U.S. patent application number 14/840978 was filed with the patent office on 2015-12-17 for antibodies directed against pyroglutamate monocyte chemoattractant protein-1 (mcp-1 n1pe).
The applicant listed for this patent is Probiodrug AG. Invention is credited to Holger Cynis, Hans-Ulrich Demuth, Kathrin Gans, Sonja Kampfer, Jens-Ulrich Rahfeld, Stephan Schilling.
Application Number | 20150361167 14/840978 |
Document ID | / |
Family ID | 41259004 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150361167 |
Kind Code |
A1 |
Cynis; Holger ; et
al. |
December 17, 2015 |
ANTIBODIES DIRECTED AGAINST PYROGLUTAMATE MONOCYTE CHEMOATTRACTANT
PROTEIN-1 (MCP-1 N1PE)
Abstract
Monoclonal antibodies which bind specifically to the
proinflammatory cytokine pyroglutamate MCP-1 (MCP-1 N1pE).
Inventors: |
Cynis; Holger; (Halle/Saale,
DE) ; Demuth; Hans-Ulrich; (Halle/Saale, DE) ;
Rahfeld; Jens-Ulrich; (Lieskau, DE) ; Schilling;
Stephan; (Halle/Saale, DE) ; Gans; Kathrin;
(Halle/Saale, DE) ; Kampfer; Sonja; (Germering,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Probiodrug AG |
Halle/Saale |
|
DE |
|
|
Family ID: |
41259004 |
Appl. No.: |
14/840978 |
Filed: |
August 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13948363 |
Jul 23, 2013 |
9157918 |
|
|
14840978 |
|
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|
|
12544319 |
Aug 20, 2009 |
8518406 |
|
|
13948363 |
|
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61090264 |
Aug 20, 2008 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 19/02 20180101; A61P 31/18 20180101; A61P 9/10 20180101; C07K
2317/30 20130101; A61P 9/12 20180101; A61P 25/00 20180101; C07K
16/24 20130101; A61P 35/00 20180101; C07K 2317/33 20130101; A61P
25/04 20180101; C07K 2317/56 20130101; A61P 29/00 20180101; A61P
13/12 20180101; C07K 2317/34 20130101; C07K 2317/76 20130101; A61P
1/16 20180101; A61P 43/00 20180101; C07K 2317/92 20130101; A61P
15/00 20180101; A61P 11/00 20180101; A61P 25/28 20180101; A61P 1/18
20180101; A61P 35/04 20180101; G01N 33/6854 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. A method of treating a disease or condition in a subject in need
thereof comprising: administering to the subject a composition
comprising an antibody or an antigen binding fragment that
selectively binds to a pyroglutamate-carrying amino-terminus of
MCP-1 N1pE, wherein the antibody or antigen binding fragment has
substantially no cross-reactivity with an epitope other than
pyroglutamate-carrying amino terminus of MCP-1 N1pE; and the
disease or condition is selected from the group consisting of: a. a
neurodegenerative disease; b. a neurodegenerative disease selected
from the group consisting of mild cognitive impairment (MCI),
Alzheimer's disease, neurodegeneration in Down Syndrome, Familial
British Dementia, Familial Danish Dementia, and multiple sclerosis;
c. chronic or acute inflammation; d. chronic or acute inflammation
selected from the group consisting of rheumatoid arthritis,
atherosclerosis, restenosis, and pancreatitis; e. fibrosis; f.
fibrosis selected from the group consisting of lung fibrosis, liver
fibrosis, and renal fibrosis; g. cancer; h. cancer selected from
the group consisting of hemangioendothelioma proliferation and
gastric carcinoma; i. a metabolic disease; j. a metabolic disease
selected from the group consisting of hypertension; and k. an
inflammatory disease selected from the group consisting of
neuropathic pain, graft rejection/graft failure/graft vasculopathy,
HIV infections/AIDS, gestosis, and tuberous sclerosis.
2. The method of claim 1 wherein the disease or condition is
selected from the group consisting of atheroschlerosis, rheumatoid
arthritis, asthma, delayed hypersensitivity reactions,
pancreatitis, Alzheimer's disease, lung fibrosis, renal fibrosis,
gestosis, graft rejection, neuropathic pain, AIDS, and tumors.
3. The method of claim 1 wherein the disease or condition is
selected from the group consisting of atherosclerosis, rheumatoid
arthritis, restenosis, and pancreatitis.
4. The method of claim 1 wherein the disease or condition is
selected from the group consisting of MCI and Alzheimer's
disease.
5. The method of claim 1, wherein the antibody or antigen binding
fragment: (i) is encoded by a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 35, 39, and 43; (ii) has a
light chain variable region that selectively binds to a
pyroglutamate-carrying amino terminus of MCP-1 N1pE, wherein the
antibody or the antigen binding fragment has substantially no
cross-reactivity with an epitope other than the
pyroglutamate-carrying amino terminus of MCP-1 N1pE; (iii) has a
light chain variable region encoded by the nucleotide sequence
selected from the group consisting of SEQ ID NOs: 33, 37, and 41 or
has a light chain variable region having the amino acid sequence
selected from the group consisting of SEQ ID NOs: 34, 38, and 43;
(iv) is encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 33, 37, and 41; (v) has an amino acid
sequence selected from the group consisting of SEQ ID NOs: 34, 38,
and 42; (vi) has a heavy chain variable region that selectively
binds to a pyroglutamate-carrying amino terminus of MCP-1 N1pE,
wherein the antibody or the antigen binding fragment has
substantially no cross-reactivity with an epitope other than the
pyroglutamate-carrying amino terminus of MCP-1 N1pE; (vii) has a
heavy chain variable region encoded by the nucleotide sequence
selected from the group consisting of SEQ ID NOs: 35, 39, and 43 or
has a heavy chain variable region having the amino acid sequence
selected from the group consisting of SEQ ID NOs: 36, 40, and 44;
(viii) is encoded by a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 35, 39, and 43; (ix) has an amino
acid sequence selected from the group consisting of SEQ ID NOs: 36,
40, and 44; or (x) is encoded by a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 7 to 32.
6. The method of claim 1, wherein the antibody or antigen binding
fragment comprises: (i) a light chain variable region of the
antibody or the antigen binding fragment that selectively binds to
a pyroglutamate-carrying amino terminus of MCP-1 N1pE, wherein the
antibody or the antigen binding fragment has substantially no
cross-reactivity with an epitope other than the
pyroglutamate-carrying amino terminus of MCP-1 N1pE; (ii) a
polypeptide encoded by a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 33, 37, and 41; (iii) an amino acid
sequence selected from the group consisting of SEQ ID NOs: 34, 38,
and 42; (iv) a heavy chain variable region of the antibody or the
antigen binding fragment that selectively binds to a
pyroglutamate-carrying amino terminus of MCP-1 N1pE, wherein the
antibody or the antigen binding fragment has substantially no
cross-reactivity with an epitope other than the
pyroglutamate-carrying amino terminus of MCP-1N1pE; (v) a
polypeptide encoded by a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 35, 39, and 43; or (vi) an amino
acid sequence selected from the group consisting of SEQ ID NOs: 36,
40, and 44.
7. The method of claim 1, wherein the antibody or antigen binding
fragment thereof is encoded by a polynucleotide that selectively
binds to a pyroglutamate-carrying amino terminus of MCP-1 N1pE,
wherein the antibody or the antigen binding fragment has
substantially no cross-reactivity with an epitope other than the
pyroglutamate-carrying amino terminus of MCP-1 N1pE.
8. The method of claim 1, wherein the disease or disorder is
associated with increased expression of MCP-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. Nonprovisional
application Ser. No. 13/948,363 filed on 23 Jul. 2013 which is a
Divisional of U.S. Nonprovisional application Ser. No. 12/544,319
filed on 20 Aug. 2009, which issued as U.S. Pat. No. 8,518,406 on
27 Aug. 2013, which claims priority to U.S. Provisional Application
Ser. No. 61/090,264 filed on 20 Aug. 2008, which is incorporated
herein by reference in its entirety to the extent permitted by
law.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The Sequence Listing, which is a part of the present
disclosure, includes a computer readable form comprising nucleotide
and/or amino acid sequences of the present invention. The subject
matter of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to monoclonal antibodies which
bind specifically to the proinflammatory cytokine pyroglutamate
MCP-1 (MCP-1 N1pE).
BACKGROUND OF THE INVENTION
[0004] Chemotactic cytokines (chemokines) are proteins that attract
and activate leukocytes and are thought to play a fundamental role
in inflammation. Chemokines are divided into four groups
categorized by the appearance of N-terminal cysteine residues
("C"-; "CC"-; "CXC"- and "CX3C"-chemokines). "CXC"-chemokines
preferentially act on neutrophils. In contrast, "CC"-chemokines
attract preferentially monocytes to sites of inflammation. Monocyte
infiltration is considered to be a key event in a number of disease
conditions (Gerard, C. and Rollins, B. J. (2001) Nat. Immunol 2,
108-115; Bhatia, M., et al., (2005) Pancreatology. 5, 132-144;
Kitamoto, S., Egashira, K., and Takeshita, A. (2003) J Pharmacol
Sci. 91, 192-196).
[0005] The MCP family, as one family of chemokines, consists of
four members (MCP-1 to 4), displaying a preference for attracting
monocytes but showing differences in their potential (Luini, W., et
al., (1994) Cytokine 6, 28-31; Uguccioni, M., et al., (1995) Eur J
Immunol 25, 64-68).
[0006] MCP-1 is a member of the .beta. (C--C) subfamily of
chemokines. In this family, the 2 cysteins nearest to the amino
terminus are adjacent to each other (thus C--C proteins). As with
many other C--C chemokines, the MCP-1 gene is located on chromosome
17 in humans. The cell surface receptors that bind MCP-1 are CCR2
and CCR5.
[0007] In the following both cDNA as well as amino acid sequences
of MCP-1 are indicated:
TABLE-US-00001 Human MCP-1 (CCL2) (GeneBank Accession: M24545) cDNA
(300 bp) SEQ ID NO: 2 1 atgaaagtct ctgccgccct tctgtgcctg ctgctcatag
cagccacctt cattccccaa 61 gggctcgctc agccagatgc aatcaatgcc
ccagtcacct gctgttataa cttcaccaat 121 aggaagatct cagtgcagag
gctcgcgagc tatagaagaa tcaccagcag caagtgtccc 181 aaagaagctg
tgatcttcaa gaccattgtg gccaaggaga tctgtgctga ccccaagcag 241
aagtgggttc aggattccat ggaccacctg gacaagcaaa cccaaactcc gaagacttga
Protein (Signal Sequence in bold: 23 aa; Mature MCP-1: 76 aa) SEQ
ID NO: 1
KVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCP
KEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT
[0008] Consistent with it being a member of the chemokine .beta.
family, MCP-1 has been shown to chemoattract and activate monocytes
in vitro at subnanomolar concentrations. Elevated MCP-1 expression
has been detected in a variety of pathologic conditions that
involve monocyte accumulation and activation, including a number of
inflammatory and non-inflammatory disease states, like rheumatoid
arthritis, atherosclerosis, asthma and delayed hypersensitivity
reactions.
[0009] A number of studies have underlined in particular the
crucial role of MCP-1 for the development of atherosclerosis (Gu,
L., et al., (1998) Mol. Cell 2, 275-281; Gosling, J., et al.,
(1999) J Clin. Invest 103, 773-778); rheumatoid arthritis (Gong, J.
H., et al., (1997) J Exp. Med 186, 131-137; Ogata, H., et al.,
(1997) J Pathol. 182, 106-114); pancreatitis (Bhatia, M., et al.,
(2005) Am. J Physiol Gastrointest. Liver Physiol 288, G1259-G1265);
Alzheimer's disease (Yamamoto, M., et al., (2005) Am. J Pathol.
166, 1475-1485); lung fibrosis (Inoshima, I., et al., (2004) Am. J
Physiol Lung Cell Mol. Physiol 286, L1038-L1044); renal fibrosis
(Wada, T., et al., (2004) J Am. Soc. Nephrol. 15, 940-948), and
graft rejection (Saiura, A., et al., (2004) Arterioscler. Thromb.
Vasc. Biol. 24, 1886-1890). Furthermore, MCP-1 might also play a
role in gestosis (Katabuchi, H., et al., (2003) Med Electron
Microsc. 36, 253-262), as a paracrine factor in tumor development
(Ohta, M., et al., (2003) Int. J Oncol. 22, 773-778; Li, S., et
al., (2005) J Exp. Med 202, 617-624), neuropathic pain (White, F.
A., et al., (2005) Proc. Natl. Acad. Sci. U.S.A) and AIDS (Park, I.
W., Wang, J. F., and Groopman, J. E. (2001) Blood 97, 352-358;
Coll, B., et al., (2006) Cytokine 34, 51-55).
[0010] The mature form of human and rodent MCP-1 is
post-translationally modified by Glutaminyl Cyclase (QC) to possess
an N-terminal pyroglutamyl (pGlu) residue.
[0011] Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the
intramolecular cyclization of N-terminal glutaminyl residues into
pyroglutamic acid (5-oxo-proline, pGlu*) under liberation of
ammonia and the intramolecular cyclization of N-terminal glutamyl
residues into pyroglutamic acid under liberation of water.
[0012] The N-terminal pGlu modification makes the protein resistant
against N-terminal degradation by aminopeptidases, which is of
importance, since chemotactic potency of MCP-1 is mediated by its
N-terminus (Van Damme, J., et al., (1999) Chem Immunol 72, 42-56).
Artificial elongation or degradation leads to a loss of function
although MCP-1 still binds to its receptor (CCR2) (Proost, P., et
al., (1998), J Immunol 160, 4034-4041; Zhang, Y. J., et al., 1994,
J Biol. Chem 269, 15918-15924; Masure, S., et al., 1995, J
Interferon Cytokine Res. 15, 955-963; Hemmerich, S., et al., (1999)
Biochemistry 38, 13013-13025).
[0013] Due to the major role of MCP-1 in a number of disease
conditions, a potent diagnostic tool and an anti-MCP-1 strategy is
required.
[0014] As mentioned above, compelling evidence points to a role of
MCP 1 in Alzheimer's disease (AD) (Xia, M. Q. and Hyman, B. T.
(1999) J Neurovirol. 5, 32-41). The presence of MCP-1 in senile
plaques and in reactive microglia, the residential macrophages of
the CNS, has been observed in brains of patients suffering from AD
(Ishizuka, K., et al., (1997) Psychiatry Clin. Neurosci. 51,
135-138. Stimulation of monocytes and microglia with Amyloid-.beta.
protein (A.beta.) induces chemokine secretion in vitro (Meda, L.,
et al., (1996) J Immunol 157, 1213-1218; Szczepanik, A. M., et al.,
(2001) J Neuroimmunol. 113, 49-62) and intracerebroventricular
infusion of A.beta. (1-42) into murine hippocampus significantly
increases MCP-1 in vivo. Moreover, A.beta. deposits attract and
activate microglial cells and force them to produce inflammatory
mediators such as MCP-1, which in turn leads to a feed back to
induce further chemotaxis, activation and tissue damage. At the
site of A.beta. deposition, activated microglia also phagocytose
A.beta. peptides leading to an amplified activation (Rogers, J. and
Lue, L. F. (2001) Neurochem. Int. 39, 333-340).
[0015] Examination of chemokine expression in the 3.times.Tg mouse
model for AD revealed that neuronal inflammation precedes plaque
formation and MCP-1 is upregulated by a factor of 11. Furthermore,
the upregulation of MCP-1 seems to correlate with the occurrence of
first intracellular A.beta. deposits (Janelsins, M. C., et al.,
(2005) J Neuroinflammation. 2, 23). Cross-breeding of the Tg2575
mouse model for AD with a MCP-1 overexpressing mouse model has
shown an increased microglia accumulation around A.beta. deposits
and that this accumulation was accompanied by increased amount of
diffuse plaques compared to single-transgenic Tg2576 littermates
(Yamamoto, M., et al. (2005) Am. J Pathol. 166, 1475-1485).
[0016] MCP-1 levels are increased in CSF of AD patients and
patients showing mild cognitive impairment (MCI) (Galimberti, D.,
et al., (2006) Arch. Neurol. 63, 538-543). Furthermore, MCP-1 shows
an increased level in serum of patients with MCI and early AD
(Clerici, F., et al., (2006) Neurobiol. Aging 27, 1763-1768).
[0017] Atherosclerotic lesions, which limit or obstruct coronary
blood flow, are the major cause of ischemic heart disease related
mortality, resulting in 500,000-600,000 deaths annually.
Percutaneous transluminal coronary angioplasty (PTCA) to open the
obstructed artery was performed in over 550,000 patients in the U.
S. and 945,000+ patients worldwide in 1996 (Lemaitre et al., 1996).
A major limitation of this technique is the problem of post-PTCA
closure of the vessel, both immediately after PTCA (acute
occlusion) and in the long term (restenosis): 30% of patients with
subtotal lesions and 50% of patients with chronic total lesions
will go on to restenosis after angioplasty. Additionally,
restenosis is a significant problem in patients undergoing
saphenous vein bypass graft. The mechanism of acute occlusion
appears to involve several factors and may result from vascular
recoil with resultant closure of the artery and/or deposition of
blood platelets along the damaged length of the newly opened blood
vessel followed by formation of a fibrin/red blood cell
thrombus.
[0018] Restenosis after angioplasty is a more gradual process and
involves initial formation of a subcritical thrombosis with release
from adherent platelets of cell derived growth factors with
subsequent proliferation of intimal smooth muscle cells and local
infiltration of inflammatory cells contributing to vascular
hyperplasia. It is important to note that multiple processes, among
those thrombosis, cell proliferation, cell migration and
inflammation each seem to contribute to the restenotic process.
[0019] In the U.S., a 30-50% restenosis rate translates to
120,000-200,000 U.S. patients at risk from restenosis. If only 80%
of such patients elect repeated angioplasty (with the remaining 20%
electing coronary artery bypass graft) and this is added to the
cost of coronary artery bypass graft for the remaining 20%, the
total cost for restenosis easily reaches into billions of dollars.
Thus, successful prevention of restenosis could result not only in
significant therapeutic benefit but also in significant health care
savings.
[0020] Although it is not clear whether elevated MCP-1 expression
is the cause or consequence of the above diseases, therapeutic
benefit resulted from the application of neutralizing antibodies in
a number of animal models.
[0021] So far, prior art monoclonal antibodies were screened for
their ability to act as receptor antagonists. None of those target
the immediate pyroglutamate carrying amino terminus of MCP-1 (=MCP1
N1pE). In this context, it is important to note that deletion of
amino acids 1-8 from the N-terminal region completely destroyed
MCP-1 activity, suggesting that the amino-terminal region is
essential for activity.
[0022] It consequently follows, that antibodies directed against
the MCP-1 N1pE can play a role not only when investigating the
expression and function of MCP-1 but also in therapeutic and
diagnostic applications in connection with diseases or disturbances
in which MCP-1 might be involved.
[0023] In view of the above, one object underlying the present
invention is to provide antibodies which are selectively binding to
MCP-1 N1pE.
SUMMARY OF THE INVENTION
[0024] The present invention provides antibodies selectively
binding to MCP-1 N1pE, i.e. pyroglutamate MCP-1.
[0025] Preferably, monoclonal antibodies are provided.
[0026] The present invention pertains in particular to antibodies
or variants thereof, which are characterized in that they bind to
the MCP-1 N1pE peptide with a high affinity. Said high affinity
means in the context of the present invention an affinity of a
K.sub.D value of 10.sup.-6 M or better, preferably a K.sub.D value
of 10.sup.-7 M or better, and even more preferably a K.sub.D value
of 10.sup.-8 M-10.sup.-12 M.
[0027] Monoclonal antibodies of this type are preferably produced
by hybridoma cells. Hybridoma cells of this type were deposited on
6, May 2008 in the Deutsche Sammlung von Mikroorganismen and
Zellkulturen (German Collection of Microorganisms and Cell
Cultures) GmbH, DSMZ, Inhoffenstrasse 7B, 38124 Braunschweig,
Germany, in accordance with the Budapest Treaty, namely
TABLE-US-00002 DSM ACC 2905 (Hybridoma cell clone 348/1D4) 2906
(Hybridoma cell clone 348/2C9) 2907 (Hybridoma cell clone 332/4B8)
and 2908 (Hybridoma cell clone 332/4F8).
[0028] In particular, monoclonal antibodies are preferred, wherein
selective binding means a binding to the pyroglutamate carrying
amino terminus of MCP-1.
[0029] Even preferred are monoclonal antibodies, wherein selective
binding means that the antibodies do not show any cross-reactivity
with epitopes outside the pyroglutamate carrying amino terminus of
MCP-1 N1pE.
[0030] The amino terminus of MCP-1 N1pE is defined here as the
first 1 to 10 amino acids of the amino terminus of MCP-1 N1pE,
preferably the first 1 to 8 amino acids of the amino terminus of
MCP N1pE, most preferably the first 1 to 4 amino acids of the amino
terminus of MCP N1pE.
[0031] The invention relates further to the above hybridoma cell
clones per se which possess the ability to produce and release such
antibodies.
[0032] By means of the antibodies 332-4B8 (DSM ACC 2907), 332-4F8
(DSM ACC 2908), 348-2C9(DSM ACC 2906) and 348-1D4 (DSM ACC 2905),
the inventors of the present application have, for the first time,
made available monoclonal antibodies, as well as hybridoma cells
which produce and release these antibodies, which make it possible
to selectively recognize and bind to, and consequently influence
MCP-1 N1pE. The antibodies consequently provide the physician and
research worker with a versatile means, which is so far unique,
for, on the one hand, detecting MCP-1 N1pE, both in cell culture
and in the sample obtained from a patient, and, on the other hand,
for potential manipulation of MCP-1 N1pE, where appropriate, either
using the antibody itself or using specific reagents which are
coupled to it.
[0033] In this connection, the inventors of the present application
have ascertained that the above antibodies 332-4B8, 332-4F8,
348-2C9 and 348-1D4 bind selectively to polypeptides of MCP-1 N1pE
starting with amino acids pE-P-D i.e.
pyroglutamate-proline-aspartic acid.
[0034] The inventors have been able to demonstrate that MCP-1 N1pE
is also detected in blood samples (serum, plasma) of mammals,
especially of mice, rats and humans, and that MCP-1 N1pE levels are
elevated after inflammatory stimuli, which can be reversed by
application of selective glutminyl cyclase (QC) inhibitors (e.g.
1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea-hydrochlor-
ide (see particularly the glutaminyl peptide cyclase inhibitors as
disclosed in WO 2008/104580).
[0035] Consequently, these antibodies are outstandingly suitable
for diagnostic and therapeutic purposes, with it being possible to
achieve a wide variety of investigations and therapeutic
effects.
[0036] Accordingly, a further embodiment of the invention relates
to a pharmaceutical composition comprising one of the above novel
antibodies. Preferably said novel antibody is coupled to a cellular
directed therapeutic agent or diagnostic agent.
[0037] An antibody according to the invention, which is coupled to
a means for detection and thus indirectly to the relevant cells,
thereby makes it possible to detect these cells directly, for
example using X-ray diagnostic/scintigraphic methods. In a
corresponding manner, coupling to a therapeutically active agent
can also make it possible to exert a direct and selective effect on
MCP-1 N1pE carrying cells.
[0038] Further advantages will be evident from the description
given below.
[0039] It will be understood that the features which are mentioned
above, and those which are still to be explained below, can be used
not only in the combinations which are in each case specified but
also in other combinations, or on their own, without departing from
the scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Those of skill in the art will understand that the drawings,
described below, are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0041] FIG. 1A: Binding characteristics of monoclonal antibody
332-4B8 to human MCP-1 N1pE-38 determined with SPR analysis
(Biacore 3000). Measurement was performed by using HBS-EP as
running buffer. Association took place for 180 sec, followed by a
180 sec dissociation phase and 5 sec regeneration with 0.1M
HCl.
[0042] FIG. 1B: Binding characteristics of monoclonal antibody
332-4F8 to human MCP-1 N1pE-38 determined with SPR analysis
(Biacore 3000). Measurement was performed by using HBS-EP as
running buffer. Association took place for 180 sec, followed by a
180 sec dissociation phase and 5 sec regeneration with 0.1M
HCl.
[0043] FIG. 1C: Binding characteristics of monoclonal antibody
348-2C9 to human MCP-1 N1pE-38 determined with SPR analysis
(Biacore 3000). Measurement was performed by using HBS-EP as
running buffer. Association took place for 180 sec, followed by a
180 sec dissociation phase and 5 sec regeneration with 0.1M
HCl.
[0044] FIG. 1D: Binding characteristics of monoclonal antibody
348-1D4 to human MCP-1 N1pE-38 determined with SPR analysis
(Biacore 3000). Measurement was performed by using HBS-EP as
running buffer. Association took place for 180 sec, followed by a
180 sec dissociation phase and 5 sec regeneration with 0.1M
HCl.
[0045] FIG. 2A: DotBlot analysis of monoclonal antibody 332-4B8 to
human MCP-1 N1pE-38 and MCP-1 3-38.
[0046] FIG. 2B: DotBlot analysis of monoclonal antibody 332-4F8 to
human MCP-1 N1pE-38 and MCP-1 3-38.
[0047] FIG. 2C: DotBlot analysis of monoclonal antibody 348-1D4 to
human MCP-1 N1pE-38 and MCP-1 3-38.
[0048] FIG. 2D: DotBlot analysis of monoclonal antibody 348-2C9 to
human MCP-1 N1pE-38 and MCP-1 3-38.
[0049] FIG. 3: PepSpot analysis of monoclonal antibodies 332-4B8,
332-4F8, 348-1D4 and 348-2C9.
[0050] FIG. 4: Quantitative detection of recombinant human MCP-1
N1pE in an ELISA by using the monoclonal anti MCP-1 antibodies
332-4B8, 348-2C9 and 348-1D4.
[0051] FIG. 5: Detection of human MCP-1 N1pE from human serum by
monoclonal antibodies 332-4B8, 348-1D4 and 348-2C9 in ELISA.
[0052] FIG. 6: Time dependent expression of hMCP-1 N1pE in NHDF
cells, stimulated by OSM and IL1.beta..
[0053] FIG. 7: Concentration dependent reduction of hMCP-1 N1pE in
the cell culture supernatant of LPS induced THP1 cells after
application of QCI.
[0054] FIG. 8: Quantitative detection of recombinant mouse MCP-1
N1pE in an ELISA by using the monoclonal anti MCP-1 antibodies
332-4B8, 348-2C9 and 348-1D4
[0055] FIG. 9: Quantitative detection of native mouse MCP-1 N1pE in
the cell culture supernatant of untreated and LPS induced RAW 264.7
cells.
[0056] FIG. 10: Concentration dependent reduction of mMCP-1 N1pE in
the cell culture supernatant of LPS induced RAW 264.7 cells after
application of QCI.
[0057] FIG. 11: Quantitative detection of mMCP-1 N1pE in mouse
serum after different time points of LPS treatment.
[0058] FIG. 12: Dilution Linearity of the quantitative detection of
mMCP-1 N1pE in peritoneal lavage fluid from thioglycollate treated
mice by ELISA.
[0059] FIG. 13: Comparison of Western Blot signals obtained for
murine MCP1 N1pE (A) and total murine MCP1 (B) with the
corresponding ELISA Data for murine MCP1 N1pE (C).
[0060] FIG. 14: Quantitative detection of rat MCP-1 N1pE by the
anti MCP-1 N1pE antibody 348-2C9 in an ELISA.
[0061] FIG. 15: Staining of MCP-1 N1pE in brain sections of rats
with the antibodies 332-4B8, 348-1D4 and 348-2C9 after
microinjection of A.beta.(3-49), LPS or NaCl.
[0062] FIG. 16: Fitting curves of the binding heat evolved by
titration of the antigen hMCP-1 N1pE-38 to the monoclonal
antibodies A--348-1D4 and B--332-4B8.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
Definitions
[0063] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0064] The term "antibody" is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments
so long as they exhibit the desired biological activity. The
antibody may be an IgM, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), IgD,
IgA or IgE, for example. Preferably however, the antibody is not an
IgM antibody.
[0065] "Antibody fragments" comprise a portion of an intact
antibody, generally the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments: diabodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0066] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e. the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to "polyclonal antibody"
preparations, which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies can
frequently be advantageous in that they are synthesized by the
hybridoma culture, uncontaminated by other immunoglobulins. The
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by generally well known recombinant DNA
methods. The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example.
[0067] The monoclonal antibodies herein specifically include
chimeric antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity.
[0068] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain a minimal sequence
derived from a non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences.
[0069] These modifications are made to further refine and optimize
antibody performance. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986), Reichmann et
al, Nature. 332:323-329 (1988): and Presta, Curr. Op. Struct.
Biel., 2:593-596 (1992). The humanized antibody includes a
Primatized.TM. antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0070] "Single-chain Fv" or "sFv" antibody fragments comprise the
variable heavy chain (VH) and variable light chain (VL) domains of
an antibody, wherein these domains are present in a single
polypeptide chain. Generally, the Fv polypeptide further comprises
a polypeptide linker between the VH and VL domains which enables
the sFv to form the desired structure for antigen binding. For a
review of sFv see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315 (1994).
[0071] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VD) in the same polypeptide chain (VH-VD). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in Hollinger et al., Proc. Natl.
Acad. Sol. USA, 90:6444-6448 (1993).
[0072] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. The isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, the isolated antibody will be
prepared by at least one purification step.
[0073] As used herein, the expressions "cell", "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and culture derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, this will be clear from the context.
[0074] The terms "polypeptide", "peptide", and "protein", as used
herein, are interchangeable and are defined to mean a biomolecule
composed of amino acids linked by a peptide bond.
[0075] "Homology" between two sequences is determined by sequence
identity. If two sequences which are to be compared with each other
differ in length, sequence identity preferably relates to the
percentage of the nucleotide residues of the shorter sequence which
are identical with the nucleotide residues of the longer sequence.
Sequence identity can be determined conventionally with the use of
computer programs such as the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive Madison, Wis. 53711).
Bestfit utilizes the local homology algorithm of Smith and
Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in
order to find the segment having the highest sequence identity
between two sequences. When using Bestfit or another sequence
alignment program to determine whether a particular sequence has,
for example, 95% identity with a reference sequence of the present
invention, the parameters are preferably adjusted so that the
percentage of identity is calculated over the entire length of the
reference sequence and homology gaps of up to 5% of the total
number of the nucleotides in the reference sequence are permitted.
When using Bestfit, the so-called optional parameters are
preferably left at their preset ("default") values. The deviations
appearing in the comparison between a given sequence and the
above-described sequences of the invention may be caused for
instance by addition, deletion, substitution, insertion or
recombination. Such a sequence comparison can preferably also be
carried out with the program "fasta20u66" (version 2.0u66,
September 1998 by William R. Pearson and the University of
Virginia; see also W.R. Pearson (1990), Methods in Enzymology 183,
63-98, appended examples and the program "The WorkBench" (San Diego
Supercomputer Center)). For this purpose, the "default" parameter
settings may be used.
[0076] As used herein, a "conservative change" refers to
alterations that are substantially conformationally or
antigenically neutral, producing minimal changes in the tertiary
structure of the mutant polypeptides, or producing minimal changes
in the antigenic determinants of the mutant polypeptides,
respectively, as compared to the native protein. When referring to
the antibodies and antibody fragments of the invention, a
conservative change means an amino acid substitution that does not
render the antibody incapable of binding to the subject receptor.
One of ordinary skill in the art will be able to predict which
amino acid substitutions can be made while maintaining a high
probability of being conformationally and antigenically neutral.
Such guidance is provided, for example in Berzofsky, (1985) Science
229:932-940 and Bowie et al. (1990) Science 247: 1306-1310. Factors
to be considered that affect the probability of maintaining
conformational and antigenic neutrality include, but are not
limited to: (a) substitution of hydrophobic amino acids is less
likely to affect antigenicity because hydrophobic residues are more
likely to be located in a protein's interior; (b) substitution of
physiochemically similar, amino acids is less likely to affect
conformation because the substituted amino acid structurally mimics
the native amino acid; and (c) alteration of evolutionarily
conserved sequences is likely to adversely affect conformation as
such conservation suggests that the amino acid sequences may have
functional importance. One of ordinary skill in the art will be
able to assess alterations in protein conformation using well-known
assays, such as, but not limited to microcomplement fixation
methods (see, e.g. Wasserman et al. (1961) J. Immunol. 87:290-295;
Levine et al. (1967) Meth. Enzymol. 11:928-936) and through binding
studies using conformation-dependent monoclonal antibodies (see,
e.g. Lewis et al. (1983) Biochem. 22:948-954).
[0077] The terms "a" and "an" as used herein are defined to mean
"one or more" and include the plural unless the context is
inappropriate.
[0078] The invention is explained in more detail below with the aid
of application examples and implementation examples as well as the
Figures.
[0079] For diagnostic applications, the antibody typically will be
labelled with a detectable moiety. Numerous labels are available
which can be generally grouped into the following categories:
[0080] (a) Radioisotopes, such as 35S, 14C, 125I, 3H, and 131I. The
antibody can be labelled with the radioisotope using the techniques
described in Current Protocols in Immunology, Volumes 1 and 2,
Gutigen et al., Ed., Wiley-Interscience. New York, N.Y. Pubs.,
(1991) for example and radioactivity can be measured using
scintillation counting.
[0081] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra for example. Fluorescence can be quantified using a
fluorimeter.
[0082] (c) Various enzyme-substrate labels are available. Such
enzyme generally catalyses a chemical alteration of the chromogenic
substrate which can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase. O-galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym (ed Langone & H. Van Vunakis), Academic Press,
New York, 73: 147-166 (1981).
[0083] Examples of enzyme-substrate combinations include, for
example:
[0084] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g. orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0085] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and
[0086] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g. p-nitrophenyl-.beta.-D-galactosidase)
or the fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0087] Numerous other enzyme-substrate combinations are available
to those skilled in the art.
[0088] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g. anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0089] The inventive antibodies need not be labeled, and the
presence thereof can be detected using a labeled antibody, which
binds to the inventive antibodies.
[0090] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays (Zola,
Monoclonal Antibodies A Manual of Techniques, pp. 147-158 (CRC
Press. Inc., 1987).
[0091] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of MCP-1 peptide in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte which remain unbound.
[0092] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three-part complex. The second
antibody may itself be labeled with a detectable moiety (direct
sandwich assays) or may be measured using an anti-immunoglobulin
antibody that is labeled with a detectable moiety (indirect
sandwich assay). For example, one preferable type of sandwich assay
is an ELISA assay, in which case the detectable moiety is an
enzyme.
[0093] For immunohistochemistry analysis, the tissue sample may be
fresh or frozen or may be embedded in paraffin and fixed with a
preservative such as formalin, for example.
[0094] Diagnostic Kits
[0095] As a matter of convenience, the antibody of the present
invention can be provided in a kit, i.e., a packaged combination of
reagents in predetermined amounts with instructions for performing
the diagnostic assay. Where the antibody is labelled with an
enzyme, the kit will include substrates and cofactors required by
the enzyme (e.g. a substrate precursor which provides the
detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g. a
blocking buffer or lysis buffer) and the like. The relative amounts
of the various reagents may be varied widely to provide for
concentrations in solution of the reagents which substantially
optimize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients which on dissolution will provide a reagent solution
having the appropriate concentration.
[0096] The diagnostic kit according to the invention may contain a
further biologically active substance as described below.
Especially preferred for the use in the diagnostic kit are
inhibitors of glutaminyl cyclase.
[0097] The diagnostic kit of the invention is especially useful for
the detection and diagnosis of MCP-1-related diseases and
conditions selected from the group consisting of inflammatory
diseases selected from [0098] a. neurodegenerative diseases, e.g.
mild cognitive impairment (MCI), Alzheimer's disease,
neurodegeneration in Down Syndrome, Familial British Dementia,
Familial Danish Dementia, multiple sclerosis, [0099] b. chronic and
acute inflammations, e.g. rheumatoid arthritis, atherosclerosis,
restenosis, pancreatitis, [0100] c. fibrosis, e.g. lung fibrosis,
liver fibrosis, renal fibrosis, [0101] d. cancer, e.g.
cancer/hemangioendothelioma proliferation, gastric carcinomas,
[0102] e. metabolic diseases, e.g. hypertension, [0103] f. and
other inflammatory diseases, e.g. neuropathic pain, graft
rejection/graft failure/graft vasculopathy, HIV infections/AIDS,
gestosis, tuberous sclerosis.
[0104] Preferably, the antibody according to the present invention
is especially useful in a diagnostic method to detect MCP-1-related
disease, e.g. atheroschlerosis, rheumatoid arthritis, asthma,
delayed hypersensitivity reactions, pancreatitis, Alzheimer's
disease, lung fibrosis, renal fibrosis, gestosis, graft rejection,
neuropathic pain, AIDS and tumors.
[0105] Most preferably, the diagnostic kit of the invention is
useful for the detection and diagnosis of Alzheimer's disease, or
also most preferably a disease selected from atherosclerosis,
rheumatoid arthritis, restenosis and pancreatitis, in particular
Alzheimer's disease or rheumatoid arthritis.
[0106] Preferred according to the present invention is a monoclonal
antibody.
[0107] More preferably according to the present invention is a
monoclonal antibody, wherein the variable part of the light chain
of said antibody has a nucleotide sequence selected from SEQ ID
NOs: 33, 37 and 41, or an amino acid sequence selected from SEQ ID
NOs: 34, 38 and 42.
[0108] Alternatively preferred according to the present invention
is a monoclonal antibody, wherein the variable part of the heavy
chain of said antibody has a nucleotide sequence selected from SEQ
ID NOs: 35, 39 and 43, or an amino acid sequence selected from SEQ
ID NOs: 36, 40 and 44.
[0109] Further preferred according to the present invention is the
monoclonal antibody, wherein the variable part of the light chain
of said antibody has the nucleotide sequence of SEQ ID NO: 33 or
the amino acid sequence of SEQ ID NO: 34, and wherein the variable
part of the heavy chain of said antibody has the nucleotide
sequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO:
36.
[0110] Also preferred according to the present invention is the
monoclonal antibody, wherein the variable part of the light chain
of said antibody has the nucleotide sequence of SEQ ID NO: 37 or
the amino acid sequence of SEQ ID NO: 38, and wherein the variable
part of the heavy chain of said antibody has the nucleotide
sequence of SEQ ID NO: 39, or the amino acid sequence of SEQ ID NO:
40.
[0111] Even preferred according to the present invention is the
monoclonal antibody, wherein the variable part of the light chain
of said antibody has the nucleotide sequence of SEQ ID NO: 41 or
the amino acid sequence of SEQ ID NO: 42, and wherein the variable
part of the heavy chain of said antibody has the nucleotide
sequence of SEQ ID NO: 43, or the amino acid sequence of SEQ ID NO:
44.
[0112] In particular preferred is a monoclonal antibody, which is
produced by a hybridoma cell line selected from the following
group
TABLE-US-00003 348/1D4 (Deposit No. DSM ACC 2905) 348/2C9 (Deposit
No. DSM ACC 2906) 332/4B8 (Deposit No. DSM ACC 2907) 332/4F8
(Deposit No. DSM ACC 2908)
[0113] According to a further preferred embodiment, the antibody
can be humanised or is a chimeric antibody or is a human
antibody.
[0114] Further, the antibody as selected from the above-mentioned
group can also be a functional variant of said group.
[0115] In the context of the present invention a "functional
variant" of the inventive antibody is an antibody which retains the
binding capacities, in particular binding capacities with high
affinity to a MCP-1 N1pE-38 or functional variant thereof. The
provision of such functional variants is known in the art and
encompasses the above-mentioned possibilities, which were indicated
under the definition of antibodies and fragments thereof.
[0116] In a preferred embodiment, the antibody is an antibody
fragment, as defined above.
[0117] In a further preferred embodiment, the antibody of the
invention is an antibody which has the complementarity-determining
regions (CDRs) of the above-defined antibodies. Preferably, the
antibody can be labeled; possible labels are those as mentioned
above and all those known to a person skilled in the art of
diagnostic uses of antibodies in particular.
[0118] The present invention further relates to a light chain
variable region comprising an nucleic acid sequence that is 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identical to a sequence selected from SEQ ID NOs: 33, 37 or 41,
or a functional part thereof.
[0119] The present invention further relates to a heavy chain
variable region comprising an nucleic acid sequence that is 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identical to a sequence selected from SEQ ID NOs: 35, 39 or 43,
or a functional part thereof.
[0120] Further preferred according to the present invention is a
monoclonal antibody including any functionally equivalent antibody
or functional parts thereof, which antibody comprises a light chain
variable domain comprising an amino acid sequence that is 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to a sequence selected from SEQ ID NOs: 34, 38 or 42.
[0121] Even preferred according to the present invention is a
monoclonal antibody including any functionally equivalent antibody
or functional parts thereof, which antibody comprises a heavy chain
variable domain comprising an amino acid sequence that is 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to a sequence selected from SEQ ID NOs: 36, 40 or 44.
[0122] Moreover, the present invention relates to a monoclonal
antibody including any functionally equivalent antibody or
functional parts thereof, wherein the variable part of the light
chain of said antibody comprises an amino acid sequence selected
from SEQ ID NOs: 34, 38 and 42 and/or wherein the variable part of
the heavy chain of said antibody comprises an amino acid sequence
selected from SEQ ID NOs: 36, 40 and 44, wherein the antibody has
been altered by introducing at least one, at least two, or at least
3 or more conservative substitutions into at least one of the
sequences of SEQ ID NOs: 34, 36, 38, 40, 42 and 44, wherein the
antibody essentially maintains its full functionality.
[0123] The present invention further relates to an isolated
polynucleotide encoding the light chain variable region of the
monoclonal antibodies presented herein, wherein said isolated
polynucleotide comprises a nucleic acid sequence selected from SEQ
ID Nos: 33, 37 and 41.
[0124] The present invention also relates to an isolated
polynucleotide encoding the heavy chain variable region of the
monoclonal antibodies presented herein, wherein said isolated
polynucleotide comprises a nucleic acid sequence selected from SEQ
ID Nos: 35, 39 and 43.
[0125] Moreover, the present invention relates to an isolated
peptide of the light chain variable region of the monoclonal
antibodies presented herein, wherein said isolated peptide
comprises an amino acid sequence selected from SEQ ID Nos: 34, 38
and 42.
[0126] Moreover, the present invention relates to an isolated
peptide of the heavy chain variable region of the monoclonal
antibodies presented herein, wherein said isolated peptide
comprises an amino acid sequence selected from SEQ ID Nos: 36, 40
and 44.
[0127] In a further preferred embodiment, the present invention
relates to an oligonucleotide selected from the group consisting of
SEQ ID NOs: 7 to 32.
[0128] Preferably, the antibody is immobilised on a solid
phase.
[0129] The present invention also relates to a composition which
comprises the antibody as defined above. In particular, said
composition is a composition for a diagnostic use, especially for
the diagnosis of MCP-1-related diseases, in particular by detection
of MCP-1 N1pE or variants thereof in a biological sample.
[0130] In another embodiment, the antibody according to the
invention and as described herein before or a fragment thereof,
exhibits a binding affinity to MCP-1 N1pE, which is at least 2
times, particularly at least 4 times, particularly at least 10
times, particularly at least 15 times, more particularly at least
20 times, but especially at least 25 times higher than the binding
affinity of conventional antibodies.
[0131] In still another embodiment, a chimeric antibody or a
fragment thereof, or a humanized antibody or a fragment thereof is
provided as described herein before, which antibody substantially
binds to MCP-1 N1pE in the mammalian, particularly the human brain
but, preferably, does not show any significant cross-reactivity
with MCP-1 N1pE, in particular with MCP-1 N1pE 3-38.
[0132] The present invention relates also to humanized forms of the
antibodies as defined above, compositions comprising said humanized
antibodies and the use of said compositions for the treatment of
MCP-1-related diseases, especially for the treatment of Alzheimer's
disease in a mammal, in particular in a human.
[0133] The present invention is also directed to the following
hybridoma cell lines
TABLE-US-00004 DSM ACC 2905 (Hybridoma cell clone 348/1D4) DSM ACC
2906 (Hybridoma cell clone 348/2C9) DSM ACC 2907 (Hybridoma cell
clone 332/4B8) and DSM ACC 2908 (Hybridomac ell clone 332/4F8).
[0134] The present invention also pertains to the use of the
antibody or the composition comprising the antibody, both as
defined above, in an in vitro diagnostic method. In particular,
this diagnostic method is directed to diagnosis of MCP-1-related
diseases, especially by detecting an MCP-1 N1pE or variants thereof
in a biological sample.
[0135] Preferably, said sample is a serum sample.
[0136] According to another preferred embodiment, said sample is a
liquor, cerebrospinal fluid (CSF) or synovial fluid sample.
[0137] In a particularly preferred embodiment, the present
invention pertains to the following method:
[0138] In vitro or in situ diagnostic method for the diagnosis of
an MCP-1-related disease or condition, comprising the following
steps: [0139] a) contacting an antibody according to the invention
with a sample, preferably selected from a serum, liquor or CSF
sample, most preferably a serum sample; or a specific body part or
body area of a subject suspected to be afflicted with said
condition or disease, and [0140] b) detecting binding of the
antibody to an MCP-1 N1pE peptide, from the sample.
[0141] More particularly, the invention relates to a method of
diagnosis of an MCP-1-related disease or condition, comprising
detecting the immunospecific binding of an antibody or an active
fragment thereof to an MCP-1 N1pE peptide, in a sample or in situ
which includes the steps of [0142] (a) bringing the sample or a
specific body part or body area suspected to contain the MCP-1
peptide into contact with an antibody, particularly a monoclonal
antibody according to the present invention, or a chimeric antibody
or a fragment thereof, or a humanized antibody or a fragment
thereof according to the invention and as described herein before,
and/or a functional part thereof; [0143] (b) allowing the antibody
and/or a functional part thereof, to bind to the MCP-1 N1pE peptide
to form an immunological complex; [0144] (c) detecting the
formation of the immunological complex; and [0145] (d) correlating
the presence or absence of the immunological complex with the
presence or absence of MCP-1 N1pE peptide in the sample or specific
body part or area.
[0146] The aforementioned diagnostic methods are especially useful
for the detection and diagnosis of MCP-1-related diseases and
conditions selected from the group consisting of inflammatory
diseases selected from [0147] a. neurodegenerative diseases, e.g.
mild cognitive impairment (MCI), Alzheimer's disease,
neurodegeneration in Down Syndrome, Familial British Dementia,
Familial Danish Dementia, multiple sclerosis, [0148] b. chronic and
acute inflammations, e.g. rheumatoid arthritis, atherosclerosis,
restenosis, pancreatitis, [0149] c. fibrosis, e.g. lung fibrosis,
liver fibrosis, renal fibrosis, [0150] d. cancer, e.g.
cancer/hemangioendothelioma proliferation, gastric carcinomas,
[0151] e. metabolic diseases, e.g. hypertension, [0152] f. and
other inflammatory diseases, e.g. neuropathic pain, graft
rejection/graft failure/graft vasculopathy, HIV infections/AIDS,
gestosis, tuberous sclerosis.
[0153] Preferably, the aforementioned diagnostic methods are useful
to detect MCP-1-related disease, e.g. atheroschlerosis, rheumatoid
arthritis, asthma, delayed hypersensitivity reactions,
pancreatitis, Alzheimer's disease, lung fibrosis, renal fibrosis,
gestosis, graft rejection, neuropathic pain, AIDS and tumors.
[0154] Most preferably, the aforementioned diagnostic methods are
useful for the detection and diagnosis of Alzheimer's disease, or
also most preferably a disease selected from atherosclerosis,
rheumatoid arthritis, restenosis and pancreatitis, in particular
Alzheimer's disease or rheumatoid arthritis.
[0155] In still another embodiment, the invention relates to a
composition comprising the antibody according to the invention, or
a chimeric antibody or a fragment thereof, or a humanized antibody
or a fragment thereof according to the invention and as described
herein before including any functionally equivalent antibody or any
derivative or functional parts thereof, in a therapeutically
effective amount, in particular a composition which is a
pharmaceutical composition optionally further comprising a
pharmaceutically acceptable carrier.
[0156] In another embodiment of the invention, said composition
comprises the antibody in a therapeutically effective amount.
[0157] Further comprised by the invention is a mixture comprising
an antibody, particularly a monoclonal antibody according to the
invention, or a chimeric antibody or a fragment thereof, or a
humanized antibody or a fragment thereof according to the invention
and as described herein before including any functionally
equivalent antibody or any derivative or functional parts thereof,
in a therapeutically effective amount and, optionally, a further
biologically active substance and/or a pharmaceutically acceptable
carrier and/or a diluent and/or an excipient.
[0158] In particular, the invention relates to a mixture, wherein
the further biologically active substance is a compound used in the
medication of a group of diseases and disorders associated with
MCP-1, such as an inflammatory diseases selected from [0159] a.
neurodegenerative diseases, e.g. mild cognitive impairment (MCI),
Alzheimer's disease, neurodegeneration in Down Syndrome, Familial
British Dementia, Familial Danish Dementia, multiple sclerosis,
[0160] b. chronic and acute inflammations, e.g. rheumatoid
arthritis, atherosclerosis, restenosis, pancreatitis, [0161] c.
fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis,
[0162] d. cancer, e.g. cancer/hemangioendothelioma proliferation,
gastric carcinomas, [0163] e. metabolic diseases, e.g.
hypertension, and [0164] f. other inflammatory diseases, e.g.
neuropathic pain, graft rejection/graft failure/graft vasculopathy,
HIV infections/AIDS, gestosis, tuberous sclerosis.
[0165] The other biologically active substance or compound may
exert its biological effect by the same or a similar mechanism as
the antibody according to the invention or by an unrelated
mechanism of action or by a multiplicity of related and/or
unrelated mechanisms of action.
[0166] Generally, the other biologically active compound may
include neutron-transmission enhancers, psychotherapeutic drugs,
acetylcholine esterase inhibitors, calcium-channel blockers,
biogenic amines, benzodiazepine tranquillizers, acetylcholine
synthesis, storage or release enhancers, acetylcholine postsynaptic
receptor agonists, monoamine oxidase-A or -B inhibitors,
N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal
anti-inflammatory drugs, antioxidants, serotonergic receptor
antagonists, CCR2 receptor antagonists and MCP-1 antibodies. With
MCP-1 antibodies as other biologically active agent are meant such
antibodies, which are binding native MCP-1, i.e. where the
N-terminal Glu residue is not cyclized to pGlu.
[0167] More particularly, the invention relates to a mixture
comprising at least one compound selected from the group consisting
of compounds effective against oxidative stress, anti-apoptotic
compounds, metal chelators, inhibitors of DNA repair such as
pirenzepin and metabolites, 3-amino-1-propanesulfonic acid (3 APS),
1,3-propanedisulfonate (1,3PDS), .alpha.-secretase activators,
.beta.- and .gamma.-secretase inhibitors, tau proteins,
neurotransmitter, .beta.-sheet breakers, attractants for amyloid
beta clearing/depleting cellular components, inhibitors of
N-terminal truncated amyloid beta peptides including pyroglutamated
amyloid beta 3-42, such as inhibitors of glutaminyl cyclase,
anti-inflammatory molecules, or cholinesterase inhibitors (ChEIs)
such as tacrine, rivastigmine, donepezil, and/or galantamine, M1
agonists and other drugs including any amyloid or tau modifying
drug and nutritive supplements, and nutritive supplements, together
with an antibody according to the present invention and,
optionally, a pharmaceutically acceptable carrier and/or a diluent
and/or an excipient.
[0168] The invention further relates to a mixture, wherein the
compound is a cholinesterase inhibitor (ChEIs), particularly a
mixture, wherein the compound is one selected from the group
consisting of tacrine, rivastigmine, donepezil, galantamine, niacin
and memantine.
[0169] In a further embodiment, the mixtures according to the
invention may comprise niacin or memantine together with an
antibody according to the present invention and, optionally, a
pharmaceutically acceptable carrier and/or a diluent and/or an
excipient.
[0170] In a further embodiment, the mixtures according to the
invention may comprise a glutaminyl cyclase inhibitor together with
an antibody according to the present invention and, optionally, a
pharmaceutically acceptable carrier and/or a diluent and/or an
excipient.
[0171] Preferred inhibitors of glutaminyl cyclase are described in
WO 2005/075436, in particular examples 1-141 as shown on pp. 31-40.
The synthesis of examples 1-141 is shown on pp. 40-48 of WO
2005/075436. The disclosure of WO 2005/075436 regarding examples
1-141, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0172] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/055945, in particular examples 1-473 as shown
on pp. 46-155. The synthesis of examples 1-473 is shown on pp.
156-192 of WO 2008/055945. The disclosure of WO 2008/055945
regarding examples 1-473, their synthesis and their use as
glutaminyl cyclase inhibitors is incorporated herein by
reference.
[0173] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/055947, in particular examples 1-345 as shown
on pp. 53-118. The synthesis of examples 1-345 is shown on pp.
119-133 of WO 2008/055947. The disclosure of WO 2008/055947
regarding examples 1-345, their synthesis and their use as
glutaminyl cyclase inhibitors is incorporated herein by
reference.
[0174] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/055950, in particular examples 1-212 as shown
on pp. 57-120. The synthesis of examples 1-212 is shown on pp.
121-128 of WO 2008/055950. The disclosure of WO 2008/055950
regarding examples 1-212, their synthesis and their use as
glutaminyl cyclase inhibitors is incorporated herein by
reference.
[0175] Further preferred inhibitors of glutaminyl cyclase are
described in WO2008/065141, in particular examples 1-25 as shown on
pp. 56-59. The synthesis of examples 1-25 is shown on pp. 60-67 of
WO2008/065141. The disclosure of WO2008/065141 regarding examples
1-25, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0176] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/110523, in particular examples 1-27 as shown
on pp. 55-59. The synthesis of examples 1-27 is shown on pp. 59-71
of WO 2008/110523. The disclosure of WO 2008/110523 regarding
examples 1-27, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0177] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128981, in particular examples 1-18 as shown
on pp. 62-65. The synthesis of examples 1-18 is shown on pp. 65-74
of WO 2008/128981. The disclosure of WO 2008/128981 regarding
examples 1-18, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0178] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128982, in particular examples 1-44 as shown
on pp. 61-67. The synthesis of examples 1-44 is shown on pp. 68-83
of WO 2008/128982. The disclosure of WO 2008/128982 regarding
examples 1-44, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0179] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128983, in particular examples 1-30 as shown
on pp. 64-68. The synthesis of examples 1-30 is shown on pp. 68-80
of WO 2008/128983. The disclosure of WO 2008/128983 regarding
examples 1-30, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0180] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128984, in particular examples 1-36 as shown
on pp. 63-69. The synthesis of examples 1-36 is shown on pp. 69-81
of WO 2008/128984. The disclosure of WO 2008/128984 regarding
examples 1-36, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0181] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128985, in particular examples 1-71 as shown
on pp. 66-76. The synthesis of examples 1-71 is shown on pp. 76-98
of WO 2008/128985. The disclosure of WO 2008/128985 regarding
examples 1-71, their synthesis and their use as glutaminyl cyclase
inhibitors is incorporated herein by reference.
[0182] Further preferred inhibitors of glutaminyl cyclase are
described in WO 2008/128986, in particular examples 1-7 as shown on
pp. 65-66. The synthesis of examples 1-7 is shown on pp. 66-73 of
WO 2008/128986. The disclosure of WO 2008/128986 regarding examples
1-7, their synthesis and their use as glutaminyl cyclase inhibitors
is incorporated herein by reference.
[0183] In still another embodiment of the invention mixtures are
provided that comprise "atypical antipsychotics" such as, for
example clozapine, ziprasidone, risperidone, aripiprazole or
olanzapine for the treatment of positive and negative psychotic
symptoms including hallucinations, delusions, thought disorders
(manifested by marked incoherence, derailment, tangentiality), and
bizarre or disorganized behavior, as well as anhedonia, flattened
affect, apathy, and social withdrawal, together with an antibody,
particularly a monoclonal antibody according to the invention, but
particularly a chimeric antibody or a fragment thereof, or a
humanized antibody or a fragment thereof according to the invention
and as described herein and, optionally, a pharmaceutically
acceptable carrier and/or a diluent and/or an excipient.
[0184] In a specific embodiment of the invention, the compositions
and mixtures according to the invention and as described herein
before comprise the antibody and the biologically active substance,
respectively, in a therapeutically effective amount.
[0185] Other compounds that can be suitably used in mixtures in
combination with the antibody according to the present invention
are described in WO 2008/065141 (see especially pages 37/38),
including PEP-inhibitors (pp. 43/44), LiCl, inhibitors of
dipeptidyl aminopeptidases, preferably inhibitors of DP IV or DP
IV-like enzymes (see pp. 48/49); acetylcholinesterase (ACE)
inhibitors (see p. 47), PIMT enhancers, inhibitors of beta
secretases (see p. 41), inhibitors of gamma secretases (see pp.
41/42), inhibitors of neutral endopeptidase, inhibitors of
phosphodiesterase-4 (PDE-4) (see pp. 42/43), TNFalpha inhibitors,
muscarinic M1 receptor antagonists (see p. 46), NMDA receptor
antagonists (see pp. 47/48), sigma-1 receptor inhibitors, histamine
H3 antagonists (se p. 43), immunomodulatory agents,
immunosuppressive agents or an agent selected from the group
consisting of antegren (natalizumab), Neurelan (fampridine-SR),
campath (alemtuzumab), IR 208, NBI 5788/MSP 771 (tiplimotide),
paclitaxel, Anergix.MS (AG 284), SH636, Differin (CD 271,
adapalene), BAY 361677 (interleukin-4),
matrix-metalloproteinase-inhibitors (e.g. BB 76163), interferon-tau
(trophoblastin) and SAIK-MS; beta-amyloid antibodies (see p.44),
cysteine protease inhibitors (see p. 44); MCP-1 antagonists (see
pp. 44/45), amyloid protein deposition inhibitors (see p. 42) and
beta amyloid synthesis inhibitors (see p. 42), which document is
incorporated herein by reference.
[0186] In another embodiment, the invention relates to a mixture
comprising the antibody, particularly a monoclonal antibody
according to the invention, or a chimeric antibody or a fragment
thereof, or a humanized antibody or a fragment thereof according to
the invention and as described herein before and/or the
biologically active substance in a therapeutically effective
amount.
[0187] The invention further relates to the use of an antibody,
particularly a monoclonal antibody according to the invention, but
particularly a chimeric antibody or a fragment thereof, or a
humanized antibody or a fragment thereof according to the invention
and as described herein before and/or a functional part thereof
and/or a pharmaceutical composition, or a mixture comprising said
antibody, for the preparation of a medicament for treating or
alleviating the effects of a group of diseases and disorders
associated with MCP-1 such as diseases including, but not limited
to, neurological disorders such as Alzheimer's Disease (AD), Lewy
body dementia, Down's syndrome, hereditary cerebral hemorrhage with
amyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as
well as other diseases which are based on or associated with
amyloid-like proteins such as progressive supranuclear palsy,
multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease,
HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult
Onset Diabetes; senile cardiac amyloidosis; endocrine tumors, and
others, including macular degeneration.
[0188] More preferably, the present invention relates to the use of
an antibody, particularly a monoclonal antibody according to the
invention, but particularly a chimeric antibody or a fragment
thereof, or a humanized antibody or a fragment thereof according to
the invention and as described herein before and/or a functional
part thereof and/or a pharmaceutical composition, or a mixture
comprising said antibody, for the preparation of a medicament for
treating or alleviating the effects of an inflammatory diseases
selected from [0189] a. neurodegenerative diseases, e.g. mild
cognitive impairment (MCI), Alzheimer's disease, neurodegeneration
in Down Syndrome, Familial British Dementia, Familial Danish
Dementia, multiple sclerosis, [0190] b. chronic and acute
inflammations, e.g. rheumatoid arthritis, atherosclerosis,
restenosis, pancreatitis, [0191] c. fibrosis, e.g. lung fibrosis,
liver fibrosis, renal fibrosis, [0192] d. cancer, e.g.
cancer/hemangioendothelioma proliferation, gastric carcinomas,
[0193] e. metabolic diseases, e.g. hypertension, and [0194] f.
other inflammatory diseases, e.g. neuropathic pain, graft
rejection/graft failure/graft vasculopathy, HIV infections/AIDS,
gestosis, tuberous sclerosis.
[0195] Also comprised by the present invention is a method for the
preparation of an antibody, particularly a monoclonal antibody
according to the invention, but particularly a chimeric antibody or
a fragment thereof, or a humanized antibody or a fragment thereof
according to the invention and as described herein before and/or a
functional part thereof and/or a pharmaceutical composition, or a
mixture comprising said antibody and/or a functional part thereof,
particularly in a therapeutically effective amount, for use in a
method of preventing, treating or alleviating the effects of a
group of diseases and disorders associated with MCP-1 as defined
above comprising formulating an antibody, particularly a monoclonal
antibody according to the invention, but particularly a chimeric
antibody or a fragment thereof, or a humanized antibody or a
fragment thereof according to the invention in a pharmaceutically
acceptable form.
[0196] Further comprised by the present invention is a method for
preventing, treating or alleviating the effects of a group of
diseases and disorders associated with MCP-1 as defined above by
administering an antibody and/or a functional part thereof, but
particularly a humanized antibody and/or a functional part thereof,
or a composition or mixture comprising such an antibody and/or a
functional part thereof, to an animal or a human affected by such a
disorder comprising administering the antibody in a therapeutically
effective amount.
[0197] Administration and Dosage
[0198] The antibody is preferably administered to a mammal in a
carrier; preferably a pharmaceutically-acceptable carrier. Suitable
carriers and their formulations are described in Remington's
Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990; and Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000. Typically, an
appropriate amount of a pharmaceutically acceptable salt is used in
the formulation to render the formulation isotonic. Examples of the
carrier include saline, Ringer's solution and dextrose solution.
The pH of the solution is preferably from about 5 to about 8, and
more preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers may be more preferable depending upon,
for instance, the route of administration and concentration of
antibody being administered.
[0199] The antibody can be administered to the mammal by injection
(e.g., systemic, intravenous, intraperitoneal, subcutaneous,
intramuscular, intraportal, intracerebral,
intracerebral-ventricular, and intranasal), or by other methods,
such as infusion, which ensure its delivery to the bloodstream in
an effective form. The antibody may also be administered by
isolated perfusion techniques, such as isolated tissue perfusion,
to exert local therapeutic effects. Intravenous injection is
preferred.
[0200] Effective dosages and schedules for administering the
antibody may be determined empirically, and making such
determinations is within the skill in the art. Those skilled in the
art will understand that the dosage of antibody that must be
administered will vary depending on, for example, the mammal that
will receive the antibody, the route of administration, the
particular type of antibody used and other drugs being administered
to the mammal. Guidance in selecting appropriate doses for antibody
is found in the literature on therapeutic uses of antibodies, e.g.,
Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges
Publications, Park Ridge, N. J., 1985, ch. 22 and pp. 303-357;
Smith et al. Antibodies in Human Diagnosis and Therapy, Haber et
al., eds., Raven Press, New York, 1977, pp. 365-389. A typical
daily dosage of the antibody used alone might range from about 1
.mu.g/kg to up to 100 mg/kg of body weight or more per day,
depending on the factors mentioned above. Generally, any of the
following doses may be used: a dose of at least about 50 mg/kg body
weight; at least about 10 mg/kg body weight; at least about 3 mg/kg
body weight; at least about 1 mg/kg body weight; at least about 750
.mu.g/kg body weight; at least about 500 .mu.g/kg body weight; at
least about 250 ug/kg body weight; at least about 100 .mu.g/kg body
weight; at least about 50 .mu.g/kg body weight; at least about 10
ug/kg body weight; at least about 1 .mu.g/kg body weight, or more,
is administered. Antibodies may be administered at lower doses or
less frequent at the beginning of the treatment to avoid potential
side effect.
[0201] In some embodiments, more than one antibody may be present.
Such compositions may contain at least one, at least two, at least
three, at least four, at least five different antibodies (including
polypeptides) of the invention.
[0202] The antibody may also be administered to the mammal in
combination with effective amounts of one or more other therapeutic
agents. The antibody may be administered sequentially or
concurrently with the one or more other therapeutic agents. The
amounts of antibody and therapeutic agent depend, for example, on
what type of drugs are used, the pathological condition being
treated, and the scheduling and routes of administration but would
generally be less than if each were used individually.
[0203] Following administration of antibody to the mammal, the
mammal's physiological condition can be monitored in various ways
well known to the skilled practitioner. The above principles of
administration and dosage can be adapted for polypeptides described
herein.
[0204] A polynucleotide encoding an antibody or a polypeptide
described herein may also be used for delivery and expression of
the antibody or the polypeptide in a desired cell. It is apparent
that an expression vector can be used to direct expression of the
antibody. The expression vector can be administered systemically,
intraperitoneally, intravenously, intramuscularly, subcutaneously,
intrathecally, intraventricularly, orally, enterally, parenterally,
intranasally, dermally, or by inhalation. For example,
administration of expression vectors includes local or systemic
administration, including injection, oral administration, particle
gun or catheterized administration, and topical administration. One
skilled in the art is familiar with administration of expression
vectors to obtain expression of an exogenous protein in vivo. See,
e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.
[0205] Targeted delivery of therapeutic compositions comprising a
polynucleotide encoding an antibody of the invention can also be
used. Receptor-mediated DNA delivery techniques are described in,
for example, Findeis et al., Trends Biotechnol. (1993) 11:202;
Chiou et al., Gene Therapeutics: Methods And Applications Of Direct
Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem.
(1988) 263:621; Wu et al., J. Biol Chem. (1994) 269:542; Zenke et
al, Proc. Natl. Acad. Sd. (USA) (1990) 87:3655; Wu et al., J. Biol.
Chem. (1991) 266:338. Therapeutic compositions containing a
polynucleotide are administered in a range of about 100 ng to about
200 mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g
to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g
to about 100 .mu.g of DNA can also be used during a gene therapy
protocol. The therapeutic polynucleotides and polypeptides of the
present invention can be delivered using gene delivery vehicles.
The gene delivery vehicle can be of viral or non-viral origin (see
generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kirnura, Human
Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995)
1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of
such coding sequences can be induced using endogenous mammalian or
heterologous promoters. Expression of the coding sequence can be
either constitutive or regulated.
[0206] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740; 4,777,127; GB
Patent No. 2,200,651; and EP 0 345 242), alphavirus-based vectors
(e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67;
ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV)
vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769;
WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).
Administration of DNA linked to killed adenovirus as described in
Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
[0207] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J
Biol. Chem. (1 989) 264: 16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968. Additional
approaches are described in Philip, MoI Cell Biol (1994) 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
[0208] In some embodiments, the numbers expressing quantities of
ingredients, properties such as molecular weight, reaction
conditions, and so forth, used to describe and claim certain
embodiments of the invention are to be understood as being modified
in some instances by the term "about." Accordingly, in some
embodiments, the numerical parameters set forth in the written
description and attached claims are approximations that can vary
depending upon the desired properties sought to be obtained by a
particular embodiment. In some embodiments, the numerical
parameters should be construed in light of the number of reported
significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of some embodiments of the invention are
approximations, the numerical values set forth in the specific
examples are reported as precisely as practicable. The numerical
values presented in some embodiments of the invention may contain
certain errors necessarily resulting from the standard deviation
found in their respective testing measurements.
[0209] In some embodiments, the terms "a" and "an" and "the" and
similar references used in the context of describing a particular
embodiment of the invention (especially in the context of certain
of the following claims) can be construed to cover both the
singular and the plural. The recitation of ranges of values herein
is merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range.
Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided with respect to
certain embodiments herein is intended merely to better illuminate
the invention and does not pose a limitation on the scope of the
invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential
to the practice of the invention.
[0210] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member can be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. One or more members of a group can be included in, or
deleted from, a group for reasons of convenience or patentability.
When any such inclusion or deletion occurs, the specification is
herein deemed to contain the group as modified thus fulfilling the
written description of all Markush groups used in the appended
claims.
[0211] All publications, patents, patent applications, and other
references cited in this application are incorporated herein by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application or other
reference was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes.
Citation of a reference herein shall not be construed as an
admission that such is prior art to the present invention.
[0212] Having described the invention in detail, it will be
apparent that modifications, variations, and equivalent embodiments
are possible without departing the scope of the invention defined
in the appended claims. Furthermore, it should be appreciated that
all examples in the present disclosure are provided as non-limiting
examples.
EXAMPLES
[0213] The following non-limiting examples are provided to further
illustrate the present invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
that follow represent approaches the inventors have found function
well in the practice of the invention, and thus can be considered
to constitute examples of modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
Preparing and Characterizing Monoclonal Antibodies which are
Directed Against MCP-1 N1pE
[0214] The aim was the generation of monoclonal antibodies reactive
with the pE-P-D-A (SEQ ID No:3) containing amino acid sequence at
the amino terminus of the peptide MCP-1 N1pE-38 (SEQ ID No:4)
(which is MCP-1 N1pE with the first 38 amino acids starting from
the N-terminus), but not reactive with the peptide MCP-1 D3-38 (SEQ
ID No:5) which is the same molecule as MCP-1 N1pE-38 but lacking pE
and P at the amino terminus.
[0215] For immunisations the peptide pE-P-D-A-I-N-A-P-V-C-amide
(human MCP-1 N1pE-9 (SEQ ID No:6)) was used. This low molecular
weight antigen was conjugated to Bovine Serum Albumin (Purified
Fraction V BSA; Pierce) as carrier protein using Sulfo-MBS (Pierce)
as cross-linker.
[0216] To generate the monoclonal antibodies 8-week-old female
BALB/c mice were immunised with the peptide-BSA-conjugate in two
different immunisation procedures as shown in table 1:
TABLE-US-00005 TABLE 1 Immunisation protocol for generation of
monoclonal MCP-1 N1pE antibodies Long Time Short Time Immunisation
Immunisation Doses (Day) (Day) Injection (.mu.g/mouse) Adjuvant 1 1
Priming 100 TiterMax Gold (i.p.) Adjuvant (Sigma Alrich, St. Louis,
USA) 30 14 Boost 100 TiterMax Gold (i.p.) Adjuvant (Sigma) 60 21
Boost 50 Incomplete (i.p.) Freund's Adjuvant (Sigma) 90 28 Boost 50
Incomplete (i.p.) Freund's Adjuvant (Sigma) 126 35 Boost 50 PBS
(i.v.) 129 38 Fusion
[0217] For example, 100 .mu.g peptide corresponds to 50 .mu.l of
peptide-BSA-conjugate dissolved in PBS. The peptide-BSA-conjugate
was emulsified in an equal volume of TiterMax Gold Adjuvant (Sigma)
or incomplete Freund's adjuvant and injected as stable emulsion
intraperitoneally (i.p.). Three days before the fusion experiment
was performed, each mouse received a total dose of 50 .mu.g peptide
(25 .mu.l peptide-BSA-conjugate) dissolved in 25 .mu.l PBS given as
i.v. injection.
[0218] The presence of the desired antibody was detected in the
sera of the recipient prior to the final booster dose using the
enzyme linked immunosorbent assay (ELISA) with human MCP-1 N1pE-9
as immobilized antigen. The specific antibody titres were greater
than 1:200000.
[0219] For fusion procedures, 6.times.10.sup.7 spleen cells from
the immunised mice and 2.times.10.sup.7 cells from mouse myeloma
cell line SP2/0 were incubated with 1.2 ml of 50% polyethylene
glycol (Sigma) for 30 seconds at 37.degree. C. After washing, the
cells were seeded in four 96-well cell culture plates. Hybrid
clones were selected by growing in HAT medium [RPMI 1640 culture
medium (Biochrom, Berlin, DE) supplemented with 20% fetal calf
serum (PAN Biotech GmbH, Aigenbach, DE) and HAT-Supplement
(50.times.; PAN)].
[0220] The culture supernatants were primarily screened for antigen
specific IgG antibodies two weeks after fusion. The presence of an
antigen specific antibody in the culture supernatants was measured
by its binding to the following peptides: [0221] human MCP-1 1-9,
[0222] human MCP-1 N1pE-38 or [0223] human MCP-1 D3-38, especially,
attached directly to the wells of a 96-well plate. The antibody
binding was quantified by adding the relevant anti-species
immunoglobulin (rabbit anti-mouse IgG (HRP) Fc-specific antibody,
Pierce, Rockford, USA) to which an enzyme is bound, followed by a
chromogenic substrate to that enzyme. Fresh culture medium and a
dilution of the polyclonal mouse antiserum were used as negative or
as positive controls.
[0224] The specific antibody producing hybridoma colonies were
transferred into 24-well plates for cell propagation and were
tested again. The hybridomas repeated positive for human MCP-1
N1pE-38 and negative for human MCP-1 D3-38 were additionally tested
by SPR analysis (Biacore 3000).
[0225] The best clones which were selected from those showing high
association and low dissociation of bound hMCP1 N1pE-38 and
additionally showing no binding to the negative control peptide
MCP1 D3-38, were then cloned and recloned by limiting-dilution
technique, characterized and frozen. For the isotype
characterization the Mouse Monoclonal Antibody Isotyping Kit
(Roche) was used.
[0226] Two weeks after fusion of splenocytes from a Balb/c mouse
immunized with the human MCP-1 N1pE-9-BSA-conjugate (short time
immunisation protocol) 44 cell culture supernatants were primarily
tested positive for the peptide human MCP-1 N1pE-38 as well as for
IgG and negative for the peptide human MCP-1 D3-38. 33 out of 44
hybrids were repeated positive after transferring in 24-well plates
and were also tested by SPR analysis (Biacore 3000). 8 hybridomas
were cloned by limiting dilution resulting in 18 specific clones
from which 8 were recloned. Thereby, two cell lines were
established which produced antibodies with strong reactivity to the
peptide human MCP-1 N1pE-38 and good binding characteristics as
demonstrated by SPR analysis (Biacore 3000). The resulting
antibodies were designated 332-4B8 and 332-4F8 and both monoclonals
332-4B8 and 332-4F8 belong to the IgG class with the isotype
IgG1.
[0227] The fusion of the spleen cells of the long time immunized
Balb/c mice with cells from mouse myeloma cell line SP2/0 resulted
in 35 cell culture supernatants primarily tested positive for human
MCP-1 N1pE-38 as well as for IgG. Out of these 35 primary positive
hybridomas 15 were repeated positive after transferring in 24-well
plates and were further tested by SPR analysis (Biacore 3000). 4
hybridomas were cloned resulting in 8 specific clones from which 4
were recloned. Finally, two cell lines with a reactivity to the
peptide human MCP-1 N1pE-38 but not human MCP-1 D3-38 were
established as demonstrated by SPR analysis (Biacore 3000). These
two cell lines 348-1D4 and 348-2C9 belong to the IgG class with the
isotype IgG2b.
[0228] From each of the above clones 10 mg of Protein-G purified
antibody was produced and subjected to further characterization
experiments. The properties of the various MCP-1 N1pE monoclonal
antibodies which were prepared can be taken from the following
examples.
Example 2
SPR Analysis (Biacore 3000) of Generated Monoclonal Antibodies
Directed Against MCP-1 N1pE
[0229] Protein-G purified monoclonal antibodies 332-4B8, 332-4F8,
348-1D4 and 348-2C9 were characterized with regard to their binding
characteristics to human MCP1 N1pE-38 by SPR analysis. These
analyses were performed on a Biacore 3000. To this avail, a CM5
chip was coated with approximately 100 response units (RU) human
MCP1 N1pE-38 peptide on flow cell (Fc) 2. Fc 4 was coated with 100
RU human MCP1 D3-38 peptide. Fc1 and Fc3 were prepared for blank
subtraction. Monoclonal antibodies were diluted in running buffer
HBS-EP (Hepes buffered saline+3 mM EDTA+0.005% (v/v) surfactant
P20, Biacore, Freiburg, DE) at concentrations ranging from 20
.mu.g/ml to 1 .mu.g/ml. First, a basal signal was determined with
HBS-EP, followed by 180 seconds of application of antibody
dilution, to determine association of antibody to antigen. Then
pure HBS-EP was injected again for another 180 sec to determine the
dissociation rate of the corresponding antibody. Finally, the
Biacore CM5 chip was regenerated by a short injection of 0.1M HCl,
to remove all residing antibody.
[0230] Blank signals from Fc 1 and Fc 3 were subtracted from
signals of Fc 2 and Fc 4, respectively.
[0231] Results:
[0232] All tested monoclonal antibodies failed to associate to
human MCP-1 D3-38 (data not shown). Regarding association to human
MCP-1 N1pE-38, the monoclonal antibodies exhibited different
binding characteristics.
[0233] The strongest association to human MCP-1 N1pE-38 was
demonstrated by 332-4B8. At a concentration of 20 .mu.g/ml nearly
2000 RU could be monitored. Dissociation was almost 0 at a
concentration of 5 .mu.g/ml, demonstrating an extremely strong
binding of 332-4B8 to its antigen (FIG. 1A).
[0234] Also, monoclonal antibody 332-4F8 showed a very stable
binding to human MCP-1 N1pE-38. However, this antibody clone
achieved only about 150 RU within 180 seconds association time. On
the other hand, no dissociation occurred at all concentrations
tested (FIG. 1B).
[0235] Monoclonal antibody clones 348-2C9 and 348-1D4 exhibited
almost equal binding characteristics. At 20 .mu.g/ml an association
signal of approximately 500 RU was monitored for both clones.
Dissociation rate decreased with lower antibody concentrations.
Although dissociation was also observable at 1 .mu.g/ml of
antibody, the measured signals stayed well above basal line. (FIG.
1C+FIG. 1D).
[0236] Taken together the results provide evidence that all
monoclonal antibodies tested are able to interact with their
corresponding antigen MCP-1 N1pE.
Example 3
Dot Blot Analysis of Generated Monoclonal Antibodies Directed
Against MCP-1 N1pE
[0237] Next it was tested, whether the differences in binding
kinetics, as determined by SPR analysis are also evident in an
experimental situation where an employed antibody is allowed to
interact with its antigen for a prolonged period of time.
[0238] A simple DotBlot protocol was accomplished to obtain a
general idea about the sensitivity of MCP-1 N1pE antibody clones
toward the respective native peptide. Human MCP-1 N1pE-38 and human
MCP-1 D3-38 peptides in descending concentrations (1000 ng-20 ng)
were spotted onto small pieces of nitrocellulose membranes. For
analysis, membranes were blocked for two hours with TBST-M (=TBST
(Tris buffered saline+0.05% Tween-20)+5% skimmed milk) at room
temperature with gentle shaking. Membranes were incubated over
night at 4.degree. C. on a rocking platform with the individual
MCP-1 N1pE antibody clones diluted to 1 .mu.g/ml in equal volumes
of TBST-M. Secondary anti-mouse antibody conjugated with alkaline
phosphatase was used for signal detection, following standard
procedures.
[0239] Results:
[0240] As seen in FIG. 2A-2D, all antibody clones tested revealed
almost equal results in the Dot Blot analysis. MCP-1 N1pE-38
peptide concentrations down to 20 ng were clearly detected by
protein-G purified monoclonal antibodies 332-4B8, 332-4F8, 348-1D4
and 348-2C9. None of the antibody clones generated cross reactivity
with MCP-1 D3-38.
Example 4
PepSpot Analysis of Generated Monoclonal Antibodies Directed
Against MCP-1 N1pE
[0241] To determine specificity and selectivity of MCP-1 N1pE
antibody clones in more detail, PepSpot analysis was performed.
[0242] Corresponding PepSpot membranes were prepared by JPT Peptide
Technologies GmbH, Berlin (JPT). On these membranes, peptides with
the indicated amino acid sequences (see FIG. 3; Z represents pE)
were immobilized at a concentration of 1 .mu.g/spot.
[0243] For analysis, membranes were blocked for two hours with
TBST-M (=TBST+5% skimmed milk) at room temperature with gentle
shaking. Membranes were incubated over night at 4.degree. C. on a
rocking platform with the individual MCP-1 N1pE antibody clones
diluted to 1 .mu.g/ml in equal volumes of TBST-M. Secondary
anti-mouse antibody conjugated with alkaline phosphatase was used
for signal detection, following standard procedures.
[0244] Results:
[0245] As seen in FIG. 3 all four antibody clones tested are highly
selective for MCP-1 N1pE peptides. Strong signals were obtained on
spots containing MCP-1 N1pE peptides starting with the first 4
amino acids of MCP-1 N1pE (underlined sequences).
[0246] In addition, spots with peptides starting with the first
three amino acids of MCP-1 N1pE (spots 10, 13 and 14) were clearly
recognized by all four antibody clones. Antibodies failed to
recognize a peptide starting with only two of the amino terminal
amino acids of MCP-1 N1pE (spot 12). Antibodies also failed to
recognize peptide spots starting with amino acids different than pE
(Z). In case of spot 3, spontaneous formation of pE from Q cannot
be excluded. Therefore, signals obtained with this peptide most
likely reflect binding of antibody to spontaneously formed pE.
[0247] Taken together the results demonstrate that all four MCP-1
N1pE antibody clones require the first 3-4 amino acids of MCP-1
N1pE for binding to the corresponding antigen.
Example 5
Examination of Cross Reactivity to Other Peptides Possessing N1pE
Residues by SPR Analysis
[0248] In order to determine selectivity of the anti MCP-1 N1pE
antibodies, cross reactivity to other human Peptides, possessing a
N-terminal pE residue was analyzed by surface plasmon
resonance.
[0249] Therefore, the following peptides or there N-terminal
regions were immobilized on the surface of CM5-Chips: MCP-1, MCP-2,
big gastrin, gonadoliberin, neurotensin, orexin A, fibronectin,
collagen 1 and TRH. As positive control also the binding to MCP-1
N1pE-38 was analyzed. The monoclonal antibodies 332-4B8, 332-4F8,
348-1D4 and 348-2C9 were diluted in HBS-EP (Biacore) down to 25
.mu.g/ml. Cross reactivity was observed using a Biacore 3000 with
several CM5-Chips, on which the respective peptides were
immobilized. The system was run with 20 .mu.l/min. Measured bulk
effects and unspecific reactions to the chip surface were corrected
by subtraction of the signal of flow cell 2, 3 or 4, at which the
tested peptides were immobilized, with the empty flow cell 1. The
association (9 min) was obtained by injection of 180 .mu.l of the
antibody clones. The dissociation was observed over 9 min.
Remaining antibody molecules were removed by injection of 5 .mu.l
0.1 M HCL. For every interaction of the antibody with the different
peptides the association and dissociation was recorded. Cross
reactivity was determined by evaluation of the association phase
concerning rate and signal at the end.
[0250] Table 2 shows, that the monoclonal antibodies are specific
for the MCP-1 N1pE epitope. No cross reactivity with the analysed
peptides was observed.
TABLE-US-00006 TABLE 2 Investigation of cross reactivity of the
monoclonal antibodies 332-4B8, 332-4F8, 348-1D4 and 348-2C9 to
several human peptides covering a N-terminal pGlu (pE) residue by
SPR analysis. pGlu Peptides % cross reactivity MCP-1 (1-38) 100
MCP-2 <1 MCP-1 (2-38) <1 MCP-1 (3-38 <1 Abeta pE3-40 <1
Big Gastrin <1 Gonadoliberin <1 Neurotensin <1 Orexin A
<1 Fibronectin <1 Collagen 1 <1 TRH <1
Example 6
Determining K.sub.D Values of Monoclonal Antibodies Directed
Against MCP-1 N1pE
[0251] In order to analyze binding kinetics of the MCP-1 N1pE
antibody clones to hMCP-1 N1pE-38, the association constant K,
dissociation constant K.sub.D, reaction enthalpy .DELTA.H as well
as reaction entropy .DELTA.S have been determined.
[0252] The binding affinities of the anti MCP1 N1pE antibodies
348-1D4 and 332-4B8 to the antigen hMCP-1 N1pE-38 were determined
using VP-ITC microcalorimeter (MicroCal). Both antibody clones as
well as the hMCP-1 N1pE-38 peptide were dialyzed against 150 mM
NaCl, 25 mM Na.sub.2HPO.sub.4, 25 mM KH.sub.2PO.sub.4, 2 mM EDTA pH
7.4 overnight at 4.degree. C. to ensure the same buffer conditions
and avoid background heat by protonation events. Afterwards the
concentration of the antibodies and the peptide was calculated from
absorbance at 280 nm and the respective extinction coefficient. For
the titration experiment with clone 348-1D4, Antibody and hMCP-1
N1pE-38 were used at concentrations of 4.38 .mu.M and 147 .mu.M,
respectively. For the titration experiment with clone 332-4B8,
Antibody and hMCP-1 N1pE-38 were used at concentrations of 1.86
.mu.M and 64.3 .mu.M, respectively. The binding heat was recorded
at 20.degree. C. by titration of 29 injections of 10 .mu.l of
antigen into the antibody solution. In order to evaluate the heat
development originated by the dilution of the hMCP-1 N1pE-38
peptide, this value was determined by titration into the dialysis
buffer using defined conditions and instrument setup. Plotting of
data occurred by MicroCal ORIGIN software. The calculated binding
heat was corrected by the heat originated by dilution of the
antigen. The resulting curve was fitted by the "One Set of Sites"
binding model. With this model, the stoichiometry, association
constant, reaction enthalpy and reaction entropy can be
calculated.
[0253] Results:
[0254] FIG. 16 shows the resulting fitting curves and the values
calculated for stoichiometry, association constant, reaction
enthalpy and reaction entropy. In addition, Table 3 gives an
overview about the obtained data.
TABLE-US-00007 TABLE 3 Parameter obtained for stoichiometry,
association and dissociation constant, reaction enthalpy and
reaction entropy after titration of the antigen hMCP-1 N1pE-38 to
the monoclonal antibodies 348-1D4 and 332-4B8. 348-1D4 332-4B8
Stoichiometry (N) 1.98 1.60 Association Constant 3.81 .times.
10.sup.6 4.27 .times. 10.sup.6 (K) in M.sup.-1 Dissociation .sup.
2.6 .times. 10.sup.-7 .sup. 2.34 .times. 10.sup.-7 Constant
(K.sub.D) in M Reaction Enthalpy -1.123 .times. 10.sup.4 -1.823
.times. 10.sup.4 (.DELTA.H) in cal/mol Reaction Entropy -8.20 -31.8
(.DELTA.S) in cal/mol*K
Example 7
Detection of Recombinant Human MCP-1 N1pE in an ELISA by Using
Monoclonal Antibodies Directed Against MCP-1 N1pE
[0255] With the techniques presented so far, selective detection of
MCP-1 N1pE could be clearly demonstrated. Therefore the presented
antibody clones were also tested for their applicability in tools
for potential diagnostic implications, like ELISA.
[0256] Consequently, an ELISA protocol was accomplished that
allowed detection of recombinant hMCP-1 N1pE.
[0257] To capture human MCP-1, commercially available polyclonal
antiserum (goat anti-hMCP1-AF (R&D Systems, Minneapolis, USA)
as capture antibody which specifically binds human MCP-1 was
immobilized in polystyrene 96-well microtitre plates. Unbound
capture antibody was washed off the plate. After a blocking step,
recombinant hMCP-1 N1pE diluted in blocking buffer was added to the
wells. After an incubation period of 2 hours at room temperature,
plates were washed at least three times with TBS-T. For detection,
MCP-1 N1pE antibody clones (332-4B8, 348-1D4, 348-2C9,
respectively) together with HRP-conjugated anti mouse antibody were
diluted in blocking buffer, added to the micro titre plate and
incubated for 1 hour at room temperature. Following several washes
with TBS-T a colour reaction with commercially available HRP
substrate TMB (SureBlue Reserve TMB Microwell Peroxidase Substrate
(1-component) (KPL, Gaithersburg, USA) was performed (30 minutes
incubation at room temperature in the dark) and subsequently
stopped by the addition of 1.2N H.sub.2SO.sub.4. Absorption was
determined by a Tecan Sunrise plate reader.
[0258] Results:
[0259] The anti MCP-1 antibodies 332-4B8, 348-1D4 and 348-2C9 are
able to detect recombinant human MCP-1 in a concentration dependent
manner. Thereby, the antibody clones 348-2C9 and 348-1D4 turned out
to be much more sensitive in comparison to 332-4B8 (FIG. 4).
Example 8
Detection of MCP-1 N1pE by ELISA in Human Serum Using Monoclonal
Antibodies Directed Against MCP-1 N1pE
[0260] Since recombinant hMCP-1 N1pE can be quantitatively detected
in an ELISA by using the monoclonal anti MCP-1 N1pE antibodies of
the present invention, the detection of native hMCP-1 N1pE in human
serum was tested.
[0261] The ELISA protocol corresponds to Example 7, except the
usage of FBS, 0.05% Tween, 10% FBS for blocking and dilution
steps.
[0262] Results:
[0263] All MCP-1 N1pE antibody clones tested, generated very strong
signals in the established ELISA set up. Signals decreased with the
dilution factor of the serum sample (FIG. 5; Table 4).
TABLE-US-00008 TABLE 4 Detection of human MCP-1 N1pE from human
serum by monoclonal antibodies 332-4B8, 348-1D4 and 348-2C9 in
ELISA Dilution of Human Serum MCP-1 N1pE 1:5 1:10 1:20 Antibody
Absorption/SD Absorption/SD Absorption/SD Clone 2.1/0.054 1.5/0.048
0.9/0.008 332-4B8 2.4/0.012 2.1/0.048 1.4/0.033 348-1D4 2.5/0.036
2.2/0.042 1.4/0.034 348-2C9
[0264] Although clone 332-4B8 demonstrated more favourable binding
characteristics in SPR analysis (Biacore), clones 348-1D4 and
348-2C9 gave higher signals in the ELISA. According to the obtained
data, all antibody clones tested are well suitable for ELISA
applications.
[0265] Additionally, these data demonstrate that MCP-1 N1pE is
detectable also in human serum of healthy individuals.
Example 9
Spike and Recovery of hMCP-1 N1pE in Human Serum
[0266] Spike and Recovery experiments were performed in order to
validate the quantitative detection of hMCP1 N1pE in human
serum.
[0267] The ELISA protocol corresponds to Example 8, for detection
of hMCP-1 N1pE the antibody 348-2C9 was used. For validation of
Spike and Recovery various levels of recombinant hMCP-1 N1pE were
spiked in human serum. Recovery was calculated by subtracting the
hMCP-1 N1pE value measured in the unspiked serum sample from the
spiked samples.
[0268] Results:
[0269] Table 5 shows Spike and Recovery data in human serum
obtained with the 348-2C9 antibody. A Recovery of the spiked hMCP-1
N1pE peptides of 66%-79.4% was found.
TABLE-US-00009 TABLE 5 Spike and Recovery of hMCP-1 N1pE in human
serum. This table shows the expected spike level in comparison to
observed hMCP-1 N1pE concentrations. Expected Spike Level Observed
Spike Level Observed Spike of hMCP1 N1pE of hMCP1 N1pE Level of
hMCP1 [ng/ml] [ng/ml] N1pE in % 6 4.76 79.37 3 2.09 69.80 1.5 1.05
69.81 0.75 0.50 66.00 0.38 0.26 69.89
[0270] These data confirm, that the monoclonal antibody 348-2C9 can
be used for the quantitative detection of hMCP1 N1pE in human
serum.
Example 10
Detection of Human MCP-1 N1pE in Cell Culture Supernatants of
Stimulated NHDF Cells by ELISA
[0271] Following an inflammatory stimulus, the expression of hMCP-1
is enhanced in Human Normal Dermal Fibroblasts (NHDF). Hence, it
can be assumed that also MCP-1 N1pE level are elevated. If this
holds true, the amount of MCP-1 N1pE should increase after
application of Oncostatin M (OSM) and Interleukin 1.beta.
(IL1.beta.) to NHDF.
[0272] To prove this, cell culture supernatants of OSM and
IL1.beta. stimulated NHDF were subjected to an ELISA analysis as
described in Example 7. The antibody 348-1D4 was used for detection
of hMCP1 N1pE. NHDF have been stimulated over 14 days and analyzed
at different time points in order to examine time dependency of
hMCP-1 N1pE secretion.
[0273] Results:
[0274] Following the inflammatory stimulus of OSM and IL1.beta.
application, the amount of hMCP1 N1pE increases in a time dependent
manner (FIG. 6). These data show, that hMCP-1 N1pE can also be
quantitatively detected in cell culture supernatant of NHDF.
Example 11
Detection of Human MCP-1 N1pE in Cell Culture Supernatant of LPS
Stimulated Human Acute Monocytic Leukemia Cell Line (THP1) in the
Presence of QC Inhibitor QCI
[0275] As demonstrated in Examples 8-10, native hMCP-1 N1pE can be
quantitatively detected in human serum as well as in cell culture
supernatants by ELISA, using the monoclonal anti hMCP-1 N1pE
antibodies of the present invention. As glutaminyl cyclase (QC) is
a prerequisite for MCP-1 N1pE formation on cellular level,
inhibition of QC should consequently result in decreased MCP-1 N1pE
level.
[0276] To prove this, the Human Acute Monocytic Leukemia Cell Line
(THP1) was stimulated 24 h with LPS in absence or presence of
increasing concentrations of the QC inhibitor QCI. Cell culture
supernatants were subjected to ELISA analysis as described in
Example 7. The antibody 348-1D4 was used for detection of hMCP1
N1pE.
[0277] Results:
[0278] FIG. 7 shows, that the amount of hMCP-1 N1pE decreases with
increasing concentrations of QC inhibitor in cell culture
supernatant of THP1 cells.
Example 12
Detection of Recombinant Mouse MCP-1 N1pE in an ELISA by Using
Monoclonal Antibodies Directed Against MCP-1 N1pE
[0279] Example 7 shows the concentration dependent detection of
recombinant human MCP-1 N1pE by the anti MCP-1 antibodies 332-4B8,
348-1D4 and 348-2C9. Since the four N-terminal amino acids of mouse
and human MCP-1 are homologue, the quantitative detection of
recombinant mouse MCP-1 was further analysed.
[0280] Consequently, an ELISA protocol was accomplished that
allowed the detection of recombinant mouse MCP-1 N1pE.
[0281] To capture mouse MCP-1, commercially available polyclonal
antiserum (rabbit anti mJE (Peprotech, Rocky Hill, USA) as capture
antibody which specifically binds mouse MCP-1 was immobilized in
polystyrene 96-well microtitre plates. Unbound capture antibody was
washed off the plate. After a blocking step, recombinant mMCP-1
N1pE diluted in blocking buffer was added to the wells. After an
incubation period of 2 hours at room temperature, plates were
washed at least three times with TBS-T. For detection, MCP-1 N1pE
antibody clones (332-4B8, 348-1D4, 348-2C9, respectively) together
with HRP-conjugated anti mouse antibody were diluted in blocking
buffer, added to the micro titre plate and incubated for 1 hour at
room temperature. Following several washing steps with TBS-T a
colour reaction with commercially available HRP substrate TMB
(SureBlue Reserve TMB Microwell Peroxidase Substrate (1-component)
(KPL, Gaithersburg, USA) was performed (30 minutes incubation at
room temperature in the dark) and subsequently stopped by the
addition of 1.2N H.sub.2SO.sub.4. Absorption was determined by a
Tecan Sunrise plate reader.
[0282] Results:
[0283] The anti MCP-1 antibodies 332-4B8, 348-1D4 and 348-2C9 are
able to detect recombinant mouse MCP-1 in a concentration dependent
manner. Similar to the results obtained with human hMCP-1, the
antibody clones 348-2C9 and 348-1D4 turned out to be much more
sensitive in comparison to 332-4B8 (FIG. 8).
Example 13
Detection of Mouse MCP-1 N1pE in Cell Culture Supernatants of a
Stimulated Murine Macrophage Cell Line RAW 264.7 by ELISA
[0284] Following an inflammatory stimulus, the expression of mMCP-1
is enhanced in RAW 264.7 cells. Hence, it can be assumed that also
MCP-1 N1pE level are elevated. If this holds true, the amount of
mMCP-1 N1pE should increase after application of LPS.
[0285] To prove this, cell culture supernatants of LPS stimulated
RAW 264.7 were subjected to an ELISA analysis as described in
Example 12. The antibody 348-2C9 was used for detection of mMCP1
N1pE. RAW 264.7 cells have been stimulated for 24h with 10 ng
LPS.
[0286] Results:
[0287] Following the inflammatory stimulus of LPS application, the
amount of mMCP1 N1pE increases tremendously (FIG. 9). These data
show that mMCP-1 N1pE can also be quantitatively detected in cell
culture supernatant of RAW 264.7 cells.
Example 14
Detection of Murine MCP-1 N1pE by ELISA in Cell Culture
Supernatants of LPS Stimulated Murine Macrophage Cell Line RAW
264.7 in the Presence of QC Inhibitor QCI Using Monoclonal Antibody
348-2C9
[0288] As demonstrated in Example 13, the mMCP-1 N1pE level
increases respectably after an inflammatory stimulus like LPS. It
could further be shown in Example 11, that the anti MCP-1 N1pE
antibodies can be used to demonstrate that the human MCP-1 N1pE
level decreases with increasing concentrations of QCI. It has now
further been examined if this effect can also be detected in the
mouse Macrophage cell line RAW 264.7.
[0289] The mouse macrophage cell line RAW 264.7 was stimulated with
LPS in the absence or presence of increasing concentrations of the
QC inhibitor QCI. Cell culture supernatants were subjected to ELISA
analysis as described in Example 12. For detection the antibody
348-2C9 was used.
[0290] Results:
[0291] As postulated before, mMCP-1 N1pE level drops in the
presence of the QC inhibitor QCI in LPS stimulated mouse
macrophages. The decrease of the signal is strictly dependent on
the concentration of QCI (see FIG. 10).
Example 15
Detection of Murine MCP-1 N1pE in Serum of Healthy Mice Versus LPS
Treated Mice by ELISA
[0292] Examples 12-14 show the quantitative detection of
recombinant mMCP1 N1pE as well as native mMCP1 N1pE in cell culture
supernatant by ELISA. As presented in Examples 8-9, the anti MCP-1
N1pE antibodies can also be used for the detection of MCP-1 N1pE in
human serum. Further, the level of MCP-1 N1pE is now determined in
mouse serum.
[0293] The ELISA protocol corresponds to Example 12, except the
usage of FBS, 0.05% Tween, 10% FBS for blocking and dilution steps
and the usage of the antibody 348-2C9 for detection.
[0294] Results:
[0295] The mMCP-1 N1pE level in mouse serum increases by LPS
stimulation, depending on the time period of stimulation from 400
pg/ml up to 900 ng/ml (FIG. 11). This experiment shows that the
antibody 348-2C9 can also be used for the quantitative detection of
mMCP-1 N1pE in murine serum.
Example 16
Examination of Dilution Linearity for the Detection of mMCP-1 N1pE
in Murine Peritoneal Lavage Fluid by ELISA
[0296] In order to examine the applicability of the anti MCP-1 N1pE
antibodies in the established ELISA, the dilution linearity of
Peritoneal Lavage Samples from mice treated with Thioglycollate was
analyzed.
[0297] The ELISA protocol corresponds to Example 12. For detection,
the antibody 348-2C9 was used. To determine assay linearity, each
sample was serially diluted with ELISA Blocker to produce values
that are within the assay range.
[0298] Results:
[0299] FIG. 12 depicts, that the analysis of different sample
dilutions results in similar mMCP-1 N1pE levels with deviations of
15% maximum. This experiment demonstrates, that the anti MCP-1 N1pE
antibodies can be used for the analysis of MCP-1 N1pE level in
mouse peritoneal lavage fluid.
Example 17
Usage of Anti MCP-1 N1pE Antibodies in Western Blot Analysis and
Comparison to Data Obtained by ELISA
[0300] Examples 3-4 reveal, that the antibodies 332-4F8, 332-4B8,
348-1D4 and 348-2C9 recognize at least the first 4 amino acids of
MCP-1 N1pE in Dot Blot and PepSpot analysis. In this experiment, it
should be tested whether the antibodies can be used for the
detection of native mouse MCP-1 N1pE in cell culture supernatant of
RAW 264.7. Furthermore, it should be tested, whether the obtained
Western Blot data can confirm the data obtained by ELISA.
[0301] For Western Blot analysis, cell culture supernatants of RAW
264.7 cells were subjected to SDS-gelelektrophoresis. Separated
proteins were transferred electrically to a nitrocellulose
membrane. After blocking of the membrane for two hours with TBST-M
at room temperature, antibody incubation occurred over night at
4.degree. C. with the anti MCP-1 N1pE clone 332-4B8 and an antibody
recognizing total MCP-1 (goat anti MCP-1, R&D Systems) diluted
to 1 .mu.g/ml in TBST-M. Secondary goat anti mouse antibody
conjugated with horseradish peroxidase was used for signal
detection, following standard procedures.
[0302] The ELISA protocol corresponds to Example 14.
[0303] Results:
[0304] As shown in FIG. 13B, there is no change in the Western Blot
signal intensity generated by the antibody goat anti Mouse MCP-1
for the detection of total mMCP-1. However, the Western Blot signal
of mMCP-1 N1pE is concentration dependent (FIG. 13A) and correlates
with the corresponding ELISA data (FIG. 13C), showing the amount of
mMCP-1 N1pE. These data show on the one hand that the anti MCP-1
N1pE antibody 332-4B8 can be used for Western Blot analysis.
Furthermore, the correctness of the ELISA data by Western Blot
analysis was confirmed.
Example 18
Detection of Recombinant Rat MCP-1 N1pE in an ELISA by Using
Monoclonal Antibodies Directed Against MCP-1 N1pE
[0305] Example 12 shows the concentration dependent detection of
recombinant mouse MCP-1 N1pE by the anti MCP-1 antibodies 332-4B8,
348-1D4 and 348-2C9. The N-terminal sequences of mouse and rat
MCP-1 are homologue. Therefore, the quantitative detection of
recombinant rat MCP-1 was analysed.
[0306] Consequently, an ELISA protocol was accomplished that
allowed the detection of recombinant rat MCP-1 N1pE.
[0307] To capture rat MCP-1, commercially available polyclonal
antiserum (rabbit polyclonal to MCP-1 [LS-054182/13136], LifeSpan
Biosciences, Seattle, USA) as capture antibody which specifically
binds rat MCP-1 was diluted with PBS to 250 ng/ml and immobilized
in polystyrene 96-well microtitre plates. Unbound capture antibody
was washed off the plate. After a blocking step, recombinant rMCP-1
N1pE diluted in PBS, 0.05% Tween, 10% FBS was added to the wells.
After an incubation period of 2 hours at room temperature, plates
were washed at least three times with TBS-T. For detection, the
MCP-1 N1pE antibody clone 348-2C9 together with HRP-conjugated anti
mouse antibody were diluted in PBS, 0.05% Tween, 10% FBS, added to
the micro titre plate and incubated for 1 hour at 4.degree. C.
Following several washes with TBS-T a colour reaction with
commercially available HRP substrate TMB (SureBlue Reserve TMB
Microwell Peroxidase Substrate (1-component) (KPL, Gaithersburg,
USA) was performed (30 minutes incubation at room temperature in
the dark) and subsequently stopped by the addition of 1.2N
H.sub.2SO.sub.4. Absorption was determined by a Tecan Sunrise plate
reader.
[0308] Results:
[0309] The anti MCP-1 antibody 348-2C9 is able to detect
recombinant rat MCP-1 in a concentration dependent manner in an
ELISA (FIG. 14).
Example 19
Spike and Recovery of rMCP-1 N1pE in Rat Serum
[0310] Example 18 shows the quantitative detection of recombinant
rat MCP-1 by the antibody 348-2C9 in an ELISA. In order to proof
whether rMCP-1 N1pE can also be detected in rat serum and to
validate this ELISA method, Spike and Recovery experiments were
performed.
[0311] The ELISA protocol corresponds to Example 18, for validation
of Spike and Recovery various levels of recombinant rat MCP-1 N1pE
were spiked in serum of LPS treated rats. Recovery was calculated
by subtracting the rMCP-1 N1pE value measured in the unspiked serum
sample from the spiked samples.
[0312] Results:
[0313] Table 6 shows Spike and Recovery data in rat serum obtained
with the 348-2C9 antibody. A Recovery of the spiked rMCP-1 N1pE
peptides of 65.5%-96.2% was found.
TABLE-US-00010 TABLE 6 Spike and Recovery of rMCP-1 N1pE in serum
of LPS stimulated rats. This table shows the expected spike level
in comparison to observed rMCP-1 N1pE concentrations. Expected
Spike Level Observed Spike Level Observed Spike of rMCP1 N1pE of
rMCP1 N1pE Level of rMCP1 [ng/ml] [ng/ml] N1pE in % 2000 1485
74.23% 1000 962 96.20% 500 327 65.49% 250 212 84.83%
[0314] These data confirm, that the monoclonal antibody 348-2C9 can
be used for the quantitative detection of rat MCP1 N1pE in rat
serum.
Example 20
Sequencing Antibody Variable Regions
[0315] Cultivation of Hybridoma Cells:
[0316] Hybridoma cells were grown in D-MEM (+L-Glutamin,
+Na-Pyruvat, 4.5 g/l Glucose, Gibco) with the addition of 15% FBS,
1% MEM-NEA (non essential amino acids, Gibco), 50 .mu.g/ml
Gentamycin (Gibco) and 50 .mu.M .beta.-mercaptoethanol at
37.degree. C. and 5% CO.sub.2. Subcultivation occurred after 3-4
days depending on cell density. Cells were seeded in a
concentration of 0.5.times.10.sup.6 cells/ml, splitting occurred at
a cell density of 2-5.times.10.sup.6 cells/ml.
[0317] cDNA Synthesis and Reverse Transcription:
[0318] Total RNA was isolated from 2.times.10.sup.6 cells according
to the manual of the NucleospinRNA Isolation Kit (Macherey-Nagel).
100 ng RNA were applied for cDNA synthesis by using Oligo
(dT).sub.15 primer (Promega) and SuperScript III Reverse
Transcriptase (Invitrogen).
[0319] PCR-Amplification of Heavy and Light Chain Variable
Regions:
[0320] Heavy chain variable regions were amplified from the
template cDNA by using Phusion.TM. High-Fidelity DNA Polymerase
(NEW ENGLAND BioLabs) with the primer MHCG1 (in case of clone 5-5-6
and 6-1-6) and MHCG2b (clone 17-4-3 and 24-2-3) in combination with
primers MHV1-12. For amplification of light chain variable regions
the primer MKC in combination with the primers MKV1-MKV11 were
used.
[0321] Cloning of PCR Products in pJET1.2:
[0322] Heavy and light chain variable regions, amplified by PCR,
were cloned into pJET1.2/blunt vector according to the protocol of
CloneJET.TM. PCR Cloning Kit (Fermentas). Sequencing occurred with
pJET1.2 sequencing primers. The primer sequences are shown in Table
7.
TABLE-US-00011 TABLE 7 Primer sequences SEQ ID Primer Sequence NO.
MKV1 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG 7 MKV2
ATGGAGWCAGACACACTCCTGYTATGGGTG 8 MKV3
ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 9 MKV4
ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 10 MKV5
ATGGATTTWCAGGTGCAGATTWTCAGCTTC 11 MKV6 ATGAGGTKCYYTGYTSAGYTYCTGRGG
12 MKV7 ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 13 MKV8
ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 14 MKV9 ATGGTRTCCWCASCTCAGTTCCTTG
15 MKV10 ATGTATATATGTTTGTTGTCTATTTCT 16 MKV11
ATGGAAGCCCCAGCTCAGCTTCTCTTCC 17 MKC ACTGGATGGTGGGAAGATGG 18 MHV1
ATGAAATGCAGCTGGGGCATSTTCTTC 19 MHV2 ATGGGATGGAGCTRTATCATSYTCTT 20
MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT 21 MHV4 ATGRACTTTGGGYTCAGCTTGRTTT
22 MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT 23 MHV6
ATGGCTTGTCYTRGSGCTRCTCTTCTGC 24 MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT 25
MHV8 ATGAGAGTGCTGATTCTTTTGTG 26 MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG
27 MHV10 ATGGGCAGACTTACATTCTCATTCCTG 28 MHV11
ATGGATTTTGGGCTGATTTTTTTTATTG 29 MHV12 ATGATGGTGTTAAGTCTTCTGTACCTG
30 MHCG1 CAGTGGATAGACAGATGGGGG 31 MHCG2b CAGTGGATAGACTGATGGGGG
32
[0323] Results:
[0324] The following sequences were identified:
TABLE-US-00012 Clone 1D4 Light chain variable part, nucleotide
sequence (SEQ ID NO: 33)
ATGGAGTCACAGTTTCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGA
TGACCCAGACTCCCCTCAGTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTC
AAGTCAGAGCCTCTTAGATAGTGCTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGC
CAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCA
CTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGG
AGTTTATTACTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA
ATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Light chain
variable part, protein sequence (SEQ ID NO: 34)
MESQFLFLLVLWIRETNGDVVMTQTPLSLSVTIGQPASISCKSSQSLLDSAGKTYLSWLL
QRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWT
FGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variable part, nucleotide
sequence (SEQ ID NO: 35)
ATGGAATGGAGCGGGGTCTTTCTCTTCCTCTTGTCAGGAACTGCAGGTGTCCACTCTGAG
GTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCC
TGTAAGGCTTCTGGATACACATTCACTGACTACTACATGGACTGGGTGAAGCAGAGCCAT
GGAGAAAGCTTTGAGTGCATTGGACGTGTTAATCCTTACAATGGTGGTACTAGCTACAAC
CAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATG
GAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTCGGTAGT
AGCTACCGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCA
TCAGTCT Heavy chain variable part, protein sequence (SEQ ID NO: 36)
MEWSGVFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMDWVKQSH
GESFECIGRVNPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARLGS
SYRWGQGTTLTVSSAKTTPPSV Clone 2C9 Light chain variable part,
nucleotide sequence (SEQ ID NO: 37)
ATGGTGTCCTCAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGT
GATGTTGTGATGACCCAGACTCCCCTCAGTTTGTCGGTTACCATTGGACAACCAGCCTCC
ATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGCTGGAAAGACATATTTGAGTTGG
TTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGAC
TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATC
AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCTGGCAAGGTACACATTTTCCG
TGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTA
TCCATCTTCCCACCATCCAGT Light chain variable part, protein sequence
(SEQ ID NO: 38)
MVSSAQFLFLLVLWIRETNGDVVMTQTPLSLSVTIGQPASISCKSSQSLLDSAGKTYLSW
LLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP
WTFGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variable part, nucleotide
sequence (SEQ ID NO: 39)
ATGGAATGGAGCGGGATCTTTATCTTCCTCTTGTCAGGAACTGCAGGTGTCCACTCTGAG
GTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCC
TGTAAGGCTTCTGGATACACATTCACTGACTACTACATGGACTGGGTGAAGCAGAGCCAT
GGAGAAAGCTTTGAGTGCATTGGACGTGTTAATCCTTACAATGGTGGTACTAGCTACAAC
CAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATG
GAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCTCGGTAGT
AGCTACCGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCA
TCAGTCTATCCACTG Heavy chain variable part, protein sequence (SEQ ID
NO: 40)
MEWSGIFIFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMDWVKQSH
GESFECIGRVNPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARLGS
SYRWGQGTTLTVSSAKTTPPSVYPL Clone 4B8 Light chain variable part,
nucleotide sequence (SEQ ID NO: 41)
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTT
TGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAA
ATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCA
GGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTTCAACCGATTTTCTGGGGTCCCAGACAGGT
TCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCT
GGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTG
GAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Light chain
variable part, protein sequence (SEQ ID NO: 42)
MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCKSSQSIVHSNGNTYLEWY
LQKPGQSPKLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPY
TFGGGTKLEIKRADAAPTVSIFPPSS Heavy chain variable part, nucleotide
sequence (SEQ ID NO: 43)
ATGGGATGGAGCGGGGTCTTTATTTTAATCCTGTCAGTAACTACAGGTGTCCACTCTGAG
GTCCAGCTGCAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCC
TGCAAGGCTTCTGGTTACTCATTCACTGGCTACAACATGAACTGGGTGAAGCAGAACAAT
GGAAAGAGCCTTGAGTGGATTGGAAATATTACTCCTTACTATGGTAGTACTAGCTACAAC
CAGAAGTTCAAGGGCAGGGTCACATTGACTGTGGACAAATCCTCCAGCACAGCCTACATG
CAGCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGCCTCCTATGGTTACGA
CGGGGGGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCC
AAAACGACACCCCCATCTGTCTATCCACTG Heavy chain variable part, protein
sequence (SEQ ID NO: 44)
MGWSGVFILILSVTTGVHSEVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNN
GKSLEWIGNITPYYGSTSYNQKFKGRVTLTVDKSSSTAYMQLKSLTSEDSAVYFCLLWLR
RGDYAMDYWGQGTSVTVSSAKTTPPSVYPL
Example 21
Application of MCP-1 N1pE Antibody Clones for
Immunohistochemistry
[0325] With the antibodies of the present invention, MCP-1 N1pE was
stained in brain sections of rats after microinjection of
A.beta.(3-49), LPS or NaCl. The stained brain sections are shown in
FIG. 15. FIG. 15 shows that the antibodies 332-4B8, 348-1D4 and
348-2C9 of the present invention are suitable for
immunohistochemistry. The antibodies specifically detect MCP-1 N1pE
in brain of rats.
[0326] Deposits
[0327] Monoclonal antibodies specifically recognizing MCP-1 N1pE,
were generated. Currently all corresponding monoclonal antibodies
expressing hybridoma cell clones 348/1D4, 348/2C9, 332/4B8 and
332/4F8 have been deposited in accordance with the Budapest Treaty
and are available at the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen (DSMZ) (German Collection of Microorganisms and Cell
Cultures) GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany,
with a deposit date of May 6, 2008, and with the respective deposit
numbers
TABLE-US-00013 DSM ACC 2905 (Hybridoma cell clone 348/1D4) DSM ACC
2906 (Hybridoma cell clone 348/2C9) DSM ACC 2907 (Hybridoma cell
clone 332/4B8) and DSM ACC 2908 (Hybridoma cell clone 332/4F8).
[0328] Specificity of those antibodies for their respective target
sequences could be confirmed. For MCP-1 N1pE, high affinity
antibody clones could be identified that should give strong signals
in an ELISA set up with an expected detection limit in the low pg
range.
Sequence CWU 1
1
44199PRTHomo sapiens 1Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu
Leu Ile Ala Ala Thr 1 5 10 15 Phe Ile Pro Gln Gly Leu Ala Gln Pro
Asp Ala Ile Asn Ala Pro Val 20 25 30 Thr Cys Cys Tyr Asn Phe Thr
Asn Arg Lys Ile Ser Val Gln Arg Leu 35 40 45 Ala Ser Tyr Arg Arg
Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60 Ile Phe Lys
Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln 65 70 75 80 Lys
Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90
95 Pro Lys Thr 2300DNAHomo sapiens 2atgaaagtct ctgccgccct
tctgtgcctg ctgctcatag cagccacctt cattccccaa 60gggctcgctc agccagatgc
aatcaatgcc ccagtcacct gctgttataa cttcaccaat 120aggaagatct
cagtgcagag gctcgcgagc tatagaagaa tcaccagcag caagtgtccc
180aaagaagctg tgatcttcaa gaccattgtg gccaaggaga tctgtgctga
ccccaagcag 240aagtgggttc aggattccat ggaccacctg gacaagcaaa
cccaaactcc gaagacttga 30034PRTHomo
sapiensMISC_FEATURE(1)..(1)pyroglutamate 3Glu Pro Asp Ala 1
438PRTHomo sapiensMISC_FEATURE(1)..(1)pyroglutamate 4Glu Pro Asp
Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr 1 5 10 15 Asn
Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr 20 25
30 Ser Ser Lys Cys Pro Lys 35 536PRTHomo sapiens 5Asp Ala Ile Asn
Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr Asn Arg 1 5 10 15 Lys Ile
Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr Ser Ser 20 25 30
Lys Cys Pro Lys 35 610PRTHomo
sapiensMISC_FEATURE(1)..(1)pyroglutamate 6Glu Pro Asp Ala Ile Asn
Ala Pro Val Cys 1 5 10 730DNAArtificial sequenceSynthetic
oligonucleotide 7atgaagttgc ctgttaggct gttggtgctg
30830DNAArtificial sequenceSynthetic oligonucleotide 8atggagwcag
acacactcct gytatgggtg 30930DNAArtificial sequenceSynthetic
Oligonucleotide 9atgagtgtgc tcactcaggt cctggsgttg
301033DNAArtificial sequenceSynthetic Oligonucleotide 10atgaggrccc
ctgctcagwt tyttggmwtc ttg 331130DNAArtificial sequenceSynthetic
Oligonucleotide 11atggatttwc aggtgcagat twtcagcttc
301227DNAArtificial sequenceSynthetic Oligonucleotide 12atgaggtkcy
ytgytsagyt yctgrgg 271331DNAArtificial sequenceSynthetic
Oligonucleotide 13atgggcwtca agatggagtc acakwyycwg g
311431DNAArtificial sequenceSynthetic Oligonucleotide 14atgtggggay
ctktttycmm tttttcaatt g 311525DNAArtificial sequenceSynthetic
Oligonucleotide 15atggtrtccw casctcagtt ccttg 251627DNAArtificial
sequenceSynthetic Oligonucleotide 16atgtatatat gtttgttgtc tatttct
271728DNAArtificial sequenceSynthetic Oligonucleotide 17atggaagccc
cagctcagct tctcttcc 281820DNAArtificial sequenceSynthetic
Oligonucleotide 18actggatggt gggaagatgg 201927DNAArtificial
sequenceSynthetic Oligonucleotide 19atgaaatgca gctggggcat sttcttc
272026DNAArtificial sequenceSynthetic Oligonucleotide 20atgggatgga
gctrtatcat sytctt 262127DNAArtificial sequenceSynthetic
Oligonucleotide 21atgaagwtgt ggttaaactg ggttttt 272225DNAArtificial
sequenceSynthetic Oligonucleotide 22atgractttg ggytcagctt grttt
252330DNAArtificial sequenceSynthetic Oligonucleotide 23atggactcca
ggctcaattt agttttcctt 302428DNAArtificial sequenceSynthetic
Oligonucleotide 24atggcttgtc ytrgsgctrc tcttctgc
282526DNAArtificial sequenceSynthetic Oligonucleotide 25atggratgga
gckggrtctt tmtctt 262623DNAArtificial sequenceSynthetic
Oligonucleotide 26atgagagtgc tgattctttt gtg 232730DNAArtificial
sequenceSynthetic Oligonucleotide 27atggmttggg tgtggamctt
gctattcctg 302827DNAArtificial sequenceSynthetic Oligonucleotide
28atgggcagac ttacattctc attcctg 272928DNAArtificial
sequenceSynthetic Oligonucleotide 29atggattttg ggctgatttt ttttattg
283027DNAArtificial sequenceSynthetic Oligonucleotide 30atgatggtgt
taagtcttct gtacctg 273121DNAArtificial sequenceSynthetic
Oligonucleotide 31cagtggatag acagatgggg g 213221DNAArtificial
sequenceSynthetic Oligonucleotide 32cagtggatag actgatgggg g
2133435DNAMus musculus 33atggagtcac agtttctgtt tctgttagtg
ctctggattc gggaaaccaa cggtgatgtt 60gtgatgaccc agactcccct cagtttgtcg
gttaccattg gacaaccagc ctccatctct 120tgcaagtcaa gtcagagcct
cttagatagt gctggaaaga catatttgag ttggttgtta 180cagaggccag
gccagtctcc aaagcgccta atctatctgg tgtctaaact ggactctgga
240gtccctgaca ggttcactgg cagtggatca gggacagatt tcacactgaa
aatcagcaga 300gtggaggctg aggatttggg agtttattac tgctggcaag
gtacacattt tccgtggacg 360ttcggtggag gcaccaagct ggaaatcaaa
cgggctgatg ctgcaccaac tgtatccatc 420ttcccaccat ccagt 43534145PRTMus
musculus 34Met Glu Ser Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg
Glu Thr 1 5 10 15 Asn Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser
Leu Ser Val Thr 20 25 30 Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu 35 40 45 Asp Ser Ala Gly Lys Thr Tyr Leu
Ser Trp Leu Leu Gln Arg Pro Gly 50 55 60 Gln Ser Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly 65 70 75 80 Val Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95 Lys Ile
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp 100 105 110
Gln Gly Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 115
120 125 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro
Ser 130 135 140 Ser 145 35427DNAMus musculus 35atggaatgga
gcggggtctt tctcttcctc ttgtcaggaa ctgcaggtgt ccactctgag 60gtccagctgc
aacagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatgtcc
120tgtaaggctt ctggatacac attcactgac tactacatgg actgggtgaa
gcagagccat 180ggagaaagct ttgagtgcat tggacgtgtt aatccttaca
atggtggtac tagctacaac 240cagaagttca agggcaaggc cacattgact
gttgacaagt cctccagcac agcctacatg 300gagctcaaca gcctgacatc
tgaggactct gcggtctatt actgtgcaag gctcggtagt 360agctaccgct
ggggccaagg caccactctc acagtctcct cagccaaaac aacaccccca 420tcagtct
42736142PRTMus musculus 36Met Glu Trp Ser Gly Val Phe Leu Phe Leu
Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr
Met Asp Trp Val Lys Gln Ser His Gly Glu Ser Phe 50 55 60 Glu Cys
Ile Gly Arg Val Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn 65 70 75 80
Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85
90 95 Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala
Val 100 105 110 Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Tyr Arg Trp Gly
Gln Gly Thr 115 120 125 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro
Pro Ser Val 130 135 140 37441DNAMus musculus 37atggtgtcct
cagctcagtt cctgtttctg ttagtgctct ggattcggga aaccaacggt 60gatgttgtga
tgacccagac tcccctcagt ttgtcggtta ccattggaca accagcctcc
120atctcttgca agtcaagtca gagcctctta gatagtgctg gaaagacata
tttgagttgg 180ttgttacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 240tctggagtcc ctgacaggtt cactggcagt
ggatcaggga cagatttcac actgaaaatc 300agcagagtgg aggctgagga
tttgggagtt tattactgct ggcaaggtac acattttccg 360tggacgttcg
gtggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta
420tccatcttcc caccatccag t 44138147PRTMus musculus 38Met Val Ser
Ser Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg 1 5 10 15 Glu
Thr Asn Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser 20 25
30 Val Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
35 40 45 Leu Leu Asp Ser Ala Gly Lys Thr Tyr Leu Ser Trp Leu Leu
Gln Arg 50 55 60 Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr 100 105 110 Cys Trp Gln Gly Thr His
Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro 130 135 140 Pro Ser
Ser 145 39435DNAMus musculus 39atggaatgga gcgggatctt tatcttcctc
ttgtcaggaa ctgcaggtgt ccactctgag 60gtccagctgc aacagtctgg acctgagctg
gtgaagcctg gggcttcagt gaagatgtcc 120tgtaaggctt ctggatacac
attcactgac tactacatgg actgggtgaa gcagagccat 180ggagaaagct
ttgagtgcat tggacgtgtt aatccttaca atggtggtac tagctacaac
240cagaagttca agggcaaggc cacattgact gttgacaagt cctccagcac
agcctacatg 300gagctcaaca gcctgacatc tgaggactct gcggtctatt
actgtgcaag gctcggtagt 360agctaccgct ggggccaagg caccactctc
acagtctcct cagccaaaac aacaccccca 420tcagtctatc cactg 43540145PRTMus
musculus 40Met Glu Trp Ser Gly Ile Phe Ile Phe Leu Leu Ser Gly Thr
Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Asp Trp Val
Lys Gln Ser His Gly Glu Ser Phe 50 55 60 Glu Cys Ile Gly Arg Val
Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys
Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala
Tyr Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110
Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Tyr Arg Trp Gly Gln Gly Thr 115
120 125 Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
Pro 130 135 140 Leu 145 41438DNAMus musculus 41atgaagttgc
ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60gttttgatga
cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc
120tcttgcaaat ctagtcagag cattgtacat agtaatggaa acacctattt
agaatggtac 180ctgcagaaac caggccagtc tccaaagctc ctgatctaca
aagttttcaa ccgattttct 240ggggtcccag acaggttcag tggcagtgga
tcagggacag atttcacact caagatcagc 300agagtggagg ctgaggatct
gggagtttat tactgctttc aaggttcaca tgttccgtac 360acgttcggag
gggggaccaa gctggaaata aaacgggctg atgctgcacc aactgtatcc
420atcttcccac catccagt 43842146PRTMus musculus 42Met Lys Leu Pro
Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser
Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile 35
40 45 Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys
Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Phe Asn
Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser 145
43450DNAMus musculus 43atgggatgga gcggggtctt tattttaatc ctgtcagtaa
ctacaggtgt ccactctgag 60gtccagctgc agcagtctgg acctgagctg gagaagcctg
gcgcttcagt gaagatatcc 120tgcaaggctt ctggttactc attcactggc
tacaacatga actgggtgaa gcagaacaat 180ggaaagagcc ttgagtggat
tggaaatatt actccttact atggtagtac tagctacaac 240cagaagttca
agggcagggt cacattgact gtggacaaat cctccagcac agcctacatg
300cagctcaaga gcctgacatc tgaggactct gcagtctatt tctgcctcct
atggttacga 360cggggggact atgctatgga ctactggggt caaggaacct
cagtcaccgt ctcctcagcc 420aaaacgacac ccccatctgt ctatccactg
45044150PRTMus musculus 44Met Gly Trp Ser Gly Val Phe Ile Leu Ile
Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Glu Lys 20 25 30 Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Gly Tyr Asn
Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu 50 55 60 Glu Trp
Ile Gly Asn Ile Thr Pro Tyr Tyr Gly Ser Thr Ser Tyr Asn 65 70 75 80
Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Ser 85
90 95 Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala
Val 100 105 110 Tyr Phe Cys Leu Leu Trp Leu Arg Arg Gly Asp Tyr Ala
Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Ala Lys Thr Thr Pro 130 135 140 Pro Ser Val Tyr Pro Leu 145 150
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