U.S. patent application number 10/565741 was filed with the patent office on 2006-11-16 for use of soluble cd164 in inflammatory and/or autoimmune disorders.
This patent application is currently assigned to Applied Research Systems ARS Holding N.V.. Invention is credited to Yolande Chvatchko.
Application Number | 20060257402 10/565741 |
Document ID | / |
Family ID | 34112463 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257402 |
Kind Code |
A1 |
Chvatchko; Yolande |
November 16, 2006 |
Use of soluble cd164 in inflammatory and/or autoimmune
disorders
Abstract
The present invention relates to novel therapeutic uses of
soluble proteins comprising the extracellular region of humans
CD164, in particular for treating inflammatory and/or autoimmune
disorders.
Inventors: |
Chvatchko; Yolande;
(Confignon, CH) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Assignee: |
Applied Research Systems ARS
Holding N.V.
Pietermaai 15
Curacao
NL
|
Family ID: |
34112463 |
Appl. No.: |
10/565741 |
Filed: |
July 23, 2004 |
PCT Filed: |
July 23, 2004 |
PCT NO: |
PCT/EP04/51596 |
371 Date: |
January 23, 2006 |
Current U.S.
Class: |
424/144.1 ;
424/185.1 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 37/06 20180101; A61P 37/04 20180101; A61P 17/06 20180101; A61P
37/02 20180101; A61P 29/00 20180101; A61K 38/177 20130101; A61P
37/00 20180101; A61P 11/00 20180101; A61P 25/08 20180101; A61P 1/16
20180101; A61P 43/00 20180101; A61P 9/00 20180101 |
Class at
Publication: |
424/144.1 ;
424/185.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2003 |
EP |
03077316.2 |
Claims
1-23. (canceled)
24. A method of treating inflammatory and/or autoimmune diseases
comprising the administration of a composition comprising a soluble
protein comprising a sequence having at least 85% of homology with
the mature form of the extracellular domain of human CD 164 (SEQ ID
NO: 1).
25. The method according to claim 24, wherein said soluble protein
is chosen from: a) SEQ ID NO: 1; or b) SEQ ID NO: 1 fused to the
signal sequence of human CD164.
26. The method according to claim 24, wherein said soluble protein
is an active mutein or an isoform of SEQ ID NO: 1.
27. The method according to claim 26, wherein said soluble protein
is chosen from: a) MGC-24 (SEQ ID NO: 6); or b) the mature form of
the extracellular domain of any of the following human CD 164
isoforms: CD164-delta 4 (SEQ ID NO: 4), CD164-delta 5 (SEQ ID NO:
5).
28. The method according to claim 24, wherein said soluble protein
is glycosylated.
29. The method according to claim 28, wherein said soluble protein
is glycosylated at any of the positions as set forth in SEQ ID NO:
1.
30. The method according to claim 24, wherein said soluble protein
is phosphorylated.
31. The method according to claim 30, wherein said soluble protein
is phosphorylated at any of the positions as set forth in SEQ ID
NO: 1.
32. The method according to claim 24, wherein said soluble protein
is myristoylated.
33. The method according to claim 32, wherein said soluble protein
is myristoylated at any of the positions as set forth in SEQ ID NO:
1.
34. The method according to claim 24, wherein said soluble protein
is a soluble fusion protein.
35. The method according to claim 34, wherein said soluble fusion
protein comprises a signal sequence.
36. The method according to claim 34, wherein said soluble fusion
protein contains a Histidine tag.
37. The method according to claim 36, wherein said soluble fusion
protein is SEQ ID NO: 2.
38. The method according to claim 34, wherein said soluble fusion
protein comprises an Fc region of an immunoglobulin.
39. The method according to claim 24, wherein said soluble protein
is an active derivative, a proteolysis-resistant modified form, a
conjugate, a complex, a fraction, a precursor, and/or a salt.
40. The method according to claim 24, wherein said inflammatory
and/or autoimmune disease is selected from the group consisting of:
multiple sclerosis, systemic lupus erythematosus, rheumatoid
arthritis, juvenile idiopathic arthritis, psoriatic arthritis,
osteoarthritis, spondylarthropathies, inflammatory bowel disease,
endotoxemia, Crohn's disease, Still's disease, uveitis, Wegener's
granulomatosis, Behcet's disease, scleroderma, Sjogren's syndrome,
sarcoidosis, pyodema gangrenosum, polymyositis, dermatomyositis,
myocarditis, psoriasis, systemic sclerosis, hepatitis C, allergies,
allergic inflammation, allergic airway inflammation, chronic
obstructive pulmonary disease (COPD), mesenteric infarction,
stroke, ulcerative colitis, allergic asthma, bronchial asthma,
mesenteric infarction, stroke, fibrosis, post-ischemic inflammation
in muscle, kidney and heart, skin inflammation, glomerulonephritis,
juvenile onset type I diabetes mellitus, hypersensitivity diseases,
viral or acute liver diseases, alcoholic liver failures,
tuberculosis, septic shock, HIV-infection, graft-versus-host
disease (GVHD) and atherosclerosis.
41. A method of inhibiting the expression of one or more cytokines
in an individual comprising administering to said individual a
composition comprising a soluble protein comprising a sequence
having at least 85% of homology with the mature form of the
extracellular domain of human CD164 (SEQ ID NO: 1).
42. The method according to claim 41, wherein said cytokine is
TNF-.alpha., IFN-.gamma., IL-2, IL-4, IL-5, or IL-10.
43. A method for identifying compounds as inhibitors of cytokine
secretion and expression comprising: a) contacting cells with a
composition comprising said compound; b) contacting cells with a
composition comprising a soluble protein comprising a sequence
having at least 85% of homology with the mature form of the
extracellular domain of human CD164 (SEQ ID NO: 1); and c)
comparing the level of cytokine secretion and expression that is
inhibited by the composition comprising said compound with the
level of cytokine secretion and expression that is inhibited by the
composition comprising said soluble protein.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of inflammation
and autoimmune disorders, in particular the discovery of novel
proteins useful for preventing and/or treating inflammatory and/or
autoimmune disorders.
BACKGROUND OF THE INVENTION
[0002] The following discussion is intended to facilitate the
understanding of the invention, but is not intended nor admitted to
be prior art to the invention.
[0003] CD164 is a member of the mucin-like receptor or slalomucin
superfamily of glycoproteins. Slalomucins are transmembrane
glycoproteins ranging from 50-3000 kD exhibiting limited similarity
at the cDNA and amino acid levels. Mucin-like expressed proteins
share the common characteristic of bearing numerous
O-glycosylations linked to serine and threonine residues, which
infer multiple kinds of cell-cell or cell-extracellular matrix
Interactions. The dense array of O-linked side chains are
characterized by an extended structure that makes many of the
mucin-like molecules long enough to protrude beyond the
polysaccharide glycocalyx that surrounds the cell and also by the
optimal exposure and high multiplicity of the terminal sugars. By
virtue of the structural configuration as well as negative charge,
mucin-like glycoproteins may act as a repulsive barrier unless
cells bear specific receptors for mucin (adhesion). Functions of
mucin receptors depend on cell types and states of activation
correlated with the core mucin peptide and with the cell-specific
expression of glycosyl transferases, which in turn regulate the
structure and presentation of the O-linked oligosaccharide
sidechains, membrane anchorage, signal transduction abilities and
or/the trafficking of the mucin to the correct cellular domain.
[0004] Human CD164 is an ortholog of murine MGC-24v (M. musculus)
and rat endolyn (R. norvegicus), a membrane protein found In
lysosomal and endosomal compartment of mammalian cells. The
relationships amongst different isoforms, together with
functionally important domains and subcellular distribution of
CD164/endolyn, have been described (Chan Y H et al., J. Biol. Chem,
276:2139-2152, 2001).
[0005] In its native state, human CD164 is a disulphide-linked
homodimer of two 80-85 kDa subunits. CD164 is highly glycosylated,
containing both O- and N-linked glycans. The extracellular region
is comprised of two mucin domains (I and II) linked by a non-mucin
domain containing intra-disulphide bridges as well as a
cysteine-rich motif that resembles a consensus pattern previously
found in growth factor and cytokine receptors. CD164 also contains
a single-pass transmembrane domain and a 13-amino acid
intracellular region that include a C-terminal motif (i.e. YHTL)
able to target the protein to endosomes and lysosomes.
[0006] Four human CD164 mRNA species have been described arising by
alternative splicing of six bona fide exons from a single genomic
transcription unit located on human chromosome 6q21 (Zannettino A,
J Biol Regul Homeost Agents, 15: 394-396, 2001; Watt and Chan, Leuk
Lymph, 37(:1-25. 2000). There are probably 4 alternative promoters,
two non-overlapping alternative last exons and one internal intron
which is not always spliced out. The predominant CD164 (E1-6)
isoform represents a 178 amino acid type I transmembrane
glycoprotein. The other described isoforms are a slalomucin CD164
or CD164 isoform delta 5 containing 178 amino acids; a 184 residues
CD164 isoform delta 4; and a 200 kD principally soluble isoform
termed MGC-24 (for Multi-Glycosylated Core protein of 24 kD)
lacking the transmembrane anchoring motif and having 189 residues.
All isoforms are highly glycosylated proteins with O- and N-linked
glycosylation sites (FIG. 1).
[0007] CD164 functions include mediating, or regulating,
haematopoletic progenitor cell adhesion and the negative regulation
of their growth and/or differentiation. CD164 is usually expressed
by CD34+ and CD34lo/-haematopoietic stem cells and associated
microenvironmental cells (Watt et al., Blood, 92: 849-866, 1998).
CD164 is also expressed by committed myeloid and erythroid colony
forming cells, on bone marrow stromal and endothelial cells, weakly
on lymphocytes, and on mesenchymal stem cells. CD164 may play a key
role in haematopoisesis by facilitating the adhesion of human CD34+
cells to bone marrow stroma and by suppressing CD34+
CD38lo/-haematopoietic progenitor cell proliferation, acting as a
potent signaling molecule (Zannettino et al. Blood, 92: 2613-2628,
1998).
[0008] These effects involve the CD164 class I and/or II epitopes
recognized by the monoclonal antibodies (mAbs) 105A5 and
103B2/9E10. The epitopes are carbohydrate-dependent and are located
on the N-terminal mucin domain I (Watt et al., Blood, 95,
3113-3124, 2000; Doyonnas et al., J Immunol, 165: 840-851, 2000).
The Interaction of haemotopoietic cells with stromal/endothelial
cells in their immediate microenvironment is thought to be of major
importance in the regulation of haematopoietic stem self-renewal,
quiescence, commitment and migration. These interactions involve
cooperation between adhesion receptors, their cognate ligands and
cytokines. A range of cell adhesion molecules (CAMS) including the
Ig, integrin, cadherin, selectin and mucin-like protein families,
participate in these processes.
[0009] In vitro, CD164 showed a role In myogenic differentiation
(Lee et al., Mol Cell Biol, 21: 7696-7706, 2001). Overexpression of
CD164 in myoblast cell lines accelerated expression of biochemical
markers of differentiation and enhanced formation of multinucleate
myotubes, whereas antisense CD164 or soluble extracellular regions
of CD164 inhibited myogenesis.
[0010] The peanut agglutinin (PNA)-binding site of soluble MGC-24
represents a tumor associated carbohydrate marker expressed in many
carcinomas. Total MGC-24 mRNA was found to be lower in human
colorectal carcinomas as compared with normal adjacent mucosal
tissues (Matsui et al., J Biochem, 127: 1103-1107, 2000). Lymphatic
vessel invasion by the carcinoma was correlated to low levels of
MGC-24 mRNA in colon carcinomas, whereas high levels did correlate
with less venous invasion and less remote metastasis. Monoclonal
antibodies specific for CD164 could prove useful for cancer
diagnosis or therapy and haematopoiesis inhibition (EP889054,
EP761814).
[0011] Other CD164-like proteins have been disclosed (NOV25, WO
02/098917; SEQ ID NO: 7852, EP1033401; FIG. 1), but their
biological properties have not been analyzed.
SUMMARY OF THE INVENTION
[0012] It has been surprisingly found that a soluble protein
comprising the mature form of the extracellular domain of human
CD164, has an inhibitory effect on the expression of cytokines
(namely interferon-.gamma., IL-2, IL-4, IL-5, IL-10 and
TNF-.alpha.) in cells that normally produce cytokines when they are
stimulated with agents such as concavalin A. Moreover, this soluble
fragment of CD164 inhibits relevant physiological responses (such
as lymphocytes or macrophages migration) In animal models relevant
for inflammatory and/or autoimmune diseases.
[0013] Therefore, soluble proteins comprising a sequence having at
least 85% of homology with the mature form of the extracellular
domain of human CD164 can be used for the manufacture of a
medicament for the treatment and/or prevention of inflammatory
and/or autoimmune disorders. Pharmaceutical compositions comprising
any of these soluble proteins are suitable for treatment and/or
prevention of inflammatory and/or autoimmune disorders, and in
general can be administered to an individual for inhibiting the
expression of cytokines.
[0014] Other features and advantages of the invention will become
evident from the following detailed description.
DESCRIPTION OF THE FIGURES
[0015] FIG. 1: (A) amino acid alignment of full length, human CD164
(hCD164; NCBI Acc. No. NP.sub.--006007; SEQ ID NO: 3), human
CD164-delta4 (hCD164-DELTA4; NCBI Acc. No. AAG53908; SEQ ID NO: 4),
CD164-delta5 (hCD164-DELTA5; NCBI Acc. No. AAG53907; SEQ ID NO: 5),
and MGC-24 (hMGC-24; NCBI Aoc. No. Q04900; SEQ ID NO: 6). Signal
sequences are boxed. The end of the extracellular region is
indicated by an arrow. The glycosylation sites are indicated by an
asterisk. (B) amino acid alignment of the mature form of the
extracellular domains of CD164 (amino acids 1-140 of SEQ ID NO: 1,
corresponding to amino acids 24-163 of SEQ ID NO: 3 and to to amino
adds 1-140 of SEQ ID NO: 2), MGC-24 (amino acids 24-163 of SEQ ID
NO: 6), CD164-delta4 (amino acids 24-150 of SEQ ID NO: 4),
CD164-delta5 (amino acids 24-145 of SEQ ID NO: 5), SEQ ID NO: 7852
(EP1033401; amino acids 24-163 of SEQ ID NO: 7), and NOV25 (WO
02/098917; amino adds 24-161 of SEQ ID NO: 8). The positions In
NOV25 different from SEQ ID NO: 1 are underlined.
[0016] FIG. 2: effect of sf-CD164 administration to
ConA-stimulated, human PBMC cells-mixture on the expression of IL-2
(A) and TNF-.alpha. (B). The X-axis represents the sf-CD164
concentration in .mu.g/ml. The Y-axis represents the percentage of
cytokine released by secretion.
[0017] FIG. 3: effect of sf-CD164 administration to
ConA-stimulated, human CD4 T cells on the expression of IL-2 (A)
and TNF-.alpha. (B). The X-axis represents the sf-CD164
concentration in .mu.g/ml. The Y-axis represents the percentage of
cytokine release by secretion.
[0018] FIG. 4: effect of sf-CD164 administration on TNF-.alpha.
release in the animal model for LPS-induced, TNF-.alpha. release.
The asterisks Indicate the statistical significance.
[0019] FIG. 5: effect of sf-CD164 administration on the cell
migration In the animal model for the Thioglycolate- (A) or
LPS-induced (B) cell recruitment In the peritoneum. The Y-axis
represents the concentration of cells per .mu.l (macrophages in A,
activated lymphocytes in B). The asterisks indicate the statistical
significance.
[0020] FIG. 6: effect of sf-CD164 administration on the
proliferation of autoantigenic MBP specific T cells. The Y-axis
represents the radioactivity (CPM, counts per minute) related to
the incorporation of radiolabeled nucleotides (.sup.3H thymidine)
by dividing cells. The asterisks Indicate the statistical
significance.
[0021] FIG. 7: effect of sf-CD164 administration to the
ConA-induced hepatis animal model on transaminase levels (ALAT; A)
IL-6 release (B), and IFN-.gamma. (C) release. Dexa stands for
Dexamethasone. The asterisks indicate the statistical
significance.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In accordance with the present invention, it has been found
that the mature form of the extracellular domain of human CD164
(SEQ ID NO: 1) has an inhibitory effect on cellular expression of
various cytokines (namely Interferon-.gamma., IL-2, IL-4, IL-5,
IL-10 and TNF-.alpha.) following the stimulation of these cells
with agents such as concanavalin A (ConA). Further confirmations of
the therapeutic utility of this protein sequence were obtained In
animal models for diseases, wherein the soluble protein
demonstrated valuable biological properties in vivo such as the
reduction of lymphocyte migration or the inhibition of MBP-(Myelin
Basic Protein) specific T cells proliferation.
[0023] There is no indication in the prior art that the
extracellular domain of human CD164, when isolated from the rest of
the molecule as a soluble protein, has any effect on the expression
of cytokines or on any other phenomena related to autoimmune and/or
inflammatory diseases.
[0024] The main object of the present invention is the use of a
soluble protein comprising a sequence having at least 85% of
homology with the mature form of the extracellular domain of human
CD164 (SEQ ID NO: 1) for the manufacture of a medicament for
treatment and/or prevention of inflammatory or/and autoimmune
disorders.
[0025] Amongst the soluble proteins that can be used accordingly to
the present invention, the most preferred soluble proteins are the
mature form of the extracellular domain of human CD164 (SEQ ID NO:
1), or this latter sequence fused to the signal sequence of human
CD 164.
[0026] Other preferred soluble proteins that can be used
accordingly to the present Invention, are variants of SEQ ID NO: 1
in the form of active muteins or isoforms of SEQ ID NO: 1.
[0027] Isoforms of human CD164 having at least 85% of homology with
the mature form of the extracellular domain of human CD164 (SEQ ID
NO: 1) are known In the literature (Chan Y H et al., J Biol Chem,
276: 2139-2152, 2001; FIG. 1). One of them called MGC-24 (SEQ ID
NO: 6) is known to be soluble since it lacks a functional
transmembrane domain, while two others called CD164-delta 4 (SEQ ID
NO: 4) and CD164-delta 5 (SEQ ID NO: 5) still conserve a
transmembrane domain. Therefore, the mature form of the
extracellular domain of these latter membrane-bound isoforms can be
considered useful according to this invention.
[0028] As "soluble proteins", the present invention intends protein
sequences not containing any sequences allowing the integration in
a cellular membrane, such as the transmembrane domain in human full
length CD164. These soluble proteins, when expressed by cells, are
therefore expected to be localized in the cells or, preferably,
secreted in the extracellular space if fused to a signal
sequence.
[0029] Soluble proteins comprising a sequence having at least 85%
of homology with the mature form of the extracellular domain of
human CD164 (SEQ ID NO: 1) are known in the literature (Lee Y N et
al., Mol Cell Biol, 21: 7696-7706, 2001) but there is no indication
of any utility for the treatment and/or prevention of inflammatory
and/or autoimmune disorders.
[0030] The soluble protein sequences defined in the present
invention as being useful for the treatment and/or prevention of
inflammatory and/or autoimmune disorders are also clearly distinct
from any other human sequence having, or supposed to have, similar
properties.
[0031] WO 02/098917 discloses the protein NOV25 (SEQ ID NO: 8; FIG.
1B) comprising a sequence homologous at 80% with the mature form of
the extracellular domain of human CD164 (SEQ ID NO: 1), and
suggests that it can be useful in a variety of diseases, including
autoimmune disease. However, this use is merely speculative, and
moreover the document fails to recognize the therapeutic utility of
the soluble fragment that can be isolated from the potential
extracellular domain of this protein, that is predicted to be
localized on a cellular membrane.
[0032] EP1033401 discloses a protein (SEQ ID NO: 7582) comprising a
sequence Identical to the mature form of the extracellular domain
of human CD164 (SEQ ID NO: 7; FIG. 1B). Even though it is suggested
therein a hypothetical therapeutic use of this protein in medicine
for any sorts of disease, this document also fails to recognize the
therapeutic utility of the soluble fragment that can be isolated
from the potential extracellular domain of this protein.
[0033] As "active", the present invention defines any variant of
the mature form of the extracellular domain of human CD164 (SEQ ID
NO: 1) having at least 85% of homology with this sequence that,
according to any of the assay presented in the examples, has a
comparable, or even increased, activity when compared to SEQ ID NO:
1, and should be as well accepted for any of the claimed uses and
methods.
[0034] By the activity being "comparable" is meant that the
activity measured in any of the described assays for the variant of
the soluble protein is at least of the same order of magnitude, and
preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%,
and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or
125% of the activity measured using a soluble protein as defined by
SEQ ID NO: 1.
[0035] By the activity being "increased" Is meant that the activity
measured in any of the described assays for the variant of the
soluble protein is at least 125%, 130%, 135%, 140%, 145%, 150%,
155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%,
350%, 375%, 400%, 450%, or 500% of the activity measured using a
soluble protein as defined by SEQ ID NO: 1.
[0036] As used herein the term "muteins" refers to any sequence
having at least 85% of homology with the mature form of the
extracellular domain of human CD164 (SEQ ID NO: 1) that can be
generated by inserting, deleting, and/or substituting one or more
amino add residues in SEQ ID NO: 1. Similar active muteins can be
natural, as the ones corresponding to an orthologous protein (i.e.
encoded by a non-human gene that has evolved from the common
ancestor for CD164) or from polymorphisms in human genome. In cases
where the nucleotide substitutions result in one or more amino acid
changes, preferred soluble proteins include those that retain one
or more anti-inflammatory-or/and anti-autoimmune-related
activity.
[0037] Alternatively, these sequences are synthetic or artificial,
which can be prepared by known chemical synthesis, recombinant DNA
technology, site-directed mutagenesis, or any other known technique
suitable thereof, which provide a finite set of substantially
corresponding mutated or shortened peptides or polypeptides which
can be routinely obtained and tested by one of ordinary skill in
the art using the teachings presented in the prior art and in the
Examples of the present invention.
[0038] Preferred changes in these active muteins are commonly known
as "conservative" or "safe" substitutions. Conservative amino acid
substitutions are those with amino acids having sufficiently
similar chemical properties, in order to preserve the structure and
the biological function of the molecule. It is clear that
insertions and deletions of amino acids may also be made in the
above defined sequences without altering their function,
particularly if the insertions or deletions only involve a few
amino acids, e.g., under ten, and preferably under three, and do
not remove or displace amino acids which are critical to the
functional conformation of a protein or a peptide.
[0039] The literature provide many models on which the selection of
conservative amino acids substitutions can be performed on the
basis of statistical and physico-chemical studies on the sequence
and/or the structure of natural protein (Rogov S I and Nekrasov A
N, Protein Eng, 14: 459-463, 2001). Protein design experiments have
shown that the use of specific subsets of amino acids can produce
foldable and active proteins, helping in the classification of
amino acid "synonymous" substitutions that can be more easily
accommodated in protein structure (Murphy L R et al., Protein Eng,
13:149-52, 2000). The synonymous amino acid groups and more
preferred synonymous groups are those defined in Table I.
[0040] Alternatively, amino acids in the soluble proteins of the
invention that are essential for function can also be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (see, e.g., Cunningham, et al.,
Science, 244:1081-5, 1989). Of special interest are substitutions
of charged amino acids with other charged or neutral amino adds
that may produce proteins with highly desirable improved
characteristics, such as less aggregation. Aggregation may not only
reduce activity but also be problematic when preparing
pharmaceutical or physiologically acceptable formulations, because
aggregates can be immunogenic (Cleland et al., Crit Rev Ther Drug
Carrier Syst, 10: 307-77, 1993).
[0041] Other examples of production of amino add substitutions in
proteins which can be used for obtaining muteins of soluble
proteins for the uses of the present invention include any known
method steps, such as presented in U.S. Pat. Nos. 4,959,314,
4,588,585, 4,737,462, 5,116,943, 4,965,195, 4,879,111, 5,017,691,
and 4,904,584.
[0042] Alternatively, the active mutein may result from sequence
alterations reducing the immunogenicity of said soluble protein
when administered to a mammal. The literature provides many example
on these sequence alterations that can be designed and introduced
at this scope or for other functional optimizations that allow a
safe and effective administration of a therapeutic protein,
especially when it is non-human, non-mammalian, or non-natural
protein (Vasserot A P et al., Drug Disc Today, 8: 118-126, 2003;
Marshall S A et al., Drug Disc Today, 8: 212-221, 2003; Schellekens
H, Nat Rev Drug Disc, 1: 457-462, 2002; Gendel S M, Ann NY Acad
SCI, 964: 87-98, 2002; Graddis T J et al., Curr Pharm Biotechnol,
3: 285-97, 2002; WO 03/104263; WO 03/006047; WO 02/98454; WO
02/96454; WO 02/79415; WO 02/79232; WO 02/66514; WO 01/40281; WO
98/52976; WO 96/40792; WO 94/11028).
[0043] It is clear that insertions and deletions of amino acids may
also be made in the above-defined sequences without altering their
function, particularly if the insertions or deletions only involve
a few amino acids, e.g., under thirty, and preferably under ten,
and do not remove or displace amino acids which are critical to a
functional conformation, e.g., cysteines or prolines. These
alterations may occur at the amino or carboxy termini or anywhere
between those terminal positions, interspersed either individually
among residues in the sequence or in one or more contiguous groups
within the sequence.
[0044] As a practical matter, whether any particular polypeptide is
a percentage homologous to the mature form of the extracellular
domain of human CD164 (SEQ ID NO: 1) can be determined
conventionally using known computer programs. Such algorithms and
programs include, but are by no means limited to, TBLASTN, BLASTP,
FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl
Acad Sci USA 85(8):2444-8; Altschul et al., (1990) J Mol Biol
215(3):403-410; Thompson et al., (1994) Nucleic Acids Res
22(2):4673-4680; Higgins et al., (1996) Meth Enzymol 266:383-402;
Altschul et al., (1997) Nuc Acids Res 25:3389-3402; Altschul et
al., (1993) Nature Genetics 3:266-272). In a particularly preferred
embodiment, protein and nucleic acid sequence homologies are
evaluated using the Basic Local Alignment Search Tool ("BLAST"),
which is well known in the art (See, e.g., Karlin and Altschul
(1990) Proc Natl Acad Sci USA 87(6):2264-8; Altschul et al., 1990,
1993, 1997, all supra).
[0045] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic add
sequence and a test sequence which is preferably obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are preferably identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al.,
(1992) Science 256(5062):1443-5; Henikoff and Henikoff (1993)
Proteins 17(1):49-61). Less preferably, the PAM or PAM250 matrices
may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978)
Matrices for Detecting Distance Relationships: Atlas of Protein
Sequence and Structure, Washington: National Biomedical Research
Foundation). The BLAST programs evaluate the statistical
significance of all high-scoring segment pairs identified, and
preferably selects those segments which satisfy a user-specified
threshold of significance, such as a user-specified percent
homology. Preferably, the statistical significance of a
high-scoring segment pair is evaluated using the statistical
significance formula of Karlin (See, e.g., Karlin and Altschul,
(1990) Proc Natl Acad Sci USA 87(6):2264-8). The BLAST programs may
be used with the default parameters or with modified parameters
provided by the user. Preferably, the parameters are default
parameters.
[0046] A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (1990) Comp. App. Biosci.
6:237-245. In a sequence alignment the query and subject sequences
are both amino acid sequences. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group=25 Length=0,
Cutoff Score=1, Window Size=sequence length, Gap Penalty-5, Gap
Size Penalty=0.05, Window Size=247 or the length of the subject
amino acid sequence, whichever is shorter.
[0047] If the subject sequence is shorter than the query sequence
due to N-or C-terminal deletions, not because of Internal
deletions, the results, In percent identity, must be manually
corrected because the FASTDB program does not account for N- and
C-terminal truncations of the subject sequence when calculating
global percent identity. For subject sequences truncated at the N-
and C-termini, relative to the query sequence, the percent identity
is corrected by calculating the number of residues of the query
sequence that are N- and C-terminal of the subject sequence, that
are not matched/aligned with a corresponding subject residue, as a
percent of the total bases of the query sequence. Whether a residue
is matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query amino acid residues
outside the farthest N- and C-terminal residues of the subject
sequence.
[0048] For example, a 90 amino acid residue subject sequence is
aligned with a 100-residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not match/align
with the first residues at the N-terminus. The 10 unpaired residues
represent 10% of the sequence (number of residues at the N- and
C-termini not matched/total number of residues in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched the final percent identity would be 90%.
[0049] In preferred embodiments, the post-translationally modified
forms of soluble proteins comprising a sequence having at least 85%
of homology with the mature form of the extracellular domain of
human CD164 (SEQ ID NO: 1) can be used for the manufacture of a
medicament for treatment and/or prevention of inflammatory or/and
autoimmune disorders. In particular, these proteins can be
acetylation, amidation, glycosylated, phosphorylated, and/or
myristoylated.
[0050] Human CD164 is known to be modified with such groups and a
series of specific positions can be indicated as set forth In SEQ
ID NO: 1: [0051] a) Potential N-glycosylation sites are located at
residues 3, 9, 18, 49, 54, 71, 81, 98 and 123; [0052] b) Potential
O-glycosylated sites are located at residues 11, 12, 17, 20, 21,
25, 26, 31, 32, 89, 90, 92, 96, 99, 100, 104, 108, 110, 111, 112,
113, 115, 117, 118, 119, 121, 122, 125, 127, 129, 130, 136. [0053]
c) Potential CAMP-and cGMP-dependent protein kinase phosphorylation
sites are located at residues 134 to 137; [0054] d) Potential
Protein Kinase C phosphorylation sites are located at residues 100
to 102 and 112 to 114; [0055] e) Potential Casein kinase II
phosphorylation sites are located at residues 73 to 76 and 136 to
139; [0056] f) Potential N-myristoylation site in sf-CD164 is
located at residue 119.
[0057] It is evident that such modifications can be also present in
the corresponding positions of the homologous soluble proteins
defined above as identified by sequence alignment (FIG. 1).
[0058] In a further preferred embodiment, the soluble protein
comprising a sequence having at least 85% of homology with the
mature form of the extracellular domain of human CD164 (SEQ ID NO:
1) is a soluble fusion protein.
[0059] These soluble fusion proteins can be obtained by cloning a
polynucleotide encoding soluble protein comprising a sequence
having at least 85% of homology with the mature form of the
extracellular domain of human CD164 (SEQ ID NO: 1) in frame to the
coding sequences for a heterologous protein sequence.
[0060] The term "heterologous", when used herein, is intended to
designate any polypeptide other than a human CD164 polypeptide.
[0061] Example of heterologous sequences, that can be comprised in
the soluble fusion proteins either at N- or at C-terminus, are the
following: extracellular domains of membrane-bound protein,
immunoglobulin constant regions (Fc region), multimerization
domains, domains of extracellular proteins, signal sequences,
export sequences, or sequences allowing purification by affinity
chromatography.
[0062] Many of these heterologous sequences are commercially
available in expression plasmids since these sequences are commonly
included in the fusion proteins in order to provide additional
properties without significantly impairing the specific biological
activity of the protein fused to them (Terpe K, Appl Microbiol
Biotechnol, 60: 523-33, 2003). Examples of such additional
properties are a longer lasting half-life in body fluids, the
extracellular localization, or an easier purification procedure as
allowed by the a stretch of Histidines forming the so-called
"histidine tag" (Gentz et al., Proc Natl Acad Sci USA, 86: 821-4,
1989) or by the "HA" tag, an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell, 37: 767-78, 1994). If
needed, the heterologous sequence can be eliinated by a proteolytic
cleavage, for example by inserting a proteolytic cleavage site
between the soluble protein and the heterologous sequence, and
exposing the purified soluble fusion protein to the appropriate
protease. These features are of particular importance for the
soluble fusion proteins since they facilitate their production and
use in the preparation of pharmaceutical compositions. For example,
the soluble protein used in the examples (sf-CD164; SEQ ID NO: 2)
was purified by means of a hexa-histidine peptide fused at the
C-terminus of the soluble CD164. When the soluble fusion protein
comprises an immunoglobulin region, the fusion may be direct, or
via a short linker peptide which can be as short as 1 to 3 amino
acid residues in length or longer, for example, 13 amino acid
residues in length. Said linker may be a tripeptide of the sequence
E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker
sequence comprising
Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced
between the sequence of the substances of the invention and the
immunoglobulin sequence. The resulting fusion protein has improved
properties, such as an extended residence time in body fluids
(half-life), increased specific activity, increased expression
level, or the purification of the fusion protein is
facilitated.
[0063] In a preferred embodiment, the soluble protein is fused to
the constant region of an Ig molecule. Preferably, it is fused to
heavy chain regions, like the CH2 and CH3 domains of human IgG1,
for example. Other isoforms of 19 molecules are also suitable for
the generation of fusion proteins according to the present
invention, such as isoforms IgG.sub.2 or IgG.sub.4, or other Ig
classes, like IgM or IgA, for example. Fusion proteins may be
monomeric or multimeric, hetero- or homomultimeric.
[0064] In a further preferred embodiment, the functional derivative
comprises at least one moiety attached to one or more functional
groups, which occur as one or more side chains on the amino acid
residues. Preferably, the moiety is a polyethylene (PEG) moiety.
PEGylation may be carried out by known methods, such as the ones
described in WO99/55377, for example.
[0065] The soluble proteins and soluble fusion proteins comprising
a sequence having at least 85% of homology with the mature form of
the extracellular domain of human CD164 (SEQ ID NO: 1) can be
extracted and isolated from bodily fluids, cells, or tissues of
human or mammalian naturally expressing them naturally. In
particular, cells whether directly isolated or cultured, can
express these soluble proteins (naturally or following the exposure
to an inducing agent) and secrete them. Methods for purifying
proteins are known in the art, and include the use of detergents or
chaotropic agents to disrupt particles followed by differential
extraction and separation of the polypeptides by ion exchange
chromatography, affinity chromatography, sedimentation according to
density, and gel electrophoresis.
[0066] In general, the soluble proteins and soluble fusion proteins
can be prepared by any procedure known in the art, including
recombinant DNA-related technologies and chemical synthesis
technologies.
[0067] Recombinant DNA-related technologies allow producing the
soluble proteins and soluble fusion proteins by first generating
polynucleotides encoding them. These nucleic adds can be obtained
by PCR from genomic DNA or, more efficiently, from a vector
containing the full sequence of human CD164 (SEQ ID NO: 3) or any
other relevant homologous sequences. The oligonucleotide primers
complementary to the desired sequence contain restriction
endonuclease sequences allowing the digestion by specific
restriction endonucleases for further cloning, taking care to
ensure that the sequence encoding the soluble protein is positioned
properly with respect to the polyA signal and the rest of the other
sequences in the expression plasmid.
[0068] Using common genetic engineering techniques, these
polynucleotides can be cloned in replicable expression vector of
viral or plasmid origin which are used to transform a prokaryotic
or eukaryotic host cell, using episomal or non-homologously
integrated vectors, as well as transformation-, infection-,
precipitation-, or transfection-based technologies. These vectors
should allow the expression of the recombinant proteins in the
prokaryotic or eukaryotic host cell under the control of their own
transcriptional initiation/termination regulatory sequences, which
are chosen to be constitutively active or inducible in said cell. A
cell line substantially enriched in such cells can be then isolated
to provide a stable cell line expressing the protein of
interest.
[0069] Many books and reviews provides teachings on how to done and
produce recombinant proteins using vectors and Prokaryotic or
Eukaryotic host cells, such as some titles in the series "A
Practical Approach" published by Oxford University Press ("DNA
Cloning 2: Expression Systems", 1995; "DNA Cloning 4: Mammalian
Systems", 1996; "Protein Expression", 1999; "Protein Purification
Techniques", 2001).
[0070] A typical expression vector should comprise: [0071] a) a DNA
sequence coding for a soluble protein or a soluble fusion protein
comprising a sequence having at least 85% of homology with the
mature form of the extracellular domain of human CD164 (SEQ ID NO:
1); and [0072] b) an expression cassette;
[0073] wherein said sequence (a) is operably associated with a
tissue-specific or a constitutive promoter included in sequence
(b).
[0074] The expression vector is any of the mammalian, yeast, insect
or bacterial expression systems known in the art. Commercially
available vectors and expression systems are available from a
variety of suppliers including Genetics Institute (Cambridge,
Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and
Invitrogen (San Diego, Calif.). If desired, to enhance expression
and facilitate proper protein folding, the codon context and codon
pairing of the sequence can be optimized for the particular
expression organism into which the expression vector is introduced
(U.S. Pat. No. 5,082,767; Gustafsson C et al., Trends Biotechnol,
22: 346-53, 2004).
[0075] Factors of importance in selecting a particular plasmid or
viral vector include: the ease with which recipient cells that
contain the vector, may be recognized and selected from those
recipient cells which do not contain the vector; the number of
copies of the vector which are desired in a particular host; and
whether it is desirable to be able to "shuttle" the vector between
host cells of different species. A recombinant vector according to
the invention comprises, but is not limited to, a YAC (Yeast
Artificial Chromosome), a BAG (Bacterial Artificial Chromosome), a
phage, a phagemid, a cosmid, a plasmid, or even a linear DNA
molecule which may consist of a chromosomal, non-chromosomal,
semi-synthetic or synthetic DNA.
[0076] Generally, recombinant expression vectors will include
origins of replication, selectable markers permitting
transformation of the host cell, and a promoter derived from a
highly expressed gene to direct transcription of a downstream
structural sequence. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably a leader sequence capable of
directing secretion of the translated protein into the periplasmic
space or the extracellular medium. In a specific embodiment wherein
the vector is adapted for transfecting and expressing desired
sequences in mammalian host cells, preferred vectors will comprise
an origin of replication in the desired host, a suitable promoter
and enhancer, and also any necessary ribosome binding sites,
polyadenylation sites, splice donor and acceptor sites,
transcriptional termination sequences, and 5'-flanking
non-transcribed sequences. DNA sequences derived from the SV40
viral genome, for example SV40 origin, early promoter, enhancer,
splice and polyadenylation sites may be used to provide the
required non-transcribed genetic elements.
[0077] The suitable promoter regions used in the expression vectors
of the present invention are chosen taking into account the cell
host in which the heterologous gene is expressed. The particular
promoter employed to control the expression of a nucleic add
sequence of interest is not believed to be important, so long as it
is capable of directing the expression of the nucleic acid in the
targeted cell. Thus, where a human cell is targeted, it is
preferable to position the nucleic acid coding region adjacent to
and under the control of a promoter that is capable of being
expressed in a human cell, such as, for example, a human or a viral
promoter. The promoter used may be constitutive or inducible.
[0078] A suitable promoter may be heterologous with respect to the
nucleic acid for which it controls the expression or alternatively
can be endogenous to the native polynucleotide containing the
coding sequence to be expressed. Additionally, the promoter is
generally heterologous with respect to the recombinant vector
sequences within which the construct promoter/coding sequence has
been inserted.
[0079] Promoter regions can be selected from any desired gene
using, for example, CAT (chloramphenicol transferase) vectors and
more preferably pKK232-8 and pCM7 vectors. Preferred bacterial
promoters are the Lacl, LacZ, the T3 or T7 bacteriophage RNA
polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP
0036776), the polyhedrin promoter, or the p10 protein promoter from
baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol
3(12):2156-65; O'Reilly et al., 1992), the lambda PR promoter or
also the trc promoter. Eukaryotic promoters Include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-L. In addition, promoters
specific for a particular cell type may be chosen, such as those
facilitating expression in adipose tissue, muscle tissue, or liver.
Selection of a convenient vector and promoter is well within the
level of ordinary skill in the art.
[0080] Where a cDNA Insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed such as human growth hormone and SV40 polyadenylation
signals. Also contemplated as an element of the expression cassette
is a terminator. These elements can serve to enhance message levels
and to minimize read through from the cassette into other
sequences.
[0081] The vectors may also contain additional, non-coding
sequences, including for example, but not limited to non-coding 5'
and 3' sequences, vector sequence, sequences used for purification,
probing, or priming. For example, heterologous sequences include
transcribed, non-translated sequences that may play a role in
transcription, and mRNA processing, for example, ribosome binding
and stability of mRNA.
[0082] Selectable markers confer an identifiable change to the cell
permitting easy identification of cells containing the expression
construct. The selectable marker genes for selection of transformed
host cells are preferably dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or
tetracycline, rifampicin or ampicilin resistance in E. coli, or
levan saccharase for mycobacteria, this latter marker being a
negative selection marker.
[0083] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and a bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of
pBR322 (ATCC 37017). Such commercial vectors include, but are not
limited to, pKK223-3 (Pharmacia, Uppsala, Sweden) and pGEMI
(Promega Blotec, Madison, Wis., USA).
[0084] Large numbers of other suitable vectors are known to those
of skill in the art, and are commercially available, such as the
following bacterial vectors: pTrc-His, pET30-His, pQE70, pQE60,
pQE-9 (Qiagen), pbs, pD10, phagescript, psIX174, pbluescript SK,
pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT,
pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmada);
pQE-30 (QIAexpress).
[0085] A suitable vector for the expression of polypeptides is a
baculovirus vector that can be propagated in insect cells and in
insect cell lines. A specific suitable host vector system is the
pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used
to transfect the SF9 cell line (ATCC No CRL 1711) which is derived
from Spodoptera frugiperda. Further suitable baculovirus vectors
are known to those skilled in the art, for example, FastBacHT.
Other suitable vectors for the expression of an APM1 globular head
polypeptide in a baculovirus expression system include, but are not
limited to, those described by Chai et al. (1993; Biotechnol Appl
Biochem. December; 18 (Pt 3):259-73); Viasak et al. (1983; Eur J
Biochem September 1;135(1):123-6); and Lenhard et al. (1996; Gene
March 9;169(2):187-90).
[0086] Further suitable vectors for the expression of polypeptides
are mammalian vectors. A number of suitable vector systems are
known to those skilled in the art, for example, pcDNA4HisMax,
pcDNA3.1Hygro-His and pcDNA3.1Hygro.
[0087] Further suitable vectors for the expression of polypeptides
are viral vector, such as the ones derived from an adenovirus.
Preferred adenovirus vectors according to the invention are those
described by Feldman and Steg (1996; Semin Interv Cardiol
1(3):203-8) or Ohno et al. (1994; Science 265(5173):781-4).
[0088] Retrovirus vectors and adeno-associated virus vectors are
generally understood to be the recombinant gene delivery systems of
choice for the transfer of exogenous polynucleotides in vivo,
particularly to mammals, including humans. These vectors provide
efficient delivery of genes into cells, and the transferred nucleic
acids are stably integrated into the chromosomal DNA of the
host.
[0089] Another possibility to express polypeptides is to activate
endogenously the genes by introducing regulatory sequence into the
right locus of the genome by homologous recombination, thus
operably linking the regulatory sequence with the gene, the
expression of which is required to be induced (WO 91/09955; WO
02/10372).
[0090] Host cells may be either prokaryotic or eukaryotic.
Preferred are eukaryotic hosts, e.g. mammalian cells, such as
human, monkey, mouse, and Chinese Hamster Ovary (CHO) cells,
because they provide post-translational modifications to protein
molecules, including correct folding or glycosylation at correct
sites. Also yeast cells can carry out post-translational peptide
modifications including glycosylation. A number of recombinant DNA
strategies exist which utilize strong promoter sequences and high
copy number of plasmids which can be utilized for production of the
desired proteins in yeast. Yeast recognizes leader sequences in
cloned mammalian gene products and secretes peptides bearing leader
sequences (i.e., pre-peptides).
[0091] Preferred host cells used as recipients for expressing the
soluble proteins are the following: [0092] a) Prokaryotic host
cells: Escherichia coli strains (I.E. DH5-.alpha. strain), Bacillus
subtilis, Salmonella typhimurium, and strains from species like
Pseudomonas, Streptomyces and Staphylococcus; [0093] b) Eukaryotic
host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1
cells (ATCC NoCCL70), COS cells (ATCC NoCRL1650; NoCRL1651), Sf-9
cells (ATCC NoCRL1711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No
CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504;
No CRL-1573), BHK (ECACC No 84100501; No 84111301), PLC cells,
HepG2, and Hep3B.
[0094] For Eukaryotic hosts (e.g. yeasts, insect or mammalian
cells), different transcriptional and translational regulatory
sequences may be employed, depending on the nature of the host.
They may be derived form viral sources, such as adenovirus, bovine
papilloma virus, Simian virus or the like, where the regulatory
signals are associated with a particular gene which has a high
level of expression. Examples are the TK promoter of the Herpes
virus, the SV40 early promoter, the yeast gal4 gene promoter, etc.
Transcriptional initiation regulatory signals may be selected which
allow for repression and activation, so that expression of the
genes can be modulated. The cells which have been stably
transformed by the introduced DNA can be selected by also
introducing one or more markers which allow for selection of host
cells which contain the expression vector. The marker may also
provide for phototrophy to an auxotropic host, biocide resistance,
e.g. antibiotics, or heavy metals such as copper, or the like. The
selectable marker gene can either be directly linked to the DNA
gene sequences to be expressed, or introduced into the same cell by
co-transfection. Additional elements may also be needed for optimal
synthesis of proteins of the invention.
[0095] If the nucleic acid encoding the soluble protein lacks a
methionine to serve as the initiation site, an initiating
methionine can be introduced next to the first codon of the nucleic
acid using conventional techniques. Similarly, if the insert from
the soluble CD164 polypeptide cDNA lacks a poly A signal, this
sequence can be added to the construct by, for example, splicing
out the Poly A signal from pSG5 (Stratagene) using BglI and SalI
restriction endonuclease enzymes and incorporating it into the
mammalian expression vector pXT1 (Stratagene). pXT1 contains the
LTRs and a portion of the gag gene from Moloney Murine Leukemia
Virus. The position of the LTRs in the construct allow efficient
stable transfection. The vector includes the Herpes Simplex
Thymidine Kinase promoter and the selectable neomycin gene.
[0096] Depending upon the host employed in a recombinant production
procedure, the polypeptides of the present invention may be
glycosylated or may be non-glycosylated. In addition, the soluble
proteins may also include an initial modified methionine residue,
in some cases as a result of host-mediated processes. Thus, it is
well known in the art that the N-terminal methionine encoded by the
translation initiation codon generally is removed with high
efficiency from any protein after translation in all eukaryotic
cells. While the N-terminal methionine on most proteins also is
efficiently removed in most prokaryotes, for some proteins, this
prokaryotic removal process is inefficient, depending on the nature
of the amino acid to which the N-terminal methionine is covalently
linked.
[0097] The soluble proteins, given their limited length, can be
also produced by chemical synthesis technologies, for example by
solid phase synthesis and liquid phase synthesis. As a solid phase
synthesis, for example, the amino acid corresponding to the
C-terminus of the peptide to be synthetized is bound to a support
which is insoluble in organic solvents, and by alternate repetition
of reactions, one wherein amino acids with their amino groups and
side chain functional groups protected with appropriate protective
groups are condensed one by one in order from the C-terminus to the
N-terminus, and one where the amino acids bound to the resin or the
protective group of the amino groups of the peptides are released,
the peptide chain is thus extended in this manner.
[0098] Solid phase synthesis methods are largely classified by the
tBoc method and the Fmoc method, depending on the type of
protective group used. Typically used protective groups include
tBoc (t-butoxycarbonyl), Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z
(2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc
(9-fluorenylmethoxycarbonyl), Mbh (4,4'-dimethoxydibenzhydryl), Mtr
(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos
(tosyl), Z (benzyloxycarbonyl) and C12-Bzl (2.6-dichlorobenzyl) for
the amino groups; NO2 (nitro) and Pmc
(2,2,5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino
groups); and tBu (t-butyl) for the hydroxyl groups). After
synthesis of the desired peptide, it is subjected to the
de-protection reaction and cut out from the solid support. Such
peptide cutting reaction may be carried with hydrogen fluoride or
tri-fluoromethane sulfonic add for the Boc method, and with TFA for
the Fmoc method. Totally synthetic proteins of a length comparable
to the one of the proteins of the invention are disclosed in the
literature (Brown A et al., J Pept Sci 2:4046, 1996; Muir T W, Annu
Rev Biochem, 72: 249-89, 2003; Casi G and Hilvert D, Curr Opin
Struct Biol, 13: 689-94, 2003).
[0099] The chemical synthesis of the soluble proteins allows
expanding the natural repertoire of protein structure and function
by making use of non-natural amino acids (Anthony-Cahill S J and
Magliery T J, Curr Pharm Biotechnol, 3: 285-97, 2002). These
molecules can be designed on the sequence and/or the structure of
the soluble proteins in order to select the residues can be
chemically modified at the level of amino acid side chains, of
amino acid chirality, and/or of the peptide backbone, and then to
improve relevant properties, such as potency, easiness of
purification, half-life. Preferred alternative, "synonymous" groups
for amino acids to be included are those defined in Table II. The
techniques for the synthesis and the development of these compounds
are well known in the art (Hruby V J and Balse P M, Curr Med Chem,
7:945-70, 2000; Goleblowski A et al., Curr Opin Drug Discov Devel,
4: 428-34, 2001; Villain M et al., Chem Biol, 8: 673-9, 2001, WO
02/10195;). Various methodology for incorporating unnatural amino
acids into proteins, using both in vitro and in vivo translation
systems, to probe and/or improve protein structure and function are
also disclosed in the literature (Dougherty D A, Curr Opin Chem
Bio, 4: 645-52, 2000).
[0100] The purification of synthetic or recombinant soluble
proteins that can be used according to the invention, can be
carried out by any one of the methods known for this purpose, i.e.
any conventional procedure involving precipitation, chromatography
(anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity
chromatography, hydroxylapatite chromatography and lectin
chromatography), electrophoresis, differential extraction, salt
fractionation, centrifugation or the like. See, for example,
Methods in Enzymology for a variety of methods for purifying
proteins.
[0101] A purification procedure that may be used in preference is
affinity chromatography using monoclonal antibodies, or any other
chemical groups that bind the target protein (directly soluble
CD164 or, if it is a soluble fusion protein, the heterologous
sequence such as an histidine tag) with sufficient affinity and
specificity. The binding groups are produced and immobilized on a
gel matrix contained within a column. Impure preparations
containing the proteins are passed through the column. The soluble
protein will be bound to the column by affinity while the
impurities will pass through. After washing away remaining
impurities, the soluble protein can be eluted from the gel by a
change in pH or ionic strength. Alternatively, HPLC (High
Performance Liquid Chromatography) can be used, The elution can be
carried using a water-acetonitrile-based solvent commonly employed
for protein purification.
[0102] Alternatively, the soluble proteins can be isolated from
milk of transgenic animals expressing the soluble proteins applying
any of the large number of methods disclosed in the literature
(Protein Purification Applications, A Practical Approach (New
Edition), Edited by Simon Roe, AEA Technology Products and Systems,
Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia
3:337-50; U.S. Pat. No. 6,140,552).
[0103] The soluble protein comprising a sequence having at least
85% of homology with the mature form of the extracellular domain of
human CD164 (SEQ ID NO: 1) can be produced, formulated,
administered, or generically used for the manufacture of a
medicament for treatment and/or prevention of inflammatory or/and
autoimmune disorders as an active derivative, a
proteolysis-resistant modified form, a conjugate, a complex, a
fraction, a precursor, and/or a salt.
[0104] The term "derivatives" as herein used refers to derivatives
which can be prepared from the functional groups present on the
lateral chains of the amino acid moieties or on the N- or
C-terminal groups according to known methods. Such derivatives
include for example esters or aliphatic amides of the
carboxyl-groups and N-acyl derivatives of free amino groups or
O-acyl derivatives of free hydroxyl-groups and are formed with
acyl-groups as for example alcanoyl- or aroyl-groups.
[0105] The term "fraction" refers to any fragment of the
polypeptidic chain of the compound itself, alone or in combination
with related molecules or residues bound to it, for example
residues of sugars or phosphates, or aggregates of the original
polypeptide or peptide. Such molecules can result also from other
modifications which do not normally alter primary sequence, for
example in vivo or in vitro chemical derivativization of peptides
(acetylation or carboxylation), those made by modifying the pattern
of phosphorylation (introduction of phosphotyrosine, phosphoserine,
or phosphothreonine residues) or glycosylation (by exposing the
peptide to enzymes which affect glycosylation e.g., mammalian
glycosylating or deglycosylating enzymes) of a peptide during its
synthesis and processing or in further processing steps.
[0106] The "precursors" are compounds which can be converted into
the compounds of present invention by metabolic and enzymatic
processing prior or after the administration to the cells or to the
body.
[0107] The term "salts" herein refers to both salts of carboxyl
groups and to add addition salts of amino groups of the peptides,
polypeptides, or analogs thereof, of the present invention. Salts
of a carboxyl group may be formed by means known in the art and
include inorganic salts, for example, sodium, calcium, ammonium,
ferric or zinc salts, and the like, and salts with organic bases as
those formed, for example, with amines, such as triethanolamine,
arginine or lysine, piperidine, procaine and the like. Add addition
salts include, for example, salts with mineral acids such as, for
example, hydrochloric acid or sulfuric add, and salts with organic
acids such as, for example, acetic acid or oxalic acid. Any of such
salts should have substantially similar activity to the peptides
and polypeptides of the invention or their analogs.
[0108] The conjugate or complex cane be formed with a molecule
chosen amongst radioactive labels, biotin, fluorescent labels,
cytotoxic agents, drug delivery agents. These conjugates or
complexes can be generated, using molecules and methods known in
the art, for example for allowing the detection of the interaction
eith other proteins (radioactive or fluorescent labels, biotin),
for improving therapeutic efficacy (cytotoxic agents), or for
improving drug delivery efficacy, using polymers such as
polyethylene glycol and other natural or synthetic polymers (Pillal
O and Panchagnula R, Curr Opin Chem Biol, 5: 447-451, 2001).
[0109] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0110] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the polypeptide with consideration
of effects on functional or antigenic domains of the polypeptide.
There are a number of attachment methods available to those skilled
in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al. (1992) Exp Hematol
20(8):1028-35, reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino add residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues, glutamic acid residues
and the C-terminal amino acid residue. Sulfhydryl groups may also
be used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0111] A polypeptide resistant to proteolysis, can be generated by
replacing a --CONH-- peptide bond with one or more of the
following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a
(CH2-O) methylene-oxy bond; a (CH2-S) thiomethylene bond; a
(CH2CH2) carba bond; a (CO--CH2) cetomethylene bond; a (CHOH--CH2)
hydroxyethylene bond); a (N--N) bound; a E-alcene bond; or a
--CH.dbd.CH-- bond. Thus, the invention also encompasses a soluble
CD164 or a variant thereof. In which at least one peptide bond has
been modified as described above. In addition, amino acids have
chirality within the body of either L or D. In some embodiments it
is preferable to alter the chirality of the amino acids in order to
extend half-life within the body. Thus, in some embodiments, one or
more of the amino acids are preferably in the L configuration. In
other embodiments, one or more of the amino acids are preferably in
the D configuration.
[0112] The therapeutic applications of the polypeptides of the
invention and of the related reagents can be evaluated (in terms or
safety, pharmacokinetics and efficacy) by the means of the in vivo
or in vitro assays making use of animal cell, tissues and models
allowing to detect an inhibition of cytokine release and/or
expression, as well in vivo or in vitro assays, such as the
inhibition of cellular recruitment. Further characterization of the
biological and therapeutic activities described in the present
invention can be obtained by applying various in molecular biology
technologies, such as two-dimensional gel electrophoresis or RNA
interference.
[0113] One specific embodiment for a method for delivering a
soluble protein to the interior of a cell of a vertebrate in vivo
comprises the step of introducing a preparation comprising a
physiologically acceptable carrier and a naked polynucleotide
operatively coding for the polypeptide of interest into the
interstitial space of a tissue comprising the cell, whereby the
naked polynucleotide is taken up into the interior of the cell and
has a physiological effect. This is particularly applicable for
transfer in vitro but it may be applied to in vivo as well.
[0114] A polynucleotide sequence encoding for a soluble protein
comprising a sequence having at least 85% of homology with the
mature form of the extracellular domain of human CD164 (SEQ ID NO:
1) can be used for the manufacture of a medicament for treatment
and/or prevention of inflammatory or/and autoimmune disorders.
These polynucleotides can be also used for the generation of
non-human animals and plants that express recombinant CD164
polypeptides. The animals or plants can be transgenic, i.e. each of
their cells contains a gene encoding the CD164 polypeptide, or,
alternatively, a polynucleotide encoding the polypeptide can be
introduced into somatic cells of the animal or plant, e.g. into
mammary secretory epithelial cells of a mammal. In preferred
embodiments, the non-human animal is a mammal such as a cow, sheep,
goat, pig, or rabbit. Methods of making transgenic animals such as
mammals are well known to those of skill in the art, and any such
method can be used in the present invention. Moreover, transgenic
mammals can be generated that secrete the recombinant soluble
proteins polypeptides in their milk. Typically, the encoded
polypeptide will include a signal sequence to ensure the secretion
of the protein into the milk.
[0115] Compositions for use in vitro and in vivo comprising a
"naked" polynucleotide are described in the prior art (WO 90/11092;
WO 95/11307; Tascon et al., Nature Medicine 2: 888-892, 1996). In
still another embodiment of the invention, the transfer of a naked
polynucleotide into cells may be proceeded with a particle
bombardment (biolistic), said particles being DNA-coated
microprojectiles accelerated to a high velocity allowing them to
pierce cell membranes and enter cells without killing them, such as
described by Klein et al. ((1990) Curr Genet February;
17(2):97-103). In a further embodiment, the polynucleotide of the
invention may be entrapped in a liposome (Ghosh and Bacchawat,
(1991) Targeted Diagn Ther 4:87-103; Wong et al., (1980) Gene
10:87-94; Nicoiau et al., (1987) Methods Enzymol 149:157-76). These
liposomes may further be targeted to cells expressing LSR by
incorporating leptin, triglycerides, ACRP30, or other known LSR
ligands into the liposome membrane. The amount of vector to be
injected to the desired host organism varies according to the site
of injection. As an indicative dose, it will be injected between
0.1 and 100 .mu.g of the vector in an animal body, preferably a
mammal body, for example a mouse body. In another embodiment of the
vector according to the invention, it may be introduced in vitro in
a host cell, preferably in a host cell previously harvested from
the animal to be treated and more preferably a somatic cell such as
a muscle cell. In a subsequent step, the cell that has been
transformed with the vector coding for the desired CD164
polypeptide or the desired fragment thereof is reintroduced into
the animal body in order to deliver the recombinant protein within
the body either locally or systemically.
[0116] For in vivo administration, the polynucleotides can be
administered in any suitable formulation, at any of a range of
concentrations (e.g. 1-500 .mu.g/ml, preferably 50-100 .mu.g/ml),
at any volume (e.g. 1-100 ml, preferably 1 to 20 ml), and can be
administered any number of times (e.g. 1, 2, 3, 5, or 10 times), at
any frequency (e.g. every 1, 2, 3, 5, 10, or any number of days).
Suitable concentrations, frequencies, modes of administration, etc.
will depend upon the particular polynucleotide, vector, animal,
etc., and can readily be determined by one of skill in the art.
[0117] The soluble protein comprising a sequence having at least
85% of homology with the mature form of the extracellular domain of
human CD164 (SEQ ID NO: 1)/are capable of inhibiting
proinflammatory- and/or immune-related cytokine expression, and are
thus believed to prevent and/or treat "inflammatory and/or
autoimmune disorders".
[0118] The primary function of the immune system is to protect an
individual against infection by foreign invaders such as
microorganisms, it may happen that the immune system attacks the
individual's own tissues, leading to pathologic states known as
autoimmune diseases, which are frequently associated with
inflammatory processes.
[0119] In particular, CD4+ T cells can be assigned to two different
subsets called T helper type 1 cells (Th1) and T helper type 2
cells (Th2) on the basis of distinct, non-overlapping cytokine
expression patterns. Th1 is characterized by the secretion of IL-2,
interferon-.gamma., IL-12 and TNF-.alpha., and Th2 by the secretion
of IL, IL-5, IL-9, IL-10 and IL-13. Nevertheless, these are not
strict subsets as IFN-.gamma. and IL-10 can suppress effects
associated with Th1 as well as Th2 responses, and IL-.gamma. and
IL-13 are also able to promote the production of IL-12, thereby
promoting Th1 and potentially inhibiting Th2 responses. Th1 T cells
are able to mediate macrophage activation and delayed-type
hypersensitivity (DTH), giving rise to pro-inflammatory or
cell-mediated immune responses, whereas Th2 T cells promote IgG1
and IgE secretion leading to Immediate-type hypersensitivity
reactions (humoral immunity; stimulate antibody-mediated responses,
activate mast cells, and elicit tissue eosinophilia). Th1 is a key
feature in the pathogenesis of diseases like rheumatoid arthritis,
sarcoidosis, and tuberculosis, whereas Th2 is involved in allergy,
antiparasite responses and in the asthmatic airway (e.g. role in
fibrosis).
[0120] A non-limitative list of disorders where a medicament or a
pharmaceutical composition comprising a soluble protein comprising
a sequence having at least 85% of homology with the mature form of
the extracellular domain of human CD164 (SEQ ID NO: 1) can be used,
includes: multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic
arthritis, osteoarthritis, spondylarthropathies, inflammatory bowel
disease, endotoxemia, Crohn's disease, Still's disease, uveitis,
Wegener's granulomatosis, Behcet's disease, scleroderma, Sjogren's
syndrome, sarcoidosis, pyodema gangrenosum, polymyositis,
dermatomyositis, myocarditis, psoriasis, systemic sclerosis,
hepatitis C, allergies, allergic inflammation, allergic airway
inflammation, chronic obstructive pulmonary disease (COPD),
mesenteric infarction, stroke, ulcerative colitis, allergic asthma,
bronchial asthma, mesenteric infarction, stroke, fibrosis,
post-ischemic inflammation in muscle, kidney and heart, skin
inflammation, glomerulonephritis, juvenile onset type I diabetes
mellitus, hypersensitvity diseases, viral or acute liver diseases,
alcoholic liver failures, tuberculosis, septic shock HIV-infection,
graft-versus-host disease (GVHD) and atherosclerosis.
[0121] Rheumatoid arthritis is a disease marked by signs and
symptoms of inflammation of the joints. Systemic lupus
erythematosus (SLE) is characterized by red, scaley patches on the
skin and by malfunction of the kidneys at the advanced stage of the
disease, and is associated with inflammatory reactions triggered by
deposition of immune complexes in blood vessels, particularly in
the kidneys. Multiple sclerosis is a human illness characterized by
relapsing, inflammatory conditions that can cause weakness, body
tremors and, in extreme cases, paralysis, and is associated with
immune system attack of the protective myelin sheath surrounding
peripheral nerve cells. Allergic inflammation is consistent with a
Th2-cell-based aetiology of atopic disease. For example, defective
priming of Th2 cells in the absence of IL-4 resulted in a failure
to generate allergic inflammatory responses after subsequent airway
challenge. IL-5 and IL-13 have been shown to be more directly
responsible for the characteristic eosinophil infiltrates and mucus
hypersecretion.
[0122] In multiple sclerosis, Th1 mediated immune responses are
thought to promote the disease, whereas Th2 mediated immune
responses are believed to have an ameliorating effect on the
progression of the disease. T cells expressing IL-10 have been
shown to suppress experimental autoimmune encephalomyelitis (EAE),
a rat model for multiple sclerosis. TNF-.alpha. has been
hypothesized to be responsible for the Induction of EAE
(TNF-.alpha. can be secreted by both Th1 and Th2 cultures).
[0123] Human systemic lupus erythematosus (SLE) is considered to be
driven by a Th2 response. However, IFN-.gamma. has been shown to
have a major effect on disease progression in a mouse model,
whereas IL-4 is expected to mediate disease maintenance.
[0124] Myocarditis is defined by inflammation of the heart muscle
and is thought to be mediated by an autoimmune response to a
cardiac-specific antigen after an acute upper respiratory
infection. The severity of the experimental autoimmune myocarditis
(EAM) in the mouse model is reduced by administration of anti-IL-4,
indicating a role of IL-4 in disease progression.
[0125] A further embodiment of the invention is a method of
inhibiting the expression of one or more cytokines in an individual
comprising administering to said individual a composition
comprising a soluble protein comprising a sequence having at least
85% of homology with the mature form of the extracellular domain of
human CD164 (SEQ ID NO: 1). The cytokine can be TNF-.alpha.,
IFN-.gamma., IL-2, IL-4, IL-5, or IL-10. These methods comprise
providing or administering to individuals in need thereof said
pharmaceutical or physiologically acceptable composition as
described below, and can be considered as methods for preventing
and/or treating inflammation and/or autoimmune disorders.
[0126] Still another embodiment of the present invention is
represented by pharmaceutical compositions comprising a soluble
protein comprising a sequence having at least 85% of homology with
the mature form of the extracellular domain of human CD164 (SEQ ID
NO: 1), in the presence of one or more pharmaceutically acceptable
excipients, for the treatment of inflammation and/or autoimmune
disorders. These compositions can further comprise an additional
immunosuppressant or anti-inflammatory substance. Alternatively,
the pharmaceutical compositions comprising the soluble can be
combined into a "cocktail" for use in the various treatment
regimens.
[0127] The pharmaceutical compositions of the invention may also
contain any suitable pharmaceutically acceptable carriers,
biologically compatible vehicles and additives that are suitable
for administration to an animal (for example, physiological saline)
and eventually comprising auxiliaries (like excipients, stabilizers
or diluents) that facilitate the processing of the active compounds
into preparations that can be used pharmaceutically. The
pharmaceutical compositions may be formulated in any acceptable way
to meet the needs of the mode of administration. For example, the
use of biomaterials and other polymers for drug delivery, as well
the different techniques and models to validate a specific mode of
administration, are disclosed in literature (Cleland J L et al.,
Curr Opin Biotechnol, 12: 212-9,2001; Luo B and Prestwich G D, Exp
Opin Ther Patents, 11: 1395-1410, 2001).
[0128] "Pharmaceutically acceptable" is meant to encompass any
carrier, which does not interfere with the effectiveness of the
biological activity of the active ingredient and that is not toxic
to the host to which is administered. For example, for parenteral
administration, the above active ingredients may be formulated in
unit dosage form for injection in vehicles such as saline, dextrose
solution, serum albumin and Ringer's solution.
[0129] Any accepted mode of administration can be used and
determined by those skilled in the art to establish the desired
blood levels of the active ingredients. For example, administration
may be by various parenteral routes such as subcutaneous,
intravenous, epidural, topical, intradermal, intrathecal, direct
intraventricular, intraperitoneal, transdermal (e.g. In slow
release formulations), intramuscular, intraperitoneal, intranasal,
intrapulmonary (inhaled), intraocular, oral, or buccal routes.
Parenteral administration can be by bolus injection or by gradual
perfusion over time. Other particularly preferred routes of
administration are aerosol and depot formulation. Sustained release
formulations, particularly depot, of the invented medicaments are
expressly contemplated.
[0130] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions, which
may contain auxiliary agents or excipients known in the art, and
can be prepared according to routine methods. In addition,
suspension of the active compounds as appropriate oily injection
suspensions may be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions that may contain substances increasing the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers. Pharmaceutical
compositions include suitable solutions for administration by
injection, and contain from about 0.01 to 99 percent, preferably
from about 20 to 75 percent of active compound together with the
excipient. Compositions that can be administered rectally include
suppositories.
[0131] For parenteral (e.g. intravenous, subcutaneous,
intramuscular) administration, the active protein(s) can be
formulated as a solution, suspension, emulsion or lyophilised
powder in association with a pharmaceutically acceptable parenteral
vehicle (e.g. water, saline, dextrose solution) and additives that
maintain isotonicity (e.g. mannitol) or chemical stability (e.g.
preservatives and buffers). The formulation is sterilized by
commonly used techniques. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0132] Pharmaceutical or physiologically acceptable preparations
that can be taken orally include push-fit capsules made of gelatin,
as well as soft, sealed capsules made of gelatin and a plasticizer,
such as glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with fillers such as lactose,
binders such as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers may be added. All
formulations for oral administration should be in dosages suitable
for such administration.
[0133] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner. For
administration by inhalation, the compounds for use according to
the present invention are conveniently delivered in the form of an
aerosol spray presentation from pressurized packs or a nebulizer,
with the use of a suitable gaseous propellant, e.g., carbon
dioxide. In the case of a pressurized aerosol the dosage unit may
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin, for use in an inhaler or
insufflator, may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0134] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder or lyophilized form for constitution with a suitable
vehicle, such as sterile pyrogen-free water, before use.
[0135] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Additionally, the compounds may be delivered using a
sustained-release system, such as semipermeable matrices of solid
hydrophobic polymers containing the therapeutic agent. Various
sustained-release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the compounds for a few
weeks up to over 100 days.
[0136] It is understood that the dosage administered will be
dependent upon the age, sex, health, and weight of the recipient,
kind of concurrent treatment, if any, frequency of treatment, and
the nature of the effect desired. The dosage will be tailored to
the individual subject, as is understood and determinable by one of
skill in the art. The total dose required for each treatment may be
administered by multiple doses or in a single dose. The
pharmaceutical composition of the present invention may be
administered alone or in conjunction with other therapeutics
directed to the condition, or directed to other symptoms of the
condition. Usually a daily dosage of active ingredient is comprised
between 0.01 to 100 milligrams per kilogram of body weight or more.
Ordinarily 1 to 40 milligrams per kilogram per day given in divided
doses or in sustained release form is effective to obtain the
desired results. Second or subsequent administrations can be
performed at a dosage, which is the same, less than, or greater
than the initial or previous dose administered to the
individual.
[0137] An "effective amount" refers to an amount of the active
ingredients that is sufficient to affect the course and the
severity of the disease, leading to the reduction or remission of
such pathology. The effective amount will depend on the route of
administration and the condition of the patient.
[0138] Dosage intervals can also be determined using the value for
the minimum effective concentration. Compounds should be
administered using a regimen that maintains plasma levels above the
minimum effective concentration for 10-90% of the time, preferably
between 30-90%; and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0139] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight the
severity of the affliction, the manner of administration and the
judgment of the prescribing physician. The dosage administered, as
single or multiple doses, to an individual will vary depending upon
a variety of factors, including pharmacokinetic properties, the
route of administration, patient conditions and characteristics
(sex, age, body weight, health, size), extent of symptoms,
concurrent treatments, frequency of treatment and the effect
desired.
[0140] The substances of the invention may be administered daily or
every other day, of less frequent. Preferably, one or more of the
substances of the invention are administered one, twice or three
times per week. The daily doses are usually given in divided doses
or in sustained release form effective to obtain the desired
results. Second or subsequent administrations can be performed at a
dosage which is the same, less than or greater than the initial or
previous dose administered to the individual. A second or
subsequent administration can be administered during or prior to
onset of the disease.
[0141] According to the invention, the substances of the invention
can be administered prophylactically or therapeutically to an
Individual prior to, simultaneously or sequentially with other
therapeutic regimens or agents (e.g. multiple drug regimens), in a
therapeutically effective amount. Active agents that are
administered simultaneously with other therapeutic agents can be
administered in the same or different compositions.
[0142] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
or encompasses a concentration point or range shown to decrease
cytokine expression in an in vitro system. Such information can be
used to more accurately determine useful doses in humans. A
therapeutically effective dose refers to that amount of the
compound that results in amelioration of symptoms in a patient.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50, (the dose
lethal to 50% of the test population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio between LD50 and ED50. Compounds
that exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED50, with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patients condition. (See,
e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1).
[0143] The present invention provides also provides novel screening
assays and kits including soluble proteins comprising a sequence
having at least 85% of homology with the mature form of the
extracellular domain of human CD164 (SEQ ID NO: 1), that can be
used identify and compare the properties of compounds as inhibitors
of cytokine secretion and expression. The kits and the assays
should comprise a soluble proteins comprising a sequence having at
least 85% of homology with the mature form of the extracellular
domain of human CD164 (SEQ ID NO: 1), eventually labelled or
immobilised on a solid support.
[0144] The following definitions are set forth to illustrate and
define the meaning and scope of the terms used to describe the
invention herein.
[0145] As used interchangeably herein, the terms
"oligonucleotides", and "polynucleotides" and nucleic add include
RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide
in either single chain or duplex form. The terms encompass
"modified nucleotides" which comprise at least one modification,
including by way of example and not limitation: (a) an alternative
linking group, (b) an analogous form of purine, (c) an analogous
form of pyrimidine, or (d) an analogous sugar. Examples of
analogous linking groups, purines, pyrimidines, and sugars are
known in the prior art (WO 95/04064). The polynucleotides encoding
the soluble proteins may be prepared by any known method, including
synthetic, recombinant, ex vivo generation, or a combination
thereof, as well as utilizing any purification methods known in the
art.
[0146] The terms polynucleotide construct, recombinant
polynucleotide and recombinant polypeptide are used herein
consistently with their use in the art. The terms "upstream" and
"downstream" are also used herein consistently with their use in
the art. The terms "base paired" and "Watson & Crick base
paired" are used interchangeably herein and consistently with their
use in the art. Similarly, the terms "complementary", "complement
thereof," "complement", "complementary polynucleotide",
"complementary nucleic acid" and "complementary nucleotide
sequence" are used interchangeably herein and consistently with
their use in the art.
[0147] Similarly, the term "purified" is used herein to describe a
soluble protein that has been separated from other compounds
including, but not limited to, nucleic acids, lipids, carbohydrates
and other proteins. In some preferred embodiments, a polypeptide is
substantially pure when at least about 50%, 60%, 75%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a
sample have a single amino acid sequence. In some preferred
embodiments, a substantially pure polypeptide typically comprises
about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5%
weight/weight of a protein sample. Polypeptide purity or
homogeneity is indicated by a number of methods well known in the
art, such as agarose or polyacrylamide gel electrophoresis of a
sample, followed by visualizing a single polypeptide band upon
staining the gel. For certain purposes, higher resolution can be
achieved by using HPLC or other methods well known in the art.
[0148] Further, as used herein, the term "purified" does not
require absolute purity; rather, it is intended as a relative
definition. Purification of starting material or natural material
to at least one order of magnitude, preferably two or three orders,
and more preferably four or five orders of magnitude is expressly
contemplated. Alternatively, purification may be expressed as "at
least" a percent purity relative to heterologous polynucleotides
(DNA, RNA or both) or polypeptides. As a preferred embodiment, the
CD164 polynucleotides or polypeptides are at least; 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or
100% pure relative to heterologous polynucleotides or polypeptides.
As a further preferred embodiment the polynucleotides or
polypeptides have an "at least" purity ranging from any number, to
the thousandth position, between 90% and 100% (e.g., at least
99.995% pure) relative to heterologous polynucleotides or
polypeptides. Additionally, purity of the polynucleotides or
polypeptides may be expressed as a percentage (as described above)
relative to all materials and compounds other than the carrier
solution. Each number, to the thousandth position, may be claimed
as individual species of purity.
[0149] The term "isolated" requires that the material be removed
from its original environment (e.g., the natural environment if it
is naturally occurring). For example, a naturally occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or DNA or polypeptide,
separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotide could be part of a
vector and/or such polynucleotide or polypeptide could be part of a
composition, and still be isolated in that the vector or
composition is not part of its natural environment.
[0150] The term "primer" denotes a specific oligonucleotide
sequence that is complementary to a target nucleotide sequence and
used to hybridize to the target nucleotide sequence. A primer
serves as an initiation point for nucleotide polymerization
catalyzed by DNA polymerase, RNA polymerase, or reverse
transcriptase.
[0151] The terms "protein" or "polypeptide" refer to a polymer of
amino acids without regard to the length of the polymer. Thus,
peptides, oligopeptides, and proteins are included within the
definition of polypeptide. This term also does not specify or
exclude post-expression modifications of polypeptides. For example,
polypeptides that include the covalent attachment of glycosyl
groups, acetyl groups, phosphate groups, lipid groups,
myristoylated groups and the like are expressly encompassed by the
term polypeptide. Also included within the definition are
phosphorylated or dephosphorylated polypeptides. Also included
within the definition are polypeptides which contain one or more
analogs of an amino add (including, for example, non-naturally
occurring amino acids, amino acids which only occur naturally in an
unrelated biological system, modified amino acids from mammalian
systems etc.), polypeptides with substituted linkages, as well as
other modifications known in the art, both naturally occurring and
non-naturally occurring.
[0152] The terms "comprising", "consisting of" and "consisting
essentially of" are defined according to their standard meaning. A
defined meaning set forth in the M.P.E.P. controls over a defined
meaning in the art and a defined meaning set forth in controlling
Federal Circuit case law controls over a meaning set forth in the
M.P.E.P. With this in mind, the terms may be substituted for one
another throughout the instant application in order to attach the
specific meaning associated with each term.
[0153] The term "treating" as used herein refers to administering a
compound after the onset of clinical symptoms.
[0154] The term "preventing" as used herein refers to administering
a compound before the onset of clinical symptoms.
[0155] The term "prevention" within the context of this invention
refers not only to a complete prevention of the disease or one or
more symptoms of the disease, but also to any partial or
substantial prevention, attenuation, reduction, decrease or
diminishing of the effect before or at early onset of disease.
[0156] The term "treatment" within the context of this invention
refers to any beneficial effect on progression of disease,
including attenuation, reduction, decrease or diminishing of the
pathological development after onset of disease.
[0157] All references cited herein, including journal articles or
abstracts, published or unpublished U.S. or foreign patent
application, issued U.S. or foreign patents or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures and text presented in the cited
references. Additionally, the entire contents of the references
cited within the references cited herein are also entirely
incorporated by reference.
[0158] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not any way an admission
that any aspect, description or embodiment of the present invention
is disclosed, taught or suggested in the relevant art.
[0159] The invention will now be described by means of the
following Examples, which should not be construed as in any way
limiting the present invention. The Examples will refer to the
specified Figures.
[0160] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art (including
the contents of the references cited herein), readily modify and/or
adapt for various application such specific embodiments, without
undue experimentation, and without departing from the general
concept of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning an range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one of ordinary skill in the
art.
EXAMPLES
Example 1
Cloning, High Throughput Expression, and Purification in Mammalian
Cells of the His-Tagged, sf-CD164
[0161] The cDNA sequence encoding the full extracellular region of
human CD164 (residues 1-163 of NCBI Ace. No. NP.sub.--006007; SEQ
ID NO: 3) was subcloned to generate an expression plasmid using
Gateway.TM. cloning technology (Invitrogen). This expression
plasmid allows the expression and the secretion of the mature form
of the extracellular region of human CD164 (140 amino acids) as a
soluble fusion protein having an hexa-histidine tag fused its
C-terminus (146 amino acids; sf-D164; SEQ ID NO: 2), then used for
affinity purification. The secretion is driven by the natural CD164
signal sequence (residues 1-23 of NCBI Acc. No. NP.sub.--006007;
SEQ ID NO: 3).
[0162] The mammalian cells chosen for the high throughput
expression were Human Embryonic Kidney 293 cells expressing the
Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, invitrogen).
[0163] The cells were maintained in suspension in Ex-cell VPRO
serum-free medium (seed stock, maintenance medium, JRH
Biosciences). Sixteen to 20 hours prior to transfection
(transfection day--1), cells were seeded (density of
2.times.10.sup.5 cells/ml) in 2.times.T225 flasks, each containing
50 ml DMEM (Dulbecco's modified Eagle's medium) I F12 (1:1) with 2%
FBS (fetal bovine serum) seeding medium (JRH Biosciences). The next
day (transfection day 0) the transfection took place by using the
JetPEI.TM. reagent (2 .mu.l/.mu.g plasmid; PolyPlus-transfection).
For each flask, 113 .mu.g of the sf-CD164 expression plasmid were
co-transfected with 2.3 .mu.g of a plasmid expressing Green
Fluorescent Protein (GFP). The transfection mix was then added to
the 2.times.T225 flasks and incubated at 37.degree. C. (5%
CO.sub.2) for 6 days. Confirmation of positive transfection was
done by microscopy (Axiovert 10 Zeiss) at day 1 and day 6 for
qualitatively evaluating the fluorescence due to GFP. On day 6
(harvest day), supernatants (100 ml) from the two flasks were
pooled and centrifuged (4.degree. C., 400 g) and placed into a pot
bearing a unique identifier.
[0164] The purification process was performed starting from 100 and
500 ml culture medium samples from cells expressing the C-terminal
His-tagged recombinant protein. The samples were diluted with one
volume cold buffer A (50 mM NaH.sub.2PO.sub.4; 600 mM NaCl; 8.7%
(w/v) glycerol, pH 7.5) to final volumes of 200 and 1000 ml,
respectively. The samples were filtered through a 0.22 .mu.m
sterile filter (Millipore, 500 ml filter unit) and kept at
4.degree. C. in sterile square media bottle (Nalgene).
[0165] The purification was performed at 4.degree. C. on the VISION
workstation (Applied Biosystems) connected to an automatic sample
loader (Labomatic). The purification procedure was composed of two
sequential steps, metal affinity chromatography on a Poros 20 MC
(Applied Biosystems) column charged with Ni ions (4.6.times.50 mm,
0.83 ml), followed by gel filtration on a Sephadex G-25 medium
(Amersham Pharmacia) column (1.0.times.10 cm).
[0166] For the first chromatography step the metal affinity column
(Ni-column) was regenerated with 30 column volumes of EDTA solution
(100 mM EDTA; 1 M NaCl; pH 8.0), recharged with Ni ions through
washing with 15 column volumes of a 100 mM NiSO.sub.4 solution,
washed with 10 column volumes of buffer A, followed by 7 column
volumes of buffer B (50 mM NaH.sub.2PO.sub.4; 600 mM NaCl; 8.7%
(w/v) glycerol, 400 mM; imidazole, pH 7.5), and finally
equilibrated with 15 column volumes of buffer A containing 15 mM
Imidazole. The sample was transferred, by the Labomatic sample
loader, into a 200 ml sample loop and subsequently charged onto the
Ni metal affinity column at a flow rate of 10 ml/min. For the 1000
ml sample the charging procedure was repeated 5 times. The
Ni-column was washed with 12 column volumes of buffer A, followed
by 28 column volumes of buffer A containing 20 mM imidazole. During
this wash, loosely attached contaminating proteins were eluted of
the column. The recombinant His-tagged protein was finally eluted
from Ni-column with 10 column volumes of buffer B at a flow rate of
2 ml/min, and the eluted protein was collected in a 1.6 ml
fraction.
[0167] For the second chromatography step, the Sephadex G-25
gel-filtration column was regenerated with 2 ml of buffer D (1.137
M NaCl; 2.7 mM KCl; 1.5 mM KH.sub.2PO.sub.4; 8 mM
Na.sub.2HPO.sub.4; pH 7.2), and subsequently equilibrated with 4
column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM
KH.sub.2PO.sub.4; 8 mM Na.sub.2HPO.sub.4; 20% (w/v) glycerol; pH
7.4). The peak fraction eluted from the Ni-column was
automatically, through the integrated sample loader on the VISION,
loaded onto the Sephadex G-25 column and the protein was eluted
with buffer C at a flow rate of 2 ml/min. The desalted sample was
recovered in a 2.2 ml fraction. The fraction was filtered through a
0.22 .mu.m sterile centrifugation filter (Millipore), aliquoted,
frozen and stored at -80.degree. C. An aliquot of the sample was
analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) by Coomassie
staining and Western blot with anti-His antibodies.
[0168] Coomassie staining was performed by incubating the NuPAGE
gel in a 0.1% Coomassie blue R250 staining solution (30% methanol,
10% acetic acid) at room temperature for 1 hour. The gel was
subsequently destained in 20% methanol, 7.5% acetic acid until the
background was clear and the protein bands clearly visible.
[0169] For the Western blot, the proteins were electrotransferred
from the NuPAGE gel to a nitrocellulose membrane at 290 mA for 1
hour at 4.degree. C. The membrane was blocked with 5% milk powder
in buffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH.sub.2PO.sub.4; 8 mM
Na.sub.2HPO.sub.4; 0.1% Tween 20, pH 7.4) for 1 hour at room
temperature, and subsequently incubated with a mixture of 2 rabbit
polyclonal anti-His antibodies (G-18 and H-15, 0.2 ug/ml each;
Santa Cruz) in 2.5% milk powder in buffer E overnight at 4.degree.
C. After further 1 hour incubation at room temperature, the
membrane was washed with buffer E (3.times.10 min), and then
incubated with a secondary HRP-conjugated anti-rabbit antibody
(DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk
powder for 2 hours at room temperature. After washing with buffer E
(3.times.10 minutes), the membrane was developed with the ECL kit
(Amersham Pharmacia) for 1 min. The membrane was subsequently
exposed to a Hyperfilm (Amersham Pharmacia), the film developed and
the western blot image visually analyzed.
[0170] The protein concentration in the samples was determined
using the BCA protein assay kit (Pierce) with bovine serum albumin
as standard.
Example 2
Effect of sf-CD164 an Cytokine Release Measured by Cell-Based
Assays
[0171] The following in vitro cell-based assays measure the effects
of sf-CD164 on cytokine secretion induced by Concanavalin A (Con A)
as measured by a cytokine bead array (CBA) assay for IL-2,
IFN-.gamma., TNF-.alpha., IL-5, IL-4 and IL-10.
[0172] The following equipments and software were used: [0173] 96
well microtiter plate photometer EX (Labsystem). [0174] Graph Pad
Software (Prism) [0175] Excel software (Microsoft) [0176] Flow
cytometer (Becton-Dickinson) [0177] CBA Analysis software [0178]
Hood for cell culture [0179] Incubator for cell culture [0180]
Centrifuge [0181] Pipettes
[0182] The following materials and reagents were used: [0183] Buffy
coat [0184] DMEM (GIBCO) [0185] Human serum type AB (SIGMA) [0186]
L-Glutamine (GIBCO) [0187] Penicillin-Streptomycin (GIBCO) [0188]
Ficoll (PHARMACIA) [0189] 96 well microtiter plate for cell culture
(COSTAR) [0190] Concanavalin A (SIGMA) [0191] Human Th1/Th2
Cytokine CBA Kit (Becton-Dickinson) [0192] PBS (GIBCO) [0193]
Falcon 50 ml sterile tubes (Becton-Dickinson) [0194] Bovine Serum
Albumin (BSA; SIGMA) [0195] Glycerol (MERCK) [0196] Dimethyl
Sulfoxide (DMSO; SIGMA) [0197] 96 well microtiter plate conical
bottom (NUNC) [0198] autoMACS.TM. Separator and MACS cell isolation
kit (Miltenyl Biotec)
[0199] The cells were Isolated for cell-based assays as
follows.
[0200] Human peripheral blood mononuclear cells (PBMC) were
isolated from buffy coat diluted with DMEM. 25 ml of diluted blood
was thereafter slowly added onto a 15 ml layer of Ficoll in a 50 ml
Falcon tube, and tubes were centrifuged (2000 rpm, 20 minutes, at
Room Temperature without brake). The interphase (ring) was then
collected and the cells were washed with 25 ml of DMEM followed by
a centrifuge step (1200 rpm, 5 min). This procedure was repeated
three times. A buffy coat gave approximately 600.times.10.sup.6
total cells.
[0201] Sub-populations of leukocytes CT cells, B cells, Monocytes)
were prepared from PBMC according to the isolation kit
manufacturer's instruction (MACS; Miltenyl Biotec). PBMC were
isolated from buffy coats as described above. Care was taken to
ensure a single-cell suspension. For preparation of CD4+ T cells,
the CD4+ T Cell Isolation Kit II was used (catalogue number
130-091-155, Miltenyl Biotec). PBMC were counted, centrifuged for
10 minutes and re-suspended in cold PBS buffer (phosphate buffered
saline pH 7.2, supplemented with 0.5% BSA, and 2 mM EDTA) at a
concentration of 2.5.times.10.sup.6 cells per ml (40 .mu.l of
buffer per 10.sup.7 cells). 10 .mu.l of Biotin-Antibody Cocktail
(supplied with the kit) per 10.sup.7 total cells was added. The
suspension was mixed well and incubated at 48.degree. C. for 10
minutes. 30 .mu.l of buffer was added per 10.sup.7 cells followed
by 20 .mu.l of Anti-Biotin MicroBeads per 10.sup.7 total cells. The
suspension was mixed well and incubated for an additional 15
minutes at 4-8.degree. C. The cells were washed with buffer by
adding 10-20.times. the labeling volume and centrifuged at
300.times.g for 10 minutes. The supernatant was removed completely
and the cells re-suspended up to 10.sup.8 cells in 500 .mu.l of
buffer. Magnetic separation was carried out with an autoMACS.TM.
Separator. The autoMACS.TM. Separator was prepared and primed
according to the manufacturer's instructions. The tube containing
the magnetically labeled cells was placed in the autoMACS.TM.
Separator and the program "deplete" was chosen. The negative
fraction was collected (outlet port "neg1"). This fraction
represents the enriched CD4+ T cells. Where required, the positive
fraction was subsequently collected (outlet port "pos1"). This
fraction represents the magnetically labeled non-CD4+ T cells.
[0202] The conditions appied for the cell-based assays were the
following: [0203] 100 000 cells/well in 96-well plates in 100 .mu.l
final in 2% glycerol. [0204] 5 ng/ml of the mitogen Concanavallin A
(ConA). [0205] 48 hours for each assay.
[0206] The cells were prepared in each well by mixing [0207] 80
.mu.l of 1.25.times.10.sup.6cells/ml were diluted in DMEM+2.5%
Human Serum+1% L-Glutamine+1% Penicillin-Streptomycin. [0208] 10
.mu.l of the solution containing sf-CD164 that wwas diluted in
PBS+20% Glycerol (the final dilution of the proteins is 1/10);
[0209] 10 .mu.l ConA.
[0210] After 48 hours, cell supernatants were collected and human
cytokines were measured by Human Th1/Th2 Cytokine CBA Kit
(Becton-Dickinson).
[0211] The mixed Human Th1/Th2 Capture Beads suspension were
prepared by vigorously vortexing for a few seconds before mixing
with the samples from microwell plate. For each assay to be
analysed, 10 .mu.l aliquot of each capture bead were added into a
single tube labelled "mixed capture beads". The Bead mixture was
thoroughly vortexed. The supernatants were diluted (1:4) using the
Assay Diluent (20 .mu.l of supernatants+60 .mu.l of Assay Diluent).
The sample dilution was then mixed before transferring samples into
a 96 wells microtiter plate conical bottom (Nunc).
[0212] The human Th1/Th2 Cytokine CBA Assay was performed by adding
50 .mu.l of the diluted supernatants Into a 96 wells microtiter
plate conical bottom (Nunc). 50 .mu.l of the mixed capture beads
were added followed by 50 .mu.l addition of the Human Th1/Th2 PE
Detection Reagent. The plate was then incubated for 3 hours at RT
and protected from direct exposure to light followed by
centrifugation at 1500 rpm for 5 minutes. The supernatant was then
carefully discarded. In a subsequent step, 200 .mu.l of wash buffer
were twice added to each well, centrifuged at 1500 rpm for 5
minutes and supernatant carefully discarded. 130 .mu.l of wash
buffer were thereafter added to each well to resuspend the bead
pellet. The samples were finally analysed on a flow cytometer. The
data were analysed using the CBA Application Software, Activity
Base and Microsoft Excel software.
[0213] The effect of sf-CD164 on cytokine release from human PBMC
cells (mixture) and Isolated T cells was measured for six
cytokines: TNF-.alpha., IFN-.gamma., IL-2, IL-4, IL-5 and IL-10.
The release of these cytokines was significantly diminished and in
a dose dependent manner by sf-CD164 In both cell-based assays (IC50
are summarized In Table III). Two exemplary dose-dependent curves
are shown for IL-2 and TNF-.alpha. for both human PBMC and Isolated
CD4+ T cells In FIGS. 2 and 3, respectively.
Example 3
Effect of sf-CD164 Administration on Cytokine Release Measured in
the LPS Induced TNF.alpha. Release Animal Model
[0214] The model of lipopolysaccharide (LPS)-induced TNF-.alpha.
release in mice was set up according the patent WO98/38179. LPS
(O111:B4; SIGMA) was injected (0.3 mg/kg, I.p.) in C3H/HeN mice
(Charles River, France). Ninety minutes later blood was sampled and
plasma TNF-.alpha. was determined using an ELISA kit (R&D).
Sf-CD164 and dexamethasone were diluted in PBS and injected
(Sf-CD164 at 0.03, 0.1 and 0.3 mg/kg, iv; or dexamethasone at 0.1
mg/kg, sc) 15 minutes prior to LPS administration.
[0215] Dexamethasone, the anti-inflammatory compound used as
positive control significantly (p<0.001) Inhibited LPS-induced
TNF-.alpha. release by 72%. Sf-CD164, at 0.3 mg/kg, significantly
(p<0.01) inhibited LPS-induced TNF-.alpha. release by 38% (FIG.
4). The lower doses of 0.03 and 0.1 mg/kg were capable to inhibit
TNF-.alpha. but in a manner less significant statistically.
Example 4
Effect of sf-CD164 Administration on Immune Cells Recruitment
Measured in Two Animal Models
[0216] The effect of sf-D164 administration on immune cells
recruitment was first tested using the thyoglicollate-induced
leukocyte peritoneal recruitment assay (FIG. 5).
[0217] The mice (strain C3H, 8 week old, n=6; Elevage Janvier,
France) were injected with sf-CD164 (0.03, 0.1 and 0.3 mg/kg, iv)
or dexamethasone (1 mg/kg, sc) diluted in PBS containing 0.02% BSA.
Thioglycollate (1.5%, 40 ml/kg, ip; SIGMA) was injected 15 minutes
after administration of the test molecules. A second administration
of the test molecules was done 24 hours later. Forty-eight hours
after the challenge with thioglycollate, the animals were
sacrificed and the ravage of the peritoneal cavity was conducted
using 2.times.5 ml PBS-1 mM EDTA (+4.degree. C.). After
centrifugation (10 min at 3000 rpm), the pellet was resuspended In
1 ml PBS. The peritoneal cells were counted using a Beckman/Coulter
counter.
[0218] Dexamethasone inhibited significantly (p<0.001) the
recruitment of macrophages by 69%. This effect was on a dose
dependent manner. Sf-CD164 (0.03, 0.1 and 0.3 mg/kg) significantly
inhibited thioglycollate-induced peritoneal recruitment of
macrophages by 5%, 26% (p<0.05) and 43% (p<0.001),
respectively, as well as lymphocytes (by 14%, 18% and 34%
respectively) and neutrophils peritoneal recruitment (by 3%, 9% and
23% respectively).
[0219] Similar results were obtained in the LPS-induced peritoneal
recruitment of neutrophils and lymphocytes (FIG. 5).
[0220] The same administration protocol described above was used
with LPS (O111:B4, Sigma; 0.9 mg/kg, 40 ml/kg, ip). Sf-CD164 (0.03,
0.1 and 0.3 mg/kg) significantly inhibited LPS-induced peritoneal
recruitment of neutrophils by 9%, 35% (p<0.001) and 43%
(p<0.001), respectively. At the same doses it also significantly
inhibited the recruitment of activated lymphocytes by 8%, 26%
(p<0.05) and 47% (p<0.001), respectively. Dexamethasone (0.1
mg/kg) significantly (p<0.001) inhibited the recruitment of
activated lymphocytes.
Example 5
Effect of sf-CD164 in a Cell-Based Assay for MBP-Specific Antigen
Processing and Presentation
[0221] An assay was developed to test the effect of sf-CD164 on the
proliferation of myelin basic protein (MBP)-specific T cells
induced by myelin basic protein peptide Ac1-11 (MBP(Ac1-11)). It
has been shown that epicutaneous immunization (ECi) with the
immunodominant peptide of myelin basic protein (MBP), Ac1-11,
protects mice that are transgenic for an Ac1-11-specific T cell
receptor against both the induced and spontaneous forms of
experimental allergic encephalomyelitis (EAE).
Spleens from B10.PL and MBP transgenic mice were harvested and
homogenized to obtain single cell suspensions. After erythrocyte
lysis with the Gay's solution, splenocytes were resuspended in PBS,
washed and counted.
[0222] After the isolation procedures, cellular viability was more
than 90% by trypan blue dye exclusion. The B10.PL antigen
presenting cells (APCs) were then irradiated with 25 Gy of
g-irradiation (stimulants), washed and resuspended in complete
medium at 1.9*10.sup.6 cells/ml. The responder cell population was
adjusted at 3.8*10.sup.6 cells/ml in complete medium. 80 .mu.l of
each cell suspension per well was mixed in 96 well plates. The
antigen was then added in a volume of 20 .mu.l: 10 .mu.g/ml of MBP
murine or 1 .mu.g/ml of Ac 1-11 MBP peptide per well (adequate
negative controls are BSA, MSA and an irrelevant MBP-derived
peptide respectively). The proteins or small molecules were added
in a volume of 20 .mu.l and then incubated at 37.degree. C. In a
humidified atmosphere with 5% CO2. After 3 days of culture, either
the supernatants were harvested and freezed at -80.degree. C. until
testing for cytokine production or 1 .mu.Ci of .sup.3H thymidine
was added and counted for radioactivity incorporation after 14-16
hours of additional incubation.
[0223] Sf-CD164 (50 .mu.g/ml) significantly inhibited the
proliferation of MBP specific T cells induced by Ac1-11 (1
.mu.g/ml; FIG. 6). Thus, sf-CD164 or soluble CD164 might be useful
in the treatment of multiple sclerosis.
Example 6
Effect of sf-CD164 Administration in an Animal Model of Fulminant
Liver Hepatitis
[0224] Sf-CD164 protein has been shown in vitro to inhibit
secretion of certain cytokines by ConA-stimulated human peripheral
blood mononuclear cells (PBMC). Since cytokines play a crucial role
in T cell induced ConA induced liver hepatitis (Seino et al. 2001,
Annals of surgery 234, 681; Kusters S, Gastroenterology
111(2):462-71, 1996; Toyonaga et al. 1994, PNAS 91, 614-618), we
tested sf-CD164 in this model.
[0225] Female C57/BL6 mice (8 weeks of age; IFFA CREDO) were used.
In general, 7 animals per experimental group are used. Mice were
maintained in standard conditions under a 12-hour light-dark cycle,
provided irradiated food and water ad libitum.
[0226] Concanavallin A (ConA; Sigma ref.C7275) was injected at 18
mg/kg iv and blood samples were taken at 1.30 and 8 hours
post-injection. Sf-CD164 was injected 30 minutes before ConA
injection. Positive controls were injected with Dexamethasone (0.1
mg/kg), and negative control were injected with PBS-BSA 1.8%
glycerol. At the time of sacrifice, blood was taken from the heart.
IL-6 and IFN-gamma cytokine levels were measured using the TH1/TH2
CBA assay 1.5 hours after ConA Injection. Transaminase blood
parameters were determined using the COBAS instrument
(Hitachi).
[0227] The experiment shows that sf-CD164 (1 mg/kg) protects from
liver injury in a mouse model mimicking fulminant hepatitis after
subcutaneous delivery of sf-CD164, since it decreases relevant
parameters such as transaminases levels (ALAT), IFN-.gamma., and
IL-6 cytokine levels (FIG. 7). The decrease in ALAT levels might be
due to both decreased IFN-.gamma. and IL-6 levels. Different
cytokines have been involved in the liver damage after ConA
injection. For example, anti TNF-alpha antibodies confer protection
against disease (Seino et al. 2001, Annals of surgery 234, 681) and
inhibition of IL-4 production by NKT cells was shown to be
hepato-protective in T-cell mediated hepatitis in mouse (Ajuebor et
al. 2003 J. Immunology 170, 5252-9). TABLE-US-00001 TABLE I Amino
Acid Synonymous Group More Preferred Synonymous Groups Ser Gly,
Ala, Ser, Thr, Pro Thr, Ser Arg Asn, Lys, Gln, Arg, His Arg, Lys,
His Leu Phe, Ile, Val, Leu, Met Ile, Val, Leu, Met Pro Gly, Ala,
Ser, Thr, Pro Pro Thr Gly, Ala, Ser, Thr, Pro Thr, Ser Ala Gly,
Thr, Pro, Ala, Ser Gly, Ala Val Met, Phe, Ile, Leu, Val Met, Ile,
Val, Leu Gly Ala, Thr, Pro, Ser, Gly Gly, Ala Ile Phe, Ile, Val,
Leu, Met Ile, Val, Leu, Met Phe Trp, Phe, Tyr Tyr, Phe Tyr Trp,
Phe, Tyr Phe, Tyr Cys Ser, Thr, Cys Cys His Asn, Lys, Gln, Arg, His
Arg, Lys, His Gln Glu, Asn, Asp, Gln Asn, Gln Asn Glu, Asn, Asp,
Gln Asn, Gln Lys Asn, Lys, Gln, Arg, His Arg, Lys, His Asp Glu,
Asn, Asp, Gln Asp, Glu Glu Glu, Asn, Asp, Gln Asp, Glu Met Phe,
Ile, Val, Leu, Met Ile, Val, Leu, Met Trp Trp, Phe, Tyr Trp
[0228] TABLE-US-00002 TABLE II Amino Acid Synonymous Group Ser
D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys,
D-Cys Arg D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile,
D-.Met, D-Ile, Orn, D-Orn Leu D-Leu, Val, D-Val, AdaA, AdaG, Leu,
D-Leu, Met, D-Met Pro D-Pro, L-I-thioazolidine-4-carboxylic acid,
D-or L-1- oxazolidine-4-carboxylic acid Thr D-Thr, Ser, D-Ser,
allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Ala D-Ala, Gly,
Aib, B-Ala, Acp, L-Cys, D-Cys Val D-Val, Leu, D-Leu, Ile, D-Ile,
Met, D-Met, AdaA, AdaG Gly Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala,
Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Phe
D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or
5-phenylproline, AdaA, AdaG, cis-3,4, or 5-phenylproline, Bpa,
D-Bpa Tyr D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Cys D-Cys,
S--Me--Cys, Met, D-Met, Thr, D-Thr Gln D-Gln, Asn, D-Asn, Glu,
D-Glu, Asp, D-Asp Asn D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Lys
D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile,
D-Ile,Orn, D-Orn Asp D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Glu
D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Met D-Met, S--Me--Cys,
Ile, D-Ile, Leu, D-Leu, Val, D-Val
[0229] TABLE-US-00003 TABLE III CYTOKINE IC50 in PBMC IC50 in CD4+
T CELLS TNF-.alpha. 0.66 .mu.g/ml 1.6 .mu.g/ml IFN-.gamma. 0.84
.mu.g/ml 1.6 .mu.g/ml IL-2 0.46 .mu.g/ml 1.2 .mu.g/ml IL-4 0.52
.mu.g/ml 1.5 .mu.g/ml IL-5 1.19 .mu.g/ml 3.3 .mu.g/ml IL-10 0.53
.mu.g/ml 3.0 .mu.g/ml
[0230]
Sequence CWU 1
1
8 1 140 PRT homo sapiens PUTATIVE-MUCIN-CORE-PROTEIN-24 (1)..(140)
N-LINKED-GLCNAC (3)..(3) N-LINKED-GLCNAC (9)..(9) O-LINKED
(11)..(11) O-LINKED (12)..(12) O-LINKED (17)..(17) N-LINKED-GLCNAC
(18)..(18) O-LINKED (20)..(20) O-LINKED (21)..(21) O-LINKED
(25)..(25) O-LINKED (26)..(26) O-LINKED (31)..(31) O-LINKED
(32)..(32) N-LINKED-GLCNAC (49)..(49) N-LINKED-GLCNAC (54)..(54)
N-LINKED-GLCNAC (71)..(71) CK2-PHOSPHO-SITE (73)..(76)
N-LINKED-GLCNAC (81)..(81) O-LINKED (89)..(89) O-LINKED (90)..(90)
O-LINKED (92)..(92) O-LINKED (96)..(96) N-LINKED-GLCNAC (98)..(98)
O-LINKED (99)..(99) O-LINKED (100)..(100) PKC-PHOSPHO-SITE
(100)..(102) O-LINKED (104)..(104) O-LINKED (108)..(108) O-LINKED
(110)..(110) O-LINKED (111)..(111) O-LINKED (112)..(112)
PKC-PHOSPHO-SITE (112)..(114) O-LINKED (113)..(113) O-LINKED
(115)..(115) O-LINKED (117)..(117) O-LINKED (118)..(118)
O-LINKED-GLYCOSAMINOGLYCAN (119)..(119) MYRISTYL (120)..(125)
O-LINKED (121)..(121) O-LINKED (122)..(122) N-LINKED-GLCNAC
(123)..(123) O-LINKED (125)..(125) O-LINKED (127)..(127) O-LINKED
(129)..(129) O-LINKED (130)..(130) CAMP-PHOSPHO-SITE (134)..(137)
O-LINKED (136)..(136) CK2-PHOSPHO-SITE (136)..(139) 1 Asp Lys Asn
Thr Thr Gln His Pro Asn Val Thr Thr Leu Ala Pro Ile 1 5 10 15 Ser
Asn Val Thr Ser Ala Pro Val Thr Ser Leu Pro Leu Val Thr Thr 20 25
30 Pro Ala Pro Glu Thr Cys Glu Gly Arg Asn Ser Cys Val Ser Cys Phe
35 40 45 Asn Val Ser Val Val Asn Thr Thr Cys Phe Trp Ile Glu Cys
Lys Asp 50 55 60 Glu Ser Tyr Cys Ser His Asn Ser Thr Val Ser Asp
Cys Gln Val Gly 65 70 75 80 Asn Thr Thr Asp Phe Cys Ser Val Ser Thr
Ala Thr Pro Val Pro Thr 85 90 95 Ala Asn Ser Thr Ala Lys Pro Thr
Val Gln Pro Ser Pro Ser Thr Thr 100 105 110 Ser Lys Thr Val Thr Thr
Ser Gly Thr Thr Asn Asn Thr Val Thr Pro 115 120 125 Thr Ser Gln Pro
Val Arg Lys Ser Thr Phe Asp Ala 130 135 140 2 146 PRT homo sapiens
2 Asp Lys Asn Thr Thr Gln His Pro Asn Val Thr Thr Leu Ala Pro Ile 1
5 10 15 Ser Asn Val Thr Ser Ala Pro Val Thr Ser Leu Pro Leu Val Thr
Thr 20 25 30 Pro Ala Pro Glu Thr Cys Glu Gly Arg Asn Ser Cys Val
Ser Cys Phe 35 40 45 Asn Val Ser Val Val Asn Thr Thr Cys Phe Trp
Ile Glu Cys Lys Asp 50 55 60 Glu Ser Tyr Cys Ser His Asn Ser Thr
Val Ser Asp Cys Gln Val Gly 65 70 75 80 Asn Thr Thr Asp Phe Cys Ser
Val Ser Thr Ala Thr Pro Val Pro Thr 85 90 95 Ala Asn Ser Thr Ala
Lys Pro Thr Val Gln Pro Ser Pro Ser Thr Thr 100 105 110 Ser Lys Thr
Val Thr Thr Ser Gly Thr Thr Asn Asn Thr Val Thr Pro 115 120 125 Thr
Ser Gln Pro Val Arg Lys Ser Thr Phe Asp Ala His His His His 130 135
140 His His 145 3 197 PRT homo sapiens 3 Met Ser Arg Leu Ser Arg
Ser Leu Leu Trp Ala Ala Thr Cys Leu Gly 1 5 10 15 Val Leu Cys Val
Leu Ser Ala Asp Lys Asn Thr Thr Gln His Pro Asn 20 25 30 Val Thr
Thr Leu Ala Pro Ile Ser Asn Val Thr Ser Ala Pro Val Thr 35 40 45
Ser Leu Pro Leu Val Thr Thr Pro Ala Pro Glu Thr Cys Glu Gly Arg 50
55 60 Asn Ser Cys Val Ser Cys Phe Asn Val Ser Val Val Asn Thr Thr
Cys 65 70 75 80 Phe Trp Ile Glu Cys Lys Asp Glu Ser Tyr Cys Ser His
Asn Ser Thr 85 90 95 Val Ser Asp Cys Gln Val Gly Asn Thr Thr Asp
Phe Cys Ser Val Ser 100 105 110 Thr Ala Thr Pro Val Pro Thr Ala Asn
Ser Thr Ala Lys Pro Thr Val 115 120 125 Gln Pro Ser Pro Ser Thr Thr
Ser Lys Thr Val Thr Thr Ser Gly Thr 130 135 140 Thr Asn Asn Thr Val
Thr Pro Thr Ser Gln Pro Val Arg Lys Ser Thr 145 150 155 160 Phe Asp
Ala Ala Ser Phe Ile Gly Gly Ile Val Leu Val Leu Gly Val 165 170 175
Gln Ala Val Ile Phe Phe Leu Tyr Lys Phe Cys Lys Ser Lys Glu Arg 180
185 190 Asn Tyr His Thr Leu 195 4 184 PRT homo sapiens 4 Met Ser
Arg Leu Ser Arg Ser Leu Leu Trp Ala Ala Thr Cys Leu Gly 1 5 10 15
Val Leu Cys Val Leu Ser Ala Asp Lys Asn Thr Thr Gln His Pro Asn 20
25 30 Val Thr Thr Leu Ala Pro Ile Ser Asn Val Thr Ser Ala Pro Val
Thr 35 40 45 Ser Leu Pro Leu Val Thr Thr Pro Ala Pro Glu Thr Cys
Glu Gly Arg 50 55 60 Asn Ser Cys Val Ser Cys Phe Asn Val Ser Val
Val Asn Thr Thr Cys 65 70 75 80 Phe Trp Ile Glu Cys Lys Asp Glu Ser
Tyr Cys Ser His Asn Ser Thr 85 90 95 Val Ser Asp Cys Gln Val Gly
Asn Thr Thr Asp Phe Cys Ser Ala Lys 100 105 110 Pro Thr Val Gln Pro
Ser Pro Ser Thr Thr Ser Lys Thr Val Thr Thr 115 120 125 Ser Gly Thr
Thr Asn Asn Thr Val Thr Pro Thr Ser Gln Pro Val Arg 130 135 140 Lys
Ser Thr Phe Asp Ala Ala Ser Phe Ile Gly Gly Ile Val Leu Val 145 150
155 160 Leu Gly Val Gln Ala Val Ile Phe Phe Leu Tyr Lys Phe Cys Lys
Ser 165 170 175 Lys Glu Arg Asn Tyr His Thr Leu 180 5 178 PRT homo
sapiens 5 Met Ser Arg Leu Ser Arg Ser Leu Leu Trp Ala Ala Thr Cys
Leu Gly 1 5 10 15 Val Leu Cys Val Leu Ser Ala Asp Lys Asn Thr Thr
Gln His Pro Asn 20 25 30 Val Thr Thr Leu Ala Pro Ile Ser Asn Val
Thr Ser Ala Pro Val Thr 35 40 45 Ser Leu Pro Leu Val Thr Thr Pro
Ala Pro Glu Thr Cys Glu Gly Arg 50 55 60 Asn Ser Cys Val Ser Cys
Phe Asn Val Ser Val Val Asn Thr Thr Cys 65 70 75 80 Phe Trp Ile Glu
Cys Lys Asp Glu Ser Tyr Cys Ser His Asn Ser Thr 85 90 95 Val Ser
Asp Cys Gln Val Gly Asn Thr Thr Asp Phe Cys Ser Val Ser 100 105 110
Thr Ala Thr Pro Val Pro Thr Ala Asn Ser Thr Gly Thr Thr Asn Asn 115
120 125 Thr Val Thr Pro Thr Ser Gln Pro Val Arg Lys Ser Thr Phe Asp
Ala 130 135 140 Ala Ser Phe Ile Gly Gly Ile Val Leu Val Leu Gly Val
Gln Ala Val 145 150 155 160 Ile Phe Phe Leu Tyr Lys Phe Cys Lys Ser
Lys Glu Arg Asn Tyr His 165 170 175 Thr Leu 6 189 PRT homo sapiens
SIGNAL (1)..(23) PUTATIVE-MUCIN-CORE-PROTEIN-24 (24)..(189)
N-LINKED-GLCNAC (26)..(26) N-LINKED-GLCNAC (32)..(32) O-LINKED
(34)..(34) O-LINKED (35)..(35) O-LINKED (40)..(40) N-LINKED-GLCNAC
(41)..(41) O-LINKED (43)..(43) O-LINKED (44)..(44) O-LINKED
(48)..(48) O-LINKED (49)..(49) O-LINKED (54)..(54) O-LINKED
(55)..(55) N-LINKED-GLCNAC (72)..(72) N-LINKED-GLCNAC (77)..(77)
N-LINKED-GLCNAC (94)..(94) N-LINKED-GLCNAC (104)..(104) O-LINKED
(112)..(112) O-LINKED (113)..(113) O-LINKED (115)..(115) O-LINKED
(119)..(119) N-LINKED-GLCNAC (121)..(121) O-LINKED (122)..(122)
O-LINKED (123)..(123) O-LINKED (127)..(127) O-LINKED (131)..(131)
O-LINKED (133)..(133) O-LINKED (134)..(134) O-LINKED (135)..(135)
O-LINKED (136)..(136) O-LINKED (138)..(138) O-LINKED (140)..(140)
O-LINKED (141)..(141) O-LINKED-GLYCOSAMINOGLYCAN (142)..(142)
O-LINKED (144)..(144) O-LINKED (145)..(145) N-LINKED-GLCNAC
(146)..(146) O-LINKED (148)..(148) O-LINKED (150)..(150) O-LINKED
(152)..(152) O-LINKED (153)..(153) O-LINKED (159)..(159) 6 Met Ser
Arg Leu Ser Arg Ser Leu Leu Trp Ala Ala Thr Cys Leu Gly 1 5 10 15
Val Leu Cys Val Leu Ser Ala Asp Lys Asn Thr Thr Gln His Pro Asn 20
25 30 Val Thr Thr Leu Ala Pro Ile Ser Asn Val Thr Ser Ala Pro Val
Thr 35 40 45 Ser Leu Pro Leu Val Thr Thr Pro Ala Pro Glu Thr Cys
Glu Gly Arg 50 55 60 Asn Ser Cys Val Ser Cys Phe Asn Val Ser Val
Val Asn Thr Thr Cys 65 70 75 80 Phe Trp Ile Glu Cys Lys Asp Glu Ser
Tyr Cys Ser His Asn Ser Thr 85 90 95 Val Ser Asp Cys Gln Val Gly
Asn Thr Thr Asp Phe Cys Ser Val Ser 100 105 110 Thr Ala Thr Pro Val
Pro Thr Ala Asn Ser Thr Ala Lys Pro Thr Val 115 120 125 Gln Pro Ser
Pro Ser Thr Thr Ser Lys Thr Val Thr Thr Ser Gly Thr 130 135 140 Thr
Asn Asn Thr Val Thr Pro Thr Ser Gln Pro Val Arg Lys Ser Thr 145 150
155 160 Phe Asp Ala Ala Ser Phe Ile Gly Gly Ile Val Leu Val Leu Glu
Ile 165 170 175 Arg Cys His Thr Arg Asn Tyr Ile Pro Asp Leu Lys Lys
180 185 7 197 PRT homo sapiens misc_feature (174)..(174) Xaa can be
any naturally occurring amino acid 7 Met Ser Arg Leu Ser Arg Ser
Leu Leu Trp Ala Ala Thr Cys Leu Gly 1 5 10 15 Val Leu Cys Val Leu
Ser Ala Asp Lys Asn Thr Thr Gln His Pro Asn 20 25 30 Val Thr Thr
Leu Ala Pro Ile Ser Asn Val Thr Ser Ala Pro Val Thr 35 40 45 Ser
Leu Pro Leu Val Thr Thr Pro Ala Pro Glu Thr Cys Glu Gly Arg 50 55
60 Asn Ser Cys Val Ser Cys Phe Asn Val Ser Val Val Asn Thr Thr Cys
65 70 75 80 Phe Trp Ile Glu Cys Lys Asp Glu Ser Tyr Cys Ser His Asn
Ser Thr 85 90 95 Val Ser Asp Cys Gln Val Gly Asn Thr Thr Asp Phe
Cys Ser Val Ser 100 105 110 Thr Ala Thr Pro Val Pro Thr Ala Asn Ser
Thr Ala Lys Pro Thr Val 115 120 125 Gln Pro Ser Pro Ser Thr Thr Ser
Lys Thr Val Thr Thr Ser Gly Thr 130 135 140 Thr Asn Asn Thr Val Thr
Pro Thr Ser Gln Pro Val Arg Lys Ser Thr 145 150 155 160 Phe Asp Ala
Ala Ser Phe Ile Gly Gly Ile Val Leu Val Xaa Gly Val 165 170 175 Xaa
Ala Val Ile Phe Phe Leu Tyr Lys Xaa Cys Lys Xaa Lys Glu Arg 180 185
190 Asn Tyr His Thr Leu 195 8 195 PRT homo sapiens 8 Met Ser Arg
Leu Ser Arg Ser Leu Leu Trp Ala Ala Thr Cys Leu Gly 1 5 10 15 Val
Leu Cys Val Leu Ser Ala Asp Lys Asn Thr Thr Gln His Pro Asn 20 25
30 Val Thr Thr Leu Ala Pro Ile Ser Asn Val Lys Ser Leu Ile Ser Cys
35 40 45 Ile Ser Pro Pro Asn Ser Pro Glu Thr Cys Glu Gly Arg Asn
Ser Cys 50 55 60 Val Ser Cys Phe Asn Val Ser Val Val Asn Thr Thr
Cys Phe Trp Ile 65 70 75 80 Glu Cys Pro Pro Thr Asp Glu Ser Tyr Cys
Ser His Asn Ser Thr Val 85 90 95 Ser Asp Cys Gln Val Gly Asn Thr
Thr Asp Phe Cys Ser Gly Lys Tyr 100 105 110 Ser Tyr Trp Leu Leu Gly
Ser Ile Pro Ala Lys Pro Thr Val Gln Pro 115 120 125 Ser Pro Ser Thr
Thr Ser Lys Thr Val Thr Thr Ser Gly Thr Thr Asn 130 135 140 Asn Thr
Val Thr Pro Thr Ser Gln Pro Val Arg Lys Ser Thr Phe Asp 145 150 155
160 Ala Ala Ser Phe Ile Gly Gly Ile Val Leu Val Leu Gly Val Gln Ala
165 170 175 Val Ile Phe Phe Leu Tyr Lys Phe Cys Lys Ser Lys Glu Arg
Asn Tyr 180 185 190 His Thr Leu 195
* * * * *