following
Vernier and VH/VL interfacing residues are back mutated as follows:
V2.fwdarw.I and/or Y95.fwdarw.F.
[0311] For heavy chain VH 11E6.2-GL one or more of the following
Vernier and VH/VL interfacing residues are back mutated as follows:
V2.fwdarw.I, V68.fwdarw.F, M70.fwdarw.F, R72.fwdarw.L,
Y95.fwdarw.F.
[0312] For light chain VL 11E6.1-GL one or more of the following
Vernier and VH/VL interfacing residues are back mutated as follows:
A43.fwdarw.S, Y49.fwdarw.F, Y87.fwdarw.F.
[0313] For light chain VL 11E6.2-GL one or more of the following
Vernier and VH/VL interfacing residues are back mutated as follows:
A43.fwdarw.S, Y49.fwdarw.F, I58.fwdarw.V, Y87.fwdarw.F.
[0314] Additional mutations include the following:
[0315] for heavy chain
[0316] VH 11E6.1-GL, Q1.fwdarw.E, and for
[0317] VH 11E6.2-GL, Q1.fwdarw.E, I76.fwdarw.T, R85.fwdarw.S,
D89.fwdarw.E;
[0318] for light chain
[0319] VL 11E6.1-GL, V11.fwdarw.L, and
[0320] VL 11E6.2-GL, V13.fwdarw.L, E70.fwdarw.D.
Example 9
Construction and Expression of Recombinant Humanized Anti RAGE
Antibodies
[0321] pHybE expression vectors harboring heavy and light chains
containing framework back mutations were co-transfected into 293-6E
cells to transiently produce full-length humanized antibodies.
Mutations were introduced into the CDR-grafted antibody sequences
as prepared according to Example 7, by de novo synthesis of the
variable domain and/or using mutagenic primers and PCR, and methods
well known in the art. The amino acid sequences of the VH and VL
regions of the humanized antibodies are disclosed in Table 8.
TABLE-US-00013 TABLE 8 Expression of humanized antibodies SEQ
Protein Sequence ID NO region 123456789012345678901234567890 62 VH
h11E6.1 EVQLVQSGSELKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFVFSLDTSVSTAYLQICSLKAED
TAVYYCARSRMVTAYGMDYWGQGTTVTVSS 63 VL h11E6.1
DIQMTQSPSSLSASVGDRVTITCKASQNVG TAVAWYQQKPGKAPKLLIYSASNRYTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ YSSYPLTFGQGTKLEIKR 62 VH h11E6.2
EVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFVFSLDTSVSTAYLQICSLKAED TAVYYCARSRMVTAYGMDYWGQGTTVTVSS 64 VL
h11E6.2 DIQMTQSPSSLSASVGDRVTITCKASQNVG
TAVAWYQQKPGKSPKLLIFSASNRYTGVPS RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
YSSYPLTFGQGTKLEIKR 62 VH h11E6.3 EVQLVQSGSELKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFVFSLDTSVSTAYLQICSLKAED
TAVYYCARSRMVTAYGMDYWGQGTTVTVSS 65 VL h11E6.3
EIVMTQSPATLSLSPGERATLSCKASQNVG TAVAWYQQKPGQAPRLLIYSASNRYTGIPA
RFSGSGSGTDFTLTISSLQSEDFAVYYCQQ YSSYPLTFGQGTKLEIKR 62 VH h11E6.4
EVQLVQSGSELKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFVFSLDTSVSTAYLQICSLKAED TAVYYCARSRMVTAYGMDYWGQGTTVTVSS 66 VL
h11E6.4 EIVMTQSPATLSLSPGERATLSCKASQNVG
TAVAWYQQKPGQSPRLLIFSASNRYTGVPA RFSGSGSGTDFTLTISSLQSEDFAVYFCQQ
YSSYPLTFGQGTKLEIKR 67 VH h11E6.5 EIQLVQSGSELKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFVFSLDTSVSTAYLQICSLKAED
TAVYFCARSRMVTAYGMDYWGQGTTVTVSS 63 VL h11E6.5
DIQMTQSPSSLSASVGDRVTITCKASQNVG TAVAWYQQKPGKAPKLLIYSASNRYTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ YSSYPLTFGQGTKLEIKR 67 VH h11E6.6
EIQLVQSGSELKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFVFSLDTSVSTAYLQICSLKAED TAVYFCARSRMVTAYGMDYWGQGTTVTVSS 64 VL
h11E6.6 DIQMTQSPSSLSASVGDRVTITCKASQNVG
TAVAWYQQKPGKSPKLLIFSASNRYTGVPS RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
YSSYPLTFGQGTKLEIKR 67 VH h11E6.7 EIQLVQSGSELKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFVFSLDTSVSTAYLQICSLKAED
TAVYFCARSRMVTAYGMDYWGQGTTVTVSS 65 VL h11E6.7
EIVMTQSPATLSLSPGERATLSCKASQNVG TAVAWYQQKPGQAPRLLIYSASNRYTGIPA
RFSGSGSGTDFTLTISSLQSEDFAVYYCQQ YSSYPLTFGQGTKLEIKR 67 VH h11E6.8
EIQLVQSGSELKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFVFSLDTSVSTAYLQICSLKAED TAVYFCARSRMVTAYGMDYWGQGTTVTVSS 66 VL
h11E6.8 EIVMTQSPATLSLSPGERATLSCKASQNVG
TAVAWYQQKPGQSPRLLIFSASNRYTGVPA RFSGSGSGTDFTLTISSLQSEDFAVYFCQQ
YSSYPLTFGQGTKLEIKR 68 VH h11E6.9 EVQLVQSGAEVKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRVTMTRDTSTSTAYMELSSLRSED
TAVYYCARSRMVTAYGMDYWGQGTSVTVSS 63 VL h11E6.9
DIQMTQSPSSLSASVGDRVTITCKASQNVG TAVAWYQQKPGKAPKLLIYSASNRYTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ YSSYPLTFGQGTKLEIKR 68 VH h11E6.10
EVQLVQSGAEVKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRVTMTRDTSTSTAYMELSSLRSED TAVYYCARSRMVTAYGMDYWGQGTSVTVSS 64 VL
h11E6.10 DIQMTQSPSSLSASVGDRVTITCKASQNVG
TAVAWYQQKPGKSPKLLIFSASNRYTGVPS RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
YSSYPLTFGQGTKLEIKR 68 VH h11E6.11 EVQLVQSGAEVKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRVTMTRDTSTSTAYMELSSLRSED
TAVYYCARSRMVTAYGMDYWGQGTSVTVSS 65 VL h11E6.11
EIVMTQSPATLSLSPGERATLSCKASQNVG TAVAWYQQKPGQAPRLLIYSASNRYTGIPA
RFSGSGSGTDFTLTISSLQSEDFAVYYCQQ YSSYPLTFGQGTKLEIKR 68 VH h11E6.12
EVQLVQSGAEVKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRVTMTRDTSTSTAYMELSSLRSED TAVYYCARSRMVTAYGMDYWGQGTSVTVSS 66 VL
h11E6.12 EIVMTQSPATLSLSPGERATLSCKASQNVG
TAVAWYQQKPGQSPRLLIFSASNRYTGVPA RFSGSGSGTDFTLTISSLQSEDFAVYFCQQ
YSSYPLTFGQGTKLEIKR 69 VH h11E6.13 EIQLVQSGAEVKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFTFTLDTSTSTAYMELSSLRSED
TAVYFCARSRMVTAYGMDYWGQGTSVTVSS 63 VL h11E6.13
DIQMTQSPSSLSASVGDRVTITCKASQNVG TAVAWYQQKPGKAPKLLIYSASNRYTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ YSSYPLTFGQGTKLEIKR 69 VH h11E6.14
EIQLVQSGAEVKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFTFTLDTSTSTAYMELSSLRSED TAVYFCARSRMVTAYGMDYWGQGTSVTVSS 64 VL
h11E6.14 DIQMTQSPSSLSASVGDRVTITCKASQNVG
TAVAWYQQKPGKSPKLLIFSASNRYTGVPS RFSGSGSGTDFTLTISSLQPEDFATYFCQQ
YSSYPLTFGQGTKLEIKR 69 VH h11E6.15 EIQLVQSGAEVKKPGASVKVSCKASGYTFT
NFGMNWVRQAPGQGLEWMGYINTNTGESIY SEEFKGRFTFTLDTSTSTAYMELSSLRSED
TAVYFCARSRMVTAYGMDYWGQGTSVTVSS 65 VL h11E6.15
EIVMTQSPATLSLSPGERATLSCKASQNVG TAVAWYQQKPGQAPRLLIYSASNRYTGIPA
RFSGSGSGTDFTLTISSLQSEDFAVYYCQQ YSSYPLTFGQGTKLEIKR 69 VH h11E6.16
EIQLVQSGAEVKKPGASVKVSCKASGYTFT NFGMNWVRQAPGQGLEWMGYINTNTGESIY
SEEFKGRFTFTLDTSTSTAYMELSSLRSED TAVYFCARSRMVTAYGMDYWGQGTSVTVSS 66 VL
h11E6.16 EIVMTQSPATLSLSPGERATLSCKASQNVG
TAVAWYQQKPGQSPRLLIFSASNRYTGVPA RFSGSGSGTDFTLTISSLQSEDFAVYFCQQ
YSSYPLTFGQGTKLEIKR
[0322] Specifically, for the heavy chains:
[0323] VH h11E6.1, VH h11E6.2, VH h11E6.3, and VH h11E6.4 contain
VH 11E6.1-GL, with a Q1.fwdarw.E mutation.
[0324] VH h11E6.5, VH h11E6.6, VH h11E6.7 and VH h11E6.8 contain VH
11E6.1-GL, with a Q1.fwdarw.E mutation and the following Vernier
and VH/VL interfacing residue back mutations: V2.fwdarw.I and
Y95.fwdarw.F.
[0325] VH h11E6.9, VH h11E6.10, VH h11E6.11, and VH h11E6.12
contain VH 11E6.2-GL, with a Q1.fwdarw.E, I76.fwdarw.T,
R85.fwdarw.S, and D89.fwdarw.E mutation.
[0326] VH h11E6.13, VH h11E6.14, VH h11E6.15, and VH h11E6.16
contain VH 11E6.2-GL, with a Q1.fwdarw.E, I76.fwdarw.T,
R85.fwdarw.S, D89.fwdarw.E mutation and the following Vernier and
VH/VL interfacing residue back mutations: V2.fwdarw.I,
V68.fwdarw.F, M70.fwdarw.F, R72.fwdarw.L, and Y95.fwdarw.F.
[0327] For the light chains:
[0328] VL h11E6.1, VL h11E6.5, VL h11E6.9, and VL h11E6.13 contain
VL 11E6.1-GL with a V11.fwdarw.L mutation.
[0329] VL h11E6.2, VL h11E6.6, VL h11E6.10, and VL h11E6.14 contain
VL 11E6.1-GL with a V11.fwdarw.L mutation and the following Vernier
and VH/VL interfacing residue back mutations: A43.fwdarw.S,
Y49.fwdarw.F, and Y87.fwdarw.F.
[0330] VL h11E6.3, VL h11E6.7, VL h11E6.11, and VL h11E6.15 contain
VL 11E6.2-GL with V13.fwdarw.L and E70.fwdarw.D mutations.
[0331] VL h11E6.4, VL h11E6.8, VL h11E6.12, and VL h11E6.16 contain
VL 11E6.2-GL with V13.fwdarw.L and E70.fwdarw.D mutations and the
following Vernier and VH/VL interfacing residue back mutations:
A43.fwdarw.S, Y49.fwdarw.F, I58.fwdarw.V, and V87.fwdarw.F.
Example 10
Characterization of Humanized 11E6 Antibodies Using Competition
ELISA
[0332] ELISA plates (Costar 3369) were coated overnight at
4.degree. C. with 50 .mu.l/well of 2 .mu.g/ml hRAGE (1-331) in 0.2
M sodium carbonate-bicarbonate buffer, pH 9.4, washed with Wash
Buffer (PBS containing 0.1% Tween 20), and blocked for 1 hr at room
temperature with 200 .mu.l/well of 2% nonfat dry milk in PBS. After
washing with Wash Buffer, a mixture of a biotinylated m11E6 (0.3
.mu.g/ml final concentration) and unlabelled competitor test
antibody starting at 81 .mu.g/ml final concentration and serially
diluted 3-fold) in 50 .mu.l/well of ELISA buffer was added in
duplicate. After incubating the plates for 1 hr at room
temperature, and washing with Wash Buffer, bound antibodies were
detected using 100 .mu.l/well of 1:10,000 dilution of
HRP-conjugated streptavidin (Fitzgerald) in ELISA buffer. After
incubating for 1 hr at room temperature, and washing with Wash
Buffer, color development was performed by adding 100 .mu.l/well of
TMB Buffer (Zymed). After incubating for 15 min at room
temperature, color development was stopped by adding 50 .mu.l/well
of 1N hydrochloric acid. Absorbance was read at 490 nm Table 9
shows the IC.sub.50 values of humanized 11E6 antibodies obtained
using the computer software GraphPad Prism (GraphPad Software Inc.,
San Diego, Calif.).
TABLE-US-00014 TABLE 9 IC.sub.50 values of humanized 11E6
antibodies in a competitive ELISA Antibody IC.sub.50 (nM) h11E6.1
19.7 h11E6.2 N/A h11E6.3 18.8 h11E6.4 14.1 h11E6.5 16.1 h11E6.6 N/A
h11E6.7 15.5 h11E6.8 10.8 h11E6.9 17.9 h11E6.10 N/A h11E6.11 19.9
h11E6.12 11.3 h11E6.13 16.8 h11E6.14 N/A h11E6.15 14.0 h11E6.16
9.2
Example 11
Determination of Binding Constants for Anti-RAGE mAb Interaction
with RAGE
[0333] Biacore 2000 and Biacore T100 instruments were used to
measure anti-RAGE mAb binding kinetics. The assay format for mAb
affinity analysis was Fc-based capture via immobilized anti-Fc
antibodies. A standard amine coupling protocol was employed to
immobilize Fc-specific IgG via primary amines to the carboxy-methyl
(CM) dextran surface of CM5 sensorchips (Biacore). For the study of
mouse anti-RAGE mAbs, anti-mouse Fc (Biacore, anti-mouse,
BR-1005-14) was used as the immobilized capture reagent and for the
study of humanized anti-RAGE mAbs, anti-human Fc (Pierce 31125) was
used as the immobilized capture reagent. An automated protocol,
available on the Biacore 2000 and Biacore T100, was used to
immobilize 8000-10000 RU of capture reagent in all 4 flowcells of
the sensorchip. Briefly, the CM-dextran surfaces were activated by
freshly prepared 1:1 50 mM N-hydroxysuccinimide (NHS):200 mM
3-(N,N-dimethylamino) propyl-N-ethylcarbodiimide (EDC). Then the
anti-Fc IgG capture reagent (20 ug/ml in 10 mM sodium acetate,
pH4.5) was applied to the surface followed by deactivation of the
surface and blocking of the residual reactive sites with 1M
ethanolamine (pH 8.5).
[0334] The running buffer employed was PBS-P [1.times.PBS (Sigma
P3813), pH 7.4, 0.005% P20 surfactant (Biacore)] for the humanized
antibodies. Each experimental cycle consisted of the following
steps: 1) anti-RAGE mAbs were captured in flowcells 2, 3 or 4 to a
level of 50-200 RU (depending on the antigen). All measurements
were referenced against flowcell 1 which had no captured anti-RAGE
mAb. 2) Antigen was injected through all 4 flowcells, 240 ul at 80
ul/min After the antigen injection, dissociation was monitored for
600 seconds at 80 ul/min 3) The anti-Fc capture surface was
regenerated with low pH glycine. For kinetic determinations,
antigen injections were a 3 fold dilution series from either 30
nM-0.12 nM (for sRAGE [RAGE (1-331)] and buffer only in randomized
duplicates.
[0335] Data were processed using either Biacore evaluation software
or Scrubber 2.0 software (BioLogic Software). Briefly, the data
were double referenced by first, subtracting the signal from the
reference cell and second, by subtracting the signal from
buffer-only injections. The double referenced data for the RAGE
injection series were then fit globally to a 1:1 (Langmuir) binding
model, which included a mass transfer term, to determine the
binding kinetic rate constants, k.sub.a and k.sub.d, and the
affinity, K.sub.D (k.sub.a=k.sub.on; k.sub.a=k.sub.off)
TABLE-US-00015 TABLE 10 Biacore data 11E6 mAb k.sub.on (M.sup.-1
s.sup.-1) k.sub.off (s.sup.-1) K.sub.D (pM) Resid Stdev h11E6.8
2.5E+07 8.4E-04 33 1.6 h11E6.12 2.0E+07 1.7E-03 83 1.8 h11E6.16
2.8E+07 1.4E-03 50 1.9 mouse 11E6 1.8E+07 1.3E-03 68 1.4
The K.sub.D values are double-digit pM for all three mAbs and there
does not seem to be a significant distinction between the three
mAbs regarding their binding kinetics.
[0336] In a concurrent experiment, the original mouse 11E6 mAb was
evaluated with this antigen (human sRAGE 1-331, V#400) and was also
double-digit pM K.sub.D.
[0337] The original mouse 11E6 mAb was previously reported to be
290 pM. However those early experiments used a different buffer
system (Biacore buffer HBS-EP+: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3
mM EDTA, 0.05% P20). The buffer choice can, of course, affect the
kinetics.
Example 12
Determination of Effect of RAGE Antibody on Chronic Constriction
Injury Model of Neuropathic Pain in Mice
12.1 Animals
[0338] Adult male mice, C57/B16 (Charles River Laboratories,
Wilmington, Mass., 250-300 g, n=44) were used in experiments
according to the internal Institutional Animal Care and Use
Committee guidelines. Housing conditions were
temperature-regulated, and under a controlled 12-h light-dark
cycle. Food and water were available ad libitum, except during
testing.
12.2 Chronic Constriction Injury Model of Neuropathic Pain
[0339] Chronic constriction injury (CCI) to the sciatic nerve was
performed according to the method described by Bennett and Xie
(1988). Mice were placed under isoflurane anesthesia and the right
common sciatic nerve was isolated at mid-thigh level. The exposed
sciatic nerve was loosely ligated by 4 chromic gut (5-0) ties
separated by an interval of 1 mm Behavioral testing for mechanical
threshold was performed two weeks post CCI injury.
12.3 Behavioral Testing
[0340] Mechanical threshold was measured using calibrated von Frey
monofilaments (Stoelting, Wood Dale, Ill.). Paw withdrawal
threshold (PWT) was determined by increasing and decreasing
stimulus intensity, and estimated using the Dixon's up-down method
(Dixon et al., 1980). Mice were placed individually in inverted
plastic containers (20.times.12.5.times.20 cm) on top of a
suspended wire mesh with a 1 cm.sup.2 grid to provide access to the
ventral side of the hind paws. Mice were acclimated to the chambers
prior to testing. Monofilaments were presented perpendicularly to
the plantar surface of the selected hind paw with enough force to
cause a slight bend in the filament. Threshold was defined by a
withdrawal of the hind paw from the stimulus. CCI-injured mice with
threshold scores .ltoreq.0.13 g were considered allodynic, and used
in these experiments. Data are expressed as % effect, where 0% was
equal to vehicle and 100% was equal to the response measured from
the contralateral paw.
12.4 Treatment Groups and Dosing
[0341] Monoclonal antibody mAb ML39-11E6 (in PBS) was administered
by i.p. injection to mice with allodynia from the chronic
constriction injury. Mice received either 30 .mu.g/mouse (n=11) or
100 .mu.g/mouse (n=11) of ML39-11E6 i.p. or 100 .mu.g/mouse of
control IgG-1 H35C206 (in PBS, n=11) i.p. on days 1, 3, and 5 of
the experiment. Mechanical threshold was measured sixty minutes
after dosing on days 1 and 5. Mice were re-tested again on day 8,
72 hours following the last dose of ML39-11E6. Gabapentin (positive
control, 100 mg/kg in water, i.p., n=11) was injected acutely 60
minutes prior to testing on days 1, 5, and 8.
12.5 Data Analysis
[0342] Data were analyzed and graphs were generated using Prism
GraphPad (GraphPad Software, Inc., San Diego, Calif.). Data were
analyzed using repeat measures one-way analysis of variance (ANOVA)
test. Statistical significance was assumed if P<0.05 (on graph,
*P<0.05, **P<0.01).
12.6 Results
[0343] RAGE antibody, ML39-11E6 attenuated established mechanical
allodynia in the chronic constriction injury model of neuropathic
pain. The anti-nociceptive efficacy was dose-dependent, observed in
a sub-chronic dosing paradigm, and efficacy was maintained 72 hours
following the last dose. Anti-nociceptive efficacy of RAGE antibody
in the CCI pain model was observed following systemic
administration, with limited permeation of the blood-brain barrier,
and is likely a peripheral mechanism of action. The results are
further illustrated in FIG. 5.
Example 13
Duration of Anti-Nociceptive Efficacy was Measured Following a
Single Dose (30, 100 or 300 Ug/Mouse) of Mouse 11E6 in Inflammatory
and Neuropathic Pain Models
[0344] Mice (C57BL/6--available from Charles River Laboratories,
Inc., Wilmington, MA250-300 g, N=10 per group) were injected with
complete Freund's adjuvant (CFA) and inflammation developed over 24
hours. At 24 hours post-CFA, a single dose of mouse 11E6 (also
known as ML39-11E6) (30, 100 or 300 .mu.g/mouse), or negative
control IgG (KLH mAB H35C206-100 .mu.g/mouse was injected. Paw
withdrawal threshold was measured using von Frey monofilaments
(available from Stoetling Co., Wood Dale, Ill.) sixty minutes, post
injection. Dose-dependent analgesic efficacy of mouse 11E6 was
observed in the CFA model of inflammatory pain following acute
dosing (sixty minutes). The results are shown in FIG. 6. The
results in FIG. 6 show an effect on acute neurotransmission.
Example 14
Duration of Anti-Nociceptive Efficacy was Measured Following a
Single Dose (300 Ug/Mouse) of Mouse and Human 11E6, Anti-RAGE
Antibodies, in the Chronic Constriction Injury Model Neuropathic
Pain
[0345] Chronic constriction injury (CCI)) model of neuropathic pain
was performed in mice (C57BL/6-250-300 g, N=5) and developed over 7
days. On the seventh day, mouse 11E6.50 (also known as ms11E6,
A-992401.0 and 11E6.50) (300 .mu.g/mouse), human mouse 11E6 (also
known as hu11E6; A-1175708.0 and 11E6.12hu), (300 .mu.g/mouse), or
negative control IgG was injected. Paw withdrawal threshold was
measured using von Frey monofilaments at 24 hours post injection.
Analgesic efficacy of mouse (ms11E6) and human 11E6 (hu11E6) was
observed in the CCI model of neuropathic pain at 24 hours following
a single dose treatment). The results are shown in FIG. 7.
[0346] Documents as cited herein are incorporated by reference.
Sequence CWU 1
1
971119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Glu Lys Leu Glu Glu Ser Gly Gly Gly Leu
Val Gln Leu Gly Gly 1 5 10 15 Ser Met Lys Ile Ser Cys Val Ala Ser
Gly Phe Thr Leu Ser Asn Tyr 20 25 30 Trp Met Asp Trp Val Arg Gln
Ser Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45 Ala Glu Ile Arg Leu
Lys Ser Asn Tyr Tyr Ser Thr His Tyr Ala Glu 50 55 60 Ser Val Lys
Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser Lys Gly Ser 65 70 75 80 Val
Ser Leu Gln Met Asp Asn Leu Thr Ala Glu Asp Thr Gly Ile Tyr 85 90
95 Phe Cys Ala Arg Asn Ala Tyr Trp Tyr Phe Asp Val Trp Gly Thr Gly
100 105 110 Thr Thr Val Thr Val Ser Ser 115 25PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Asn
Tyr Trp Met Asp 1 5 319PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Glu Ile Arg Leu Lys Ser Asn
Tyr Tyr Ser Thr His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly
48PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Asn Ala Tyr Trp Tyr Phe Asp Val 1 5
5108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ala Thr Cys Lys Ala
Ser Gln Asp Val Gly Thr Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Leu Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr
Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu
Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90
95 Thr Phe Gly Asp Gly Thr Lys Leu Glu Leu Lys Arg 100 105
611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala 1 5
10 77PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Trp Thr Ser Thr Arg His Thr 1 5 89PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Gln
Gln Tyr Asn Asn Tyr Pro Leu Thr 1 5 9120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1
5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Phe 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu
Lys Trp Met 35 40 45 Gly Tyr Ile Asn Thr Asn Thr Gly Glu Ser Ile
Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu
Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys
Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Ser Arg Met
Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser
Val Thr Val Ser Ser 115 120 105PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Asn Phe Gly Met Asn 1 5
1117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Tyr Ile Asn Thr Asn Thr Gly Glu Ser Ile Tyr Ser
Glu Glu Phe Lys 1 5 10 15 Gly 1211PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 12Ser Arg Met Val Thr Ala
Tyr Gly Met Asp Tyr 1 5 10 13108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 13Asp Ile Val Met Thr
Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val
Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45 Phe Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Leu Ser Asn Met
Gln Pro 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser
Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Val Gly Thr Lys Leu Glu Leu
Lys Arg 100 105 1411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Lys Ala Ser Gln Asn Val Gly Thr Ala
Val Ala 1 5 10 157PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 15Ser Ala Ser Asn Arg Tyr Thr 1 5
169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gln Gln Tyr Ser Ser Tyr Pro Leu Thr 1 5
17120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Asn Asn Tyr 20 25 30 Leu Ile Glu Trp Ile Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro
Gly Ser Gly Gly Thr Asn His Asn Glu Lys Phe 50 55 60 Lys Val Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile
Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Ser Ala Gly Thr Ala Arg Ala Arg Phe Ala Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120
185PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Asn Tyr Leu Ile Glu 1 5 1917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Val
Ile Asn Pro Gly Ser Gly Gly Thr Asn His Asn Glu Lys Phe Lys 1 5 10
15 Val 2011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Ser Ala Gly Thr Ala Arg Ala Arg Phe Ala Tyr 1 5
10 21108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala
Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Glu
Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser
Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser
Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu
Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Phe Pro Phe 85 90
95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105
2211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn 1 5
10 237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Ala Thr Ser Ser Leu Asp Ser 1 5
249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Leu Gln Tyr Ala Ser Phe Pro Phe Thr 1 5
25330PRTHomo sapiens 25Ala Ser Thr Lys Gly Pro Ser Val Phe Phe Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 26106PRTHomo sapiens 26Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10
15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 276276DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 27tggcgaatgg
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaat cgatagattg 1260tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
ctcataacac 1380cccttgtatt actgtttatg taagcagaca gttttattgt
tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag 1680aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 1860accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt 1980ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca
tatatggtgc actctcagta 2340caatctgctc tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 2460gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
agctcgttga gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg
ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt
aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg
atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca
cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca
gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga
ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt
cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg
gtggcgggac cagtgacgaa 3240ggcttgagcg agggcgtgca agattccgaa
taccgcaagc gacaggccga tcatcgtcgc 3300gctccagcga aagcggtcct
cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360gagttgcatg
ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
3420ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
ccggtgccta 3480atgagtgagc taacttacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 3540cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 3600tgggcgccag ggtggttttt
cttttcacca gtgagacggg caacagctga ttgcccttca 3660ccgcctggcc
ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
3720aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
gtatcgtcgt 3780atcccactac cgagatgtcc gcaccaacgc gcagcccgga
ctcggtaatg gcgcgcattg 3840cgcccagcgc catctgatcg ttggcaacca
gcatcgcagt gggaacgatg ccctcattca 3900gcatttgcat ggtttgttga
aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960tcggctgaat
ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
4020agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
gcgaccagat 4080gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
aatactgttg atgggtgtct 4140ggtcagagac atcaagaaat aacgccggaa
cattagtgca ggcagcttcc acagcaatgg 4200catcctggtc atccagcgga
tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260tgtgcaccgc
cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
4320tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
ggcgcgtgca 4380gggccagact ggaggtggca acgccaatca gcaacgactg
tttgcccgcc agttgttgtg 4440ccacgcggtt gggaatgtaa ttcagctccg
ccatcgccgc ttccactttt tcccgcgttt 4500tcgcagaaac gtggctggcc
tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560catactctgc
gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
4620cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980gcgaaattaa
tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
5040ataattttgt ttaactttaa gaaggagata tacatatgaa aaagacagct
atcgcgattg 5100cagtggcact ggctggtttc gctaccgtag cgcaggccgc
tcaaaacatc acagcccgga 5160ttggcgagcc actggtgctg aagtgtaagg
gggcccccaa gaaaccaccc cagcggctgg 5220aatggaaact gaacacaggc
cggacagaag cttggaaggt cctgtctccc cagggaggag 5280gcccctggga
cagtgtggct cgtgtccttc ccaacggctc cctcttcctt ccggctgtcg
5340ggatccagga tgaggggatt ttccggtgcc aggcaatgaa caggaatgga
aaggagacca 5400agtccaacta ccgagtccgt gtctaccaga ttcctgggaa
gccagaaatt gtagattctg 5460cctctgaact cacggctggt gttcccaata
aggtggggac atgtgtgtca gagggaagct 5520accctgcagg gactcttagc
tggcacttgg atgggaagcc cctggtgcct aatgagaagg 5580gagtatctgt
gaaggaacag accaggagac accctgagac agggctcttc acactgcagt
5640cggagctaat ggtgacccca gcccggggag gagatccccg tcccaccttc
tcctgtagct 5700tcagcccagg ccttccccga caccgggcct tgcgcacagc
ccccatccag ccccgtgtct 5760gggagcctgt gcctctggag gaggtccaat
tggtggtgga gccagaaggt ggagcagtag 5820ctcctggtgg aaccgtaacc
ctgacctgtg aagtccctgc ccagccctct cctcaaatcc 5880actggatgaa
ggatggtgtg cccttgcccc ttccccccag ccctgtgctg atcctccctg
5940agatagggcc tcaggaccag ggaacctaca gctgtgtggc cacccattcc
agccacgggc 6000cccaggaaag ccgtgctgtc agcatcagca tcatcgaacc
aggcgaggag gggccaactg 6060caggctctgt gggaggatca gggctgggaa
ctcaccacca ccaccaccac tgactcgagc 6120accaccacca ccaccactga
gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg 6180ctgctgccac
cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
6240ggggtttttt gctgaaagga ggaactatat ccggat 6276285617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaat cgatagattg 1260tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
ctcataacac 1380cccttgtatt actgtttatg taagcagaca gttttattgt
tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag 1680aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 1860accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt 1980ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca
tatatggtgc actctcagta 2340caatctgctc tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 2460gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
agctcgttga gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg
ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt
aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg
atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca
cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca
gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga
ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt
cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg
gtggcgggac cagtgacgaa 3240ggcttgagcg agggcgtgca agattccgaa
taccgcaagc gacaggccga tcatcgtcgc 3300gctccagcga aagcggtcct
cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360gagttgcatg
ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
3420ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
ccggtgccta 3480atgagtgagc taacttacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 3540cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 3600tgggcgccag ggtggttttt
cttttcacca gtgagacggg caacagctga ttgcccttca 3660ccgcctggcc
ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
3720aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
gtatcgtcgt 3780atcccactac cgagatatcc gcaccaacgc gcagcccgga
ctcggtaatg gcgcgcattg 3840cgcccagcgc catctgatcg ttggcaacca
gcatcgcagt gggaacgatg ccctcattca 3900gcatttgcat ggtttgttga
aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960tcggctgaat
ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
4020agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
gcgaccagat 4080gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
aatactgttg atgggtgtct 4140ggtcagagac atcaagaaat aacgccggaa
cattagtgca ggcagcttcc acagcaatgg 4200catcctggtc atccagcgga
tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260tgtgcaccgc
cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
4320tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
ggcgcgtgca 4380gggccagact ggaggtggca acgccaatca gcaacgactg
tttgcccgcc agttgttgtg 4440ccacgcggtt gggaatgtaa ttcagctccg
ccatcgccgc ttccactttt tcccgcgttt 4500tcgcagaaac gtggctggcc
tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560catactctgc
gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
4620cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
gtgtccggga 4680tctcgacgct ctcccttatg cgactcctgc attaggaagc
agcccagtag taggttgagg 4740ccgttgagca ccgccgccgc aaggaatggt
gcatgcaagg agatggcgcc caacagtccc 4800ccggccacgg ggcctgccac
catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860cgagcccgat
cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
4920gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
gatcccgcga 4980aattaatacg actcactata ggggaattgt gagcggataa
caattcccct ctagaaataa 5040ttttgtttaa ctttaagaag gagatatacc
atgggcagca gccatcatca tcatcatcac 5100agcagcggcc tggtgccgcg
cggcagccat atgcaaaaca tcacagcccg gattggcgag 5160ccactggtgc
tgaagtgtaa gggggccccc aagaaaccac cccagcggct ggaatggaaa
5220ctgaacacag gccggacaga agcttggaag gtcctgtctc cccagggagg
aggcccctgg 5280gacagtgtgg ctcgtgtcct tcccaacggc tccctcttcc
ttccggctgt cgggatccag 5340gatgagggga ttttccggtg ccaggcaatg
aacaggaatg gaaaggagac caagtccaac 5400taccgagtcc gtgtctacca
gattcctggg aagccagaaa ttgtagattc ttgactcgag 5460caccaccacc
accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
5520gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
acgggtcttg 5580aggggttttt tgctgaaagg aggaactata tccggat
5617295932DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 29tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg
gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt
ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
tgcaatttat 600tcatatcagg attatcaata ccatattttt gaaaaagccg
tttctgtaat gaaggagaaa 660actcaccgag gcagttccat aggatggcaa
gatcctggta tcggtctgcg attccgactc 720gtccaacatc aatacaacct
attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780aatcaccatg
agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
840agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac 900cgttattcat tcgtgattgc gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac 960aattacaaac aggaatcgaa tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat 1020tttcacctga atcaggatat
tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080tggtgagtaa
ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1140taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
gcaacgctac 1200ctttgccatg tttcagaaac aactctggcg catcgggctt
cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca ttatcgcgag
cccatttata cccatataaa tcagcatcca 1320tgttggaatt taatcgcggc
ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380cccttgtatt
actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680aactctgtag
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1740agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc acacagccca
gcttggagcg aacgacctac 1860accgaactga gatacctaca gcgtgagcta
tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980ccagggggaa
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280tattttctcc
ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2340caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac acccgctgac
gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg tcatcaccga
aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580gtgaagcgat
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt tctgttcatg
ggggtaatga taccgatgaa 2760acgagagagg atgctcacga tacgggttac
tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt aaacaactgg
cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880tcaatgccag
cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2940tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct cgcgtatcgg
tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct agccgggtcc
tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180catgccggcg
ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
3240ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
tcatcgtcgc 3300gctccagcga aagcggtcct cgccgaaaat gacccagagc
gctgccggca cctgtcctac 3360gagttgcatg ataaagaaga cagtcataag
tgcggcgacg atagtcatgc cccgcgccca 3420ccggaaggag ctgactgggt
tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480atgagtgagc
taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
3540cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 3600tgggcgccag ggtggttttt cttttcacca gtgagacggg
caacagctga ttgcccttca 3660ccgcctggcc ctgagagagt tgcagcaagc
ggtccacgct ggtttgcccc agcaggcgaa 3720aatcctgttt gatggtggtt
aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780atcccactac
cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
3840cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
ccctcattca 3900gcatttgcat ggtttgttga aaaccggaca tggcactcca
gtcgccttcc cgttccgcta 3960tcggctgaat ttgattgcga gtgagatatt
tatgccagcc agccagacgc agacgcgccg 4020agacagaact taatgggccc
gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080gctccacgcc
cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
4140ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
acagcaatgg 4200catcctggtc atccagcgga tagttaatga tcagcccact
gacgcgttgc gcgagaagat 4260tgtgcaccgc cgctttacag gcttcgacgc
cgcttcgttc taccatcgac accaccacgc 4320tggcacccag ttgatcggcg
cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380gggccagact
ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
4440ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
tcccgcgttt 4500tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
ggtctgataa gagacaccgg 4560catactctgc gacatcgtat aacgttactg
gtttcacatt caccaccctg aattgactct 4620cttccgggcg ctatcatgcc
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg
actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5040ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
tcatcatcac 5100agcagcggcc tggtgccgcg cggcagccat atgcaaaaca
tcacagcccg gattggcgag 5160ccactggtgc tgaagtgtaa gggggccccc
aagaaaccac cccagcggct ggaatggaaa 5220ctgaacacag gccggacaga
agcttggaag gtcctgtctc cccagggagg aggcccctgg 5280gacagtgtgg
ctcgtgtcct tcccaacggc tccctcttcc ttccggctgt cgggatccag
5340gatgagggga ttttccggtg ccaggcaatg aacaggaatg gaaaggagac
caagtccaac 5400taccgagtcc gtgtctacca gattcctggg aagccagaaa
ttgtagattc tgcctctgaa 5460ctcacggctg gtgttcccaa taaggtgggg
acatgtgtgt cagagggaag ctaccctgca 5520gggactctta gctggcactt
ggatgggaag cccctggtgc ctaatgagaa gggagtatct 5580gtgaaggaac
agaccaggag acaccctgag acagggctct tcacactgca gtcggagcta
5640atggtgaccc cagcccgggg aggagatccc cgtcccacct tctcctgtag
cttcagccca 5700ggccttcccc gacaccgggc cttgcgcaca gcccccatcc
agccccgtgt ctgggagcct 5760gtgccttgac tcgagcacca ccaccaccac
cactgagatc cggctgctaa caaagcccga 5820aaggaagctg agttggctgc
tgccaccgct gagcaataac tagcataacc ccttggggcc 5880tctaaacggg
tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at
5932306238DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 30tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg
gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt
ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
tgcaatttat 600tcatatcagg attatcaata ccatattttt gaaaaagccg
tttctgtaat gaaggagaaa 660actcaccgag gcagttccat aggatggcaa
gatcctggta tcggtctgcg attccgactc 720gtccaacatc aatacaacct
attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780aatcaccatg
agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
840agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac 900cgttattcat tcgtgattgc gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac 960aattacaaac aggaatcgaa tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat 1020tttcacctga atcaggatat
tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080tggtgagtaa
ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1140taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
gcaacgctac 1200ctttgccatg tttcagaaac aactctggcg catcgggctt
cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca ttatcgcgag
cccatttata cccatataaa tcagcatcca 1320tgttggaatt taatcgcggc
ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380cccttgtatt
actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680aactctgtag
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1740agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc acacagccca
gcttggagcg aacgacctac 1860accgaactga gatacctaca gcgtgagcta
tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980ccagggggaa
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280tattttctcc
ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2340caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac acccgctgac
gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg tcatcaccga
aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580gtgaagcgat
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt tctgttcatg
ggggtaatga taccgatgaa 2760acgagagagg atgctcacga tacgggttac
tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt aaacaactgg
cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880tcaatgccag
cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2940tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct cgcgtatcgg
tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct agccgggtcc
tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180catgccggcg
ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
3240ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
tcatcgtcgc 3300gctccagcga aagcggtcct cgccgaaaat gacccagagc
gctgccggca cctgtcctac 3360gagttgcatg ataaagaaga cagtcataag
tgcggcgacg atagtcatgc cccgcgccca 3420ccggaaggag ctgactgggt
tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480atgagtgagc
taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
3540cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 3600tgggcgccag ggtggttttt cttttcacca gtgagacggg
caacagctga ttgcccttca 3660ccgcctggcc ctgagagagt tgcagcaagc
ggtccacgct ggtttgcccc agcaggcgaa 3720aatcctgttt gatggtggtt
aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780atcccactac
cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
3840cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
ccctcattca 3900gcatttgcat ggtttgttga aaaccggaca tggcactcca
gtcgccttcc cgttccgcta 3960tcggctgaat ttgattgcga gtgagatatt
tatgccagcc agccagacgc agacgcgccg 4020agacagaact taatgggccc
gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080gctccacgcc
cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
4140ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
acagcaatgg 4200catcctggtc atccagcgga tagttaatga tcagcccact
gacgcgttgc gcgagaagat 4260tgtgcaccgc cgctttacag gcttcgacgc
cgcttcgttc taccatcgac accaccacgc 4320tggcacccag ttgatcggcg
cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380gggccagact
ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
4440ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
tcccgcgttt 4500tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
ggtctgataa gagacaccgg 4560catactctgc gacatcgtat aacgttactg
gtttcacatt caccaccctg aattgactct 4620cttccgggcg ctatcatgcc
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg
actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5040ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
tcatcatcac 5100agcagcggcc tggtgccgcg cggcagccat atgcaaaaca
tcacagcccg gattggcgag 5160ccactggtgc tgaagtgtaa gggggccccc
aagaaaccac cccagcggct ggaatggaaa 5220ctgaacacag gccggacaga
agcttggaag gtcctgtctc cccagggagg aggcccctgg 5280gacagtgtgg
ctcgtgtcct tcccaacggc tccctcttcc ttccggctgt cgggatccag
5340gatgagggga ttttccggtg ccaggcaatg aacaggaatg gaaaggagac
caagtccaac 5400taccgagtcc gtgtctacca gattcctggg aagccagaaa
ttgtagattc tgcctctgaa 5460ctcacggctg gtgttcccaa taaggtgggg
acatgtgtgt cagagggaag ctaccctgca 5520gggactctta gctggcactt
ggatgggaag cccctggtgc ctaatgagaa gggagtatct 5580gtgaaggaac
agaccaggag acaccctgag acagggctct tcacactgca gtcggagcta
5640atggtgaccc cagcccgggg aggagatccc cgtcccacct tctcctgtag
cttcagccca 5700ggccttcccc gacaccgggc cttgcgcaca gcccccatcc
agccccgtgt ctgggagcct 5760gtgcctctgg aggaggtcca attggtggtg
gagccagaag gtggagcagt agctcctggt 5820ggaaccgtaa ccctgacctg
tgaagtccct gcccagccct ctcctcaaat ccactggatg 5880aaggatggtg
tgcccttgcc ccttcccccc agccctgtgc tgatcctccc tgagataggg
5940cctcaggacc agggaaccta cagctgtgtg gccacccatt ccagccacgg
gccccaggaa 6000agccgtgctg tcagcatcag catcatcgaa ccaggcgagg
aggggccaac tgcaggctct 6060gtgggaggat catgactcga gcaccaccac
caccaccact gagatccggc tgctaacaaa 6120gcccgaaagg aagctgagtt
ggctgctgcc accgctgagc aataactagc ataacccctt 6180ggggcctcta
aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggat
6238315614DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 31tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg
gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt
ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
tgcaatttat 600tcatatcagg attatcaata ccatattttt gaaaaagccg
tttctgtaat gaaggagaaa 660actcaccgag gcagttccat aggatggcaa
gatcctggta tcggtctgcg attccgactc 720gtccaacatc aatacaacct
attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780aatcaccatg
agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
840agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac 900cgttattcat tcgtgattgc gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac 960aattacaaac aggaatcgaa tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat 1020tttcacctga atcaggatat
tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080tggtgagtaa
ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1140taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
gcaacgctac 1200ctttgccatg tttcagaaac aactctggcg catcgggctt
cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca ttatcgcgag
cccatttata cccatataaa tcagcatcca 1320tgttggaatt taatcgcggc
ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380cccttgtatt
actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680aactctgtag
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1740agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc acacagccca
gcttggagcg aacgacctac 1860accgaactga gatacctaca gcgtgagcta
tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980ccagggggaa
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280tattttctcc
ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2340caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac acccgctgac
gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg tcatcaccga
aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580gtgaagcgat
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt tctgttcatg
ggggtaatga taccgatgaa 2760acgagagagg atgctcacga tacgggttac
tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt aaacaactgg
cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880tcaatgccag
cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2940tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct cgcgtatcgg
tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct agccgggtcc
tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180catgccggcg
ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
3240ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
tcatcgtcgc 3300gctccagcga aagcggtcct cgccgaaaat gacccagagc
gctgccggca cctgtcctac 3360gagttgcatg ataaagaaga cagtcataag
tgcggcgacg atagtcatgc cccgcgccca 3420ccggaaggag ctgactgggt
tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480atgagtgagc
taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
3540cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 3600tgggcgccag ggtggttttt cttttcacca gtgagacggg
caacagctga ttgcccttca 3660ccgcctggcc ctgagagagt tgcagcaagc
ggtccacgct ggtttgcccc agcaggcgaa 3720aatcctgttt gatggtggtt
aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780atcccactac
cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
3840cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
ccctcattca 3900gcatttgcat ggtttgttga aaaccggaca tggcactcca
gtcgccttcc cgttccgcta 3960tcggctgaat ttgattgcga gtgagatatt
tatgccagcc agccagacgc agacgcgccg 4020agacagaact taatgggccc
gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080gctccacgcc
cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
4140ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
acagcaatgg 4200catcctggtc atccagcgga tagttaatga tcagcccact
gacgcgttgc gcgagaagat 4260tgtgcaccgc cgctttacag gcttcgacgc
cgcttcgttc taccatcgac accaccacgc 4320tggcacccag ttgatcggcg
cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380gggccagact
ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
4440ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
tcccgcgttt 4500tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
ggtctgataa gagacaccgg 4560catactctgc gacatcgtat aacgttactg
gtttcacatt caccaccctg aattgactct 4620cttccgggcg ctatcatgcc
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg
actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5040ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
tcatcatcac 5100agcagcggcc tggtgccgcg cggcagccat atggcctctg
aactcacggc tggtgttccc 5160aataaggtgg ggacatgtgt gtcagaggga
agctaccctg cagggactct tagctggcac 5220ttggatggga agcccctggt
gcctaatgag aagggagtat ctgtgaagga acagaccagg 5280agacaccctg
agacagggct cttcacactg cagtcggagc taatggtgac cccagcccgg
5340ggaggagatc cccgtcccac cttctcctgt agcttcagcc caggccttcc
ccgacaccgg 5400gccttgcgca cagcccccat ccagccccgt gtctgggagc
ctgtgccttg actcgagcac 5460caccaccacc accactgaga tccggctgct
aacaaagccc gaaaggaagc tgagttggct 5520gctgccaccg ctgagcaata
actagcataa ccccttgggg cctctaaacg ggtcttgagg 5580ggttttttgc
tgaaaggagg aactatatcc ggat 5614325920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
32tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaat cgatagattg 1260tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
ctcataacac 1380cccttgtatt actgtttatg taagcagaca gttttattgt
tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag 1680aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 1860accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt 1980ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca
tatatggtgc actctcagta 2340caatctgctc tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 2460gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
agctcgttga gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg
ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt
aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg
atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca
cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca
gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga
ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt
cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg
gtggcgggac cagtgacgaa 3240ggcttgagcg agggcgtgca agattccgaa
taccgcaagc gacaggccga tcatcgtcgc 3300gctccagcga aagcggtcct
cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360gagttgcatg
ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
3420ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
ccggtgccta 3480atgagtgagc taacttacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 3540cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 3600tgggcgccag ggtggttttt
cttttcacca gtgagacggg caacagctga ttgcccttca 3660ccgcctggcc
ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
3720aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
gtatcgtcgt 3780atcccactac cgagatatcc gcaccaacgc gcagcccgga
ctcggtaatg gcgcgcattg 3840cgcccagcgc catctgatcg ttggcaacca
gcatcgcagt gggaacgatg ccctcattca 3900gcatttgcat ggtttgttga
aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960tcggctgaat
ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
4020agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
gcgaccagat 4080gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
aatactgttg atgggtgtct 4140ggtcagagac atcaagaaat aacgccggaa
cattagtgca ggcagcttcc acagcaatgg 4200catcctggtc atccagcgga
tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260tgtgcaccgc
cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
4320tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
ggcgcgtgca 4380gggccagact ggaggtggca acgccaatca gcaacgactg
tttgcccgcc agttgttgtg 4440ccacgcggtt gggaatgtaa ttcagctccg
ccatcgccgc ttccactttt tcccgcgttt 4500tcgcagaaac gtggctggcc
tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560catactctgc
gacatcgtat aacgttactg
gtttcacatt caccaccctg aattgactct 4620cttccgggcg ctatcatgcc
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg
actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5040ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
tcatcatcac 5100agcagcggcc tggtgccgcg cggcagccat atggcctctg
aactcacggc tggtgttccc 5160aataaggtgg ggacatgtgt gtcagaggga
agctaccctg cagggactct tagctggcac 5220ttggatggga agcccctggt
gcctaatgag aagggagtat ctgtgaagga acagaccagg 5280agacaccctg
agacagggct cttcacactg cagtcggagc taatggtgac cccagcccgg
5340ggaggagatc cccgtcccac cttctcctgt agcttcagcc caggccttcc
ccgacaccgg 5400gccttgcgca cagcccccat ccagccccgt gtctgggagc
ctgtgcctct ggaggaggtc 5460caattggtgg tggagccaga aggtggagca
gtagctcctg gtggaaccgt aaccctgacc 5520tgtgaagtcc ctgcccagcc
ctctcctcaa atccactgga tgaaggatgg tgtgcccttg 5580ccccttcccc
ccagccctgt gctgatcctc cctgagatag ggcctcagga ccagggaacc
5640tacagctgtg tggccaccca ttccagccac gggccccagg aaagccgtgc
tgtcagcatc 5700agcatcatcg aaccaggcga ggaggggcca actgcaggct
ctgtgggagg atcatgactc 5760gagcaccacc accaccacca ctgagatccg
gctgctaaca aagcccgaaa ggaagctgag 5820ttggctgctg ccaccgctga
gcaataacta gcataacccc ttggggcctc taaacgggtc 5880ttgaggggtt
ttttgctgaa aggaggaact atatccggat 5920335605DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
33tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaat cgatagattg 1260tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
ctcataacac 1380cccttgtatt actgtttatg taagcagaca gttttattgt
tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag 1680aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 1860accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt 1980ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca
tatatggtgc actctcagta 2340caatctgctc tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 2460gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
agctcgttga gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg
ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt
aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg
atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca
cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca
gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga
ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt
cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg
gtggcgggac cagtgacgaa 3240ggcttgagcg agggcgtgca agattccgaa
taccgcaagc gacaggccga tcatcgtcgc 3300gctccagcga aagcggtcct
cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360gagttgcatg
ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
3420ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
ccggtgccta 3480atgagtgagc taacttacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 3540cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 3600tgggcgccag ggtggttttt
cttttcacca gtgagacggg caacagctga ttgcccttca 3660ccgcctggcc
ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
3720aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
gtatcgtcgt 3780atcccactac cgagatatcc gcaccaacgc gcagcccgga
ctcggtaatg gcgcgcattg 3840cgcccagcgc catctgatcg ttggcaacca
gcatcgcagt gggaacgatg ccctcattca 3900gcatttgcat ggtttgttga
aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960tcggctgaat
ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
4020agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
gcgaccagat 4080gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
aatactgttg atgggtgtct 4140ggtcagagac atcaagaaat aacgccggaa
cattagtgca ggcagcttcc acagcaatgg 4200catcctggtc atccagcgga
tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260tgtgcaccgc
cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
4320tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
ggcgcgtgca 4380gggccagact ggaggtggca acgccaatca gcaacgactg
tttgcccgcc agttgttgtg 4440ccacgcggtt gggaatgtaa ttcagctccg
ccatcgccgc ttccactttt tcccgcgttt 4500tcgcagaaac gtggctggcc
tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560catactctgc
gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
4620cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
gtgtccggga 4680tctcgacgct ctcccttatg cgactcctgc attaggaagc
agcccagtag taggttgagg 4740ccgttgagca ccgccgccgc aaggaatggt
gcatgcaagg agatggcgcc caacagtccc 4800ccggccacgg ggcctgccac
catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860cgagcccgat
cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
4920gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
gatcccgcga 4980aattaatacg actcactata ggggaattgt gagcggataa
caattcccct ctagaaataa 5040ttttgtttaa ctttaagaag gagatatacc
atgggcagca gccatcatca tcatcatcac 5100agcagcggcc tggtgccgcg
cggcagccat atgctggagg aggtccaatt ggtggtggag 5160ccagaaggtg
gagcagtagc tcctggtgga accgtaaccc tgacctgtga agtccctgcc
5220cagccctctc ctcaaatcca ctggatgaag gatggtgtgc ccttgcccct
tccccccagc 5280cctgtgctga tcctccctga gatagggcct caggaccagg
gaacctacag ctgtgtggcc 5340acccattcca gccacgggcc ccaggaaagc
cgtgctgtca gcatcagcat catcgaacca 5400ggcgaggagg ggccaactgc
aggctctgtg ggaggatcat gactcgagca ccaccaccac 5460caccactgag
atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
5520gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
gggttttttg 5580ctgaaaggag gaactatatc cggat 560534345PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1
5 10 15 Thr Val Ala Gln Ala Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu
Pro 20 25 30 Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro
Gln Arg Leu 35 40 45 Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala
Trp Lys Val Leu Ser 50 55 60 Pro Gln Gly Gly Gly Pro Trp Asp Ser
Val Ala Arg Val Leu Pro Asn 65 70 75 80 Gly Ser Leu Phe Leu Pro Ala
Val Gly Ile Gln Asp Glu Gly Ile Phe 85 90 95 Arg Cys Gln Ala Met
Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn Tyr 100 105 110 Arg Val Arg
Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp Ser 115 120 125 Ala
Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys Val 130 135
140 Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly
145 150 155 160 Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys
Glu Gln Thr 165 170 175 Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu
Gln Ser Glu Leu Met 180 185 190 Val Thr Pro Ala Arg Gly Gly Asp Pro
Arg Pro Thr Phe Ser Cys Ser 195 200 205 Phe Ser Pro Gly Leu Pro Arg
His Arg Ala Leu Arg Thr Ala Pro Ile 210 215 220 Gln Pro Arg Val Trp
Glu Pro Val Pro Leu Glu Glu Val Gln Leu Val 225 230 235 240 Val Glu
Pro Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr Leu 245 250 255
Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met Lys 260
265 270 Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile Leu
Pro 275 280 285 Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val
Ala Thr His 290 295 300 Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val
Ser Ile Ser Ile Ile 305 310 315 320 Glu Pro Gly Glu Glu Gly Pro Thr
Ala Gly Ser Val Gly Gly Ser Gly 325 330 335 Leu Gly Thr His His His
His His His 340 345 35127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 35Met Gly Ser Ser His His
His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His
Met Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu 20 25 30 Val Leu
Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu 35 40 45
Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro 50
55 60 Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro Asn
Gly 65 70 75 80 Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly
Ile Phe Arg 85 90 95 Cys Gln Ala Met Asn Arg Asn Gly Lys Glu Thr
Lys Ser Asn Tyr Arg 100 105 110 Val Arg Val Tyr Gln Ile Pro Gly Lys
Pro Glu Ile Val Asp Ser 115 120 125 36232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
36Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1
5 10 15 Arg Gly Ser His Met Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro
Leu 20 25 30 Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln
Arg Leu Glu 35 40 45 Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp
Lys Val Leu Ser Pro 50 55 60 Gln Gly Gly Gly Pro Trp Asp Ser Val
Ala Arg Val Leu Pro Asn Gly 65 70 75 80 Ser Leu Phe Leu Pro Ala Val
Gly Ile Gln Asp Glu Gly Ile Phe Arg 85 90 95 Cys Gln Ala Met Asn
Arg Asn Gly Lys Glu Thr Lys Ser Asn Tyr Arg 100 105 110 Val Arg Val
Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp Ser Ala 115 120 125 Ser
Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys Val Ser 130 135
140 Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly Lys
145 150 155 160 Pro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu
Gln Thr Arg 165 170 175 Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln
Ser Glu Leu Met Val 180 185 190 Thr Pro Ala Arg Gly Gly Asp Pro Arg
Pro Thr Phe Ser Cys Ser Phe 195 200 205 Ser Pro Gly Leu Pro Arg His
Arg Ala Leu Arg Thr Ala Pro Ile Gln 210 215 220 Pro Arg Val Trp Glu
Pro Val Pro 225 230 37334PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 37Met Gly Ser Ser His His
His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His
Met Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu 20 25 30 Val Leu
Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu 35 40 45
Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro 50
55 60 Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro Asn
Gly 65 70 75 80 Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly
Ile Phe Arg 85 90 95 Cys Gln Ala Met Asn Arg Asn Gly Lys Glu Thr
Lys Ser Asn Tyr Arg 100 105 110 Val Arg Val Tyr Gln Ile Pro Gly Lys
Pro Glu Ile Val Asp Ser Ala 115 120 125 Ser Glu Leu Thr Ala Gly Val
Pro Asn Lys Val Gly Thr Cys Val Ser 130 135 140 Glu Gly Ser Tyr Pro
Ala Gly Thr Leu Ser Trp His Leu Asp Gly Lys 145 150 155 160 Pro Leu
Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln Thr Arg 165 170 175
Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu Met Val 180
185 190 Thr Pro Ala Arg Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys Ser
Phe 195 200 205 Ser Pro Gly Leu Pro Arg His Arg Ala Leu Arg Thr Ala
Pro Ile Gln 210 215 220 Pro Arg Val Trp Glu Pro Val Pro Leu Glu Glu
Val Gln Leu Val Val 225 230 235 240 Glu Pro Glu Gly Gly Ala Val Ala
Pro Gly Gly Thr Val Thr Leu Thr 245 250 255 Cys Glu Val Pro Ala Gln
Pro Ser Pro Gln Ile His Trp Met Lys Asp 260 265 270 Gly Val Pro Leu
Pro Leu Pro Pro Ser Pro Val Leu Ile Leu Pro Glu 275 280 285 Ile Gly
Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr His Ser 290 295 300
Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile Ile Glu 305
310 315 320 Pro Gly Glu Glu Gly Pro Thr Ala Gly Ser Val Gly Gly Ser
325 330 38126PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 38Met Gly Ser Ser His His His His
His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala
Ser Glu Leu Thr Ala Gly Val Pro Asn Lys 20 25 30 Val Gly Thr Cys
Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser 35 40 45 Trp His
Leu Asp Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser 50 55 60
Val Lys Glu Gln Thr Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu 65
70 75 80 Gln Ser Glu Leu Met Val Thr Pro Ala Arg Gly Gly Asp Pro
Arg Pro 85 90 95 Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro Arg
His Arg Ala Leu 100 105 110 Arg Thr Ala Pro Ile Gln Pro Arg Val Trp
Glu Pro Val Pro 115
120 125 39228PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 39Met Gly Ser Ser His His His His
His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala
Ser Glu Leu Thr Ala Gly Val Pro Asn Lys 20 25 30 Val Gly Thr Cys
Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser 35 40 45 Trp His
Leu Asp Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser 50 55 60
Val Lys Glu Gln Thr Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu 65
70 75 80 Gln Ser Glu Leu Met Val Thr Pro Ala Arg Gly Gly Asp Pro
Arg Pro 85 90 95 Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro Arg
His Arg Ala Leu 100 105 110 Arg Thr Ala Pro Ile Gln Pro Arg Val Trp
Glu Pro Val Pro Leu Glu 115 120 125 Glu Val Gln Leu Val Val Glu Pro
Glu Gly Gly Ala Val Ala Pro Gly 130 135 140 Gly Thr Val Thr Leu Thr
Cys Glu Val Pro Ala Gln Pro Ser Pro Gln 145 150 155 160 Ile His Trp
Met Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro 165 170 175 Val
Leu Ile Leu Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser 180 185
190 Cys Val Ala Thr His Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val
195 200 205 Ser Ile Ser Ile Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala
Gly Ser 210 215 220 Val Gly Gly Ser 225 40123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1
5 10 15 Arg Gly Ser His Met Leu Glu Glu Val Gln Leu Val Val Glu Pro
Glu 20 25 30 Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr Leu Thr
Cys Glu Val 35 40 45 Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met
Lys Asp Gly Val Pro 50 55 60 Leu Pro Leu Pro Pro Ser Pro Val Leu
Ile Leu Pro Glu Ile Gly Pro 65 70 75 80 Gln Asp Gln Gly Thr Tyr Ser
Cys Val Ala Thr His Ser Ser His Gly 85 90 95 Pro Gln Glu Ser Arg
Ala Val Ser Ile Ser Ile Ile Glu Pro Gly Glu 100 105 110 Glu Gly Pro
Thr Ala Gly Ser Val Gly Gly Ser 115 120 41330PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 330 42326PRTHomo sapiens 42Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185
190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310
315 320 Ser Leu Ser Pro Gly Lys 325 4330PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20
25 30 4414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met Gly 1 5 10 4532PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 45Arg Phe Val Phe Ser Leu Asp Thr
Ser Val Ser Thr Ala Tyr Leu Gln 1 5 10 15 Ile Cys Ser Leu Lys Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 4611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 4730PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Gln 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 20
25 30 4832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 48Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Arg Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys Ala Arg 20 25 30 4923PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys 20 5015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
5132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 51Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 5211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 5323PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 53Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys 20 5415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15
5532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 55Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu
Asp Phe Ala Val Tyr Tyr Cys 20 25 30 56120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
56Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Phe 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Tyr Ile Asn Thr Asn Thr Gly Glu Ser Ile
Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp
Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Cys Ser Leu Lys
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Arg Met
Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr
Val Thr Val Ser Ser 115 120 57120PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 57Gln 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 Asn Phe 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Tyr Ile Asn Thr Asn Thr Gly Glu Ser Ile Tyr Ser Glu Glu Phe
50 55 60 Lys 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 Ser Arg Met Val Thr Ala Tyr Gly
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser
115 120 58108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 58Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 59108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 59Glu 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 Lys Ala Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Asn Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65
70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 60404PRTHomo sapiens 60Met Ala Ala Gly Thr Ala Val Gly Ala
Trp Val Leu Val Leu Ser Leu 1 5 10 15 Trp Gly Ala Val Val Gly Ala
Gln Asn Ile Thr Ala Arg Ile Gly Glu 20 25 30 Pro Leu Val Leu Lys
Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg 35 40 45 Leu Glu Trp
Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu 50 55 60 Ser
Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro 65 70
75 80 Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly
Ile 85 90 95 Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys Glu Thr
Lys Ser Asn 100 105 110 Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys
Pro Glu Ile Val Asp 115 120 125 Ser Ala Ser Glu Leu Thr Ala Gly Val
Pro Asn Lys Val Gly Thr Cys 130 135 140 Val Ser Glu Gly Ser Tyr Pro
Ala Gly Thr Leu Ser Trp His Leu Asp 145 150 155 160 Gly Lys Pro Leu
Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln 165 170 175 Thr Arg
Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu 180 185 190
Met Val Thr Pro Ala Arg Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys 195
200 205 Ser Phe Ser Pro Gly Leu Pro Arg His Arg Ala Leu Arg Thr Ala
Pro 210 215 220 Ile Gln Pro Arg Val Trp Glu Pro Val Pro Leu Glu Glu
Val Gln Leu 225 230 235 240 Val Val Glu Pro Glu Gly Gly Ala Val Ala
Pro Gly Gly Thr Val Thr 245 250 255 Leu Thr Cys Glu Val Pro Ala Gln
Pro Ser Pro Gln Ile His Trp Met 260 265 270 Lys Asp Gly Val Pro Leu
Pro Leu Pro Pro Ser Pro Val Leu Ile Leu 275 280 285 Pro Glu Ile Gly
Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr 290
295 300 His Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser
Ile 305 310 315 320 Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala Gly Ser
Val Gly Gly Ser 325 330 335 Gly Leu Gly Thr Leu Ala Leu Ala Leu Gly
Ile Leu Gly Gly Leu Gly 340 345 350 Thr Ala Ala Leu Leu Ile Gly Val
Ile Leu Trp Gln Arg Arg Gln Arg 355 360 365 Arg Gly Glu Glu Arg Lys
Ala Pro Glu Asn Gln Glu Glu Glu Glu Glu 370 375 380 Arg Ala Glu Leu
Asn Gln Ser Glu Glu Pro Glu Ala Gly Glu Ser Ser 385 390 395 400 Thr
Gly Gly Pro 61331PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 61Met Ala Ala Gly Thr Ala Val Gly
Ala Trp Val Leu Val Leu Ser Leu 1 5 10 15 Trp Gly Ala Val Val Gly
Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu 20 25 30 Pro Leu Val Leu
Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg 35 40 45 Leu Glu
Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu 50 55 60
Ser Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro 65
70 75 80 Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu
Gly Ile 85 90 95 Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys Glu
Thr Lys Ser Asn 100 105 110 Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly
Lys Pro Glu Ile Val Asp 115 120 125 Ser Ala Ser Glu Leu Thr Ala Gly
Val Pro Asn Lys Val Gly Thr Cys 130 135 140 Val Ser Glu Gly Ser Tyr
Pro Ala Gly Thr Leu Ser Trp His Leu Asp 145 150 155 160 Gly Lys Pro
Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln 165 170 175 Thr
Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu 180 185
190 Met Val Thr Pro Ala Arg Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys
195 200 205 Ser Phe Ser Pro Gly Leu Pro Arg His Arg Ala Leu Arg Thr
Ala Pro 210 215 220 Ile Gln Pro Arg Val Trp Glu Pro Val Pro Leu Glu
Glu Val Gln Leu 225 230 235 240 Val Val Glu Pro Glu Gly Gly Ala Val
Ala Pro Gly Gly Thr Val Thr 245 250 255 Leu Thr Cys Glu Val Pro Ala
Gln Pro Ser Pro Gln Ile His Trp Met 260 265 270 Lys Asp Gly Val Pro
Leu Pro Leu Pro Pro Ser Pro Val Leu Ile Leu 275 280 285 Pro Glu Ile
Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr 290 295 300 His
Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile 305 310
315 320 Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala Gly 325 330
62120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 62Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Thr
Asn Thr Gly Glu Ser Ile Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg
Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Arg Met Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120
63108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 63Asp 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 Lys Ala
Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
64108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 64Asp 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 Lys Ala
Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Phe Ser Ala Ser Asn
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
65108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala
Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn
Arg Tyr Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
66108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala
Ser Gln Asn Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Phe Ser Ala Ser Asn
Arg Tyr Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu
Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
67120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Glu Ile Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Thr
Asn Thr Gly Glu Ser Ile Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg
Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Ser Arg Met Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120
68120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Glu 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 Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Thr
Asn Thr Gly Glu Ser Ile Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Arg Met Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
69120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Glu Ile 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 Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Thr
Asn Thr Gly Glu Ser Ile Tyr Ser Glu Glu Phe 50 55 60 Lys Gly Arg
Phe Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Ser Arg Met Val Thr Ala Tyr Gly Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 7031PRTHomo
sapiens 70Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu Lys
Cys Lys 1 5 10 15 Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp
Lys Leu Asn 20 25 30 7130PRTHomo sapiens 71Lys Leu Asn Thr Gly Arg
Thr Glu Ala Trp Lys Val Leu Ser Pro Gln 1 5 10 15 Gly Gly Gly Pro
Trp Asp Ser Val Ala Arg Val Leu Pro Asn 20 25 30 7230PRTHomo
sapiens 72Leu Pro Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln
Asp Glu 1 5 10 15 Gly Ile Phe Arg Cys Gln Ala Met Asn Arg Asn Gly
Lys Glu 20 25 30 7330PRTHomo sapiens 73Gly Lys Glu Thr Lys Ser Asn
Tyr Arg Val Arg Val Tyr Gln Ile Pro 1 5 10 15 Gly Lys Pro Glu Ile
Val Asp Ser Ala Ser Glu Leu Thr Ala 20 25 30 7430PRTHomo sapiens
74Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys Val Ser Glu Gly 1
5 10 15 Ser Tyr Pro Ala Gly Thr Leu Ser Trp Lys Leu Asp Gly Lys 20
25 30 7530PRTHomo sapiens 75Asp Gly Lys Pro Leu Val Pro Asn Glu Lys
Gly Val Ser Val Lys Glu 1 5 10 15 Gln Thr Arg Arg His Pro Glu Thr
Gly Leu Phe Thr Leu Gln 20 25 30 7630PRTHomo sapiens 76Thr Leu Gln
Ser Glu Leu Met Val Thr Pro Ala Arg Gly Gly Asp Pro 1 5 10 15 Arg
Pro Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro Arg 20 25 30
7730PRTHomo sapiens 77Leu Pro Arg His Arg Ala Leu Arg Thr Ala Pro
Ile Gln Pro Arg Val 1 5 10 15 Trp Glu Pro Val Pro Leu Glu Glu Val
Gln Leu Val Val Glu 20 25 30 7830PRTHomo sapiens 78Val Val Glu Pro
Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr 1 5 10 15 Leu Thr
Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His 20 25 30
7930PRTHomo sapiens 79Gln Ile His Trp Met Lys Asp Gly Val Pro Leu
Pro Leu Pro Pro Ser 1 5 10 15 Pro Val Leu Ile Leu Pro Glu Ile Gly
Pro Gln Asp Gln Gly 20 25 30 8030PRTHomo sapiens 80Asp Gln Gly Thr
Tyr Ser Cys Val Ala Thr His Ser Ser His Gly Pro 1 5 10 15 Gln Glu
Ser Arg Ala Val Ser Ile Ser Ile Ile Glu Pro Gly 20 25 30
8128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 81ccgaattccg gaagcaggat ggcagccg
288232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 82ccctcgagcc cctcaaggcc ctcagtacta ct
328331DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 83agtaacggcc gccagtgtgc tggaattcgg a
318432DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 84ccggtaccac ctgcagttgg cccctcctcg cc
328536DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 85cgaagcttga tgaacaggaa tggaaaggag accaag
368634DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 86tcctcgagca cctgcagttg gcccctcctc gcct
348727DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 87gcaccatggc agccggaaca gcagttg
278829DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 88gagtctcgag gcagaatcta caatttctg
298990DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 89atgctacata tgaaaaagac agctatcgcg attgcagtgg
cactggctgg tttcgctacc 60gtagcgcagg ccgctcaaaa catcacagcc
909079DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 90atgctactcg agtcagtggt ggtggtggtg gtgagttccc
agccctgatc ctcccacaga 60gcctgcagtt ggcccctcc 799142DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
91gtacgatatc gagggacgaa tggatccacc gtgcccagca cc
429232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 92ctagtctaga tcatttaccc ggagacaggg ag
329342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 93gtacgatatc gagggacgaa tggatccacc gtgcccagca cc
429432DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 94ctagtctaga tcatttaccc ggagacaggg ag
32956PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 95His His His His His His 1 5 967PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 96His
His His His His His Thr 1 5 975PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 97Gly Gly Gly Gly Ser 1 5
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References