Note: Descriptions are shown in the official language in which they were submitted.
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Adiponectin fragments and conjugates
FIELD OF THE INVENTION
The present invention relates to a novel conjugate comprising an adiponectin
polypeptide, to a
novel adiponectin polypeptide fragment, to a method of preparing such
fragments or conjugates, to a
nucleotide sequence encoding the adiponectin polypeptide fragment or part of
the conjugate, to an
expression vector comprising the nucleotide sequence, to a host cell
comprising the nucleotide sequence,
to a pharmaceutical composition comprising the conjugate, to a pharmaceutical
composition comprising
the fragment, to use of the conjugate for the manufacture of a medicament for
treatment of type 1
1o diabetes; impaired glucose tolerance; type 2 diabetes; syndrome X; obesity;
cardiovascular disease, such
as atherosclerosis; septic shock; or dyslipidemia; or for lowering body weight
without reducing food
intake, and to a method of treating a mammal with type 1 diabetes; impaired
glucose tolerance; type 2
diabetes; syndrome X; obesity; dyslipidemia; cardiovascular disease, such as
atherosclerosis; or for
lowering body weight of a mammal without reducing food intake; rheumatoid
arthritis; Crohn's disease;
systemic lupus erythematosus; Sjogren's disease; cachexia; septic shock;
myasthenia gravis; post-
traumatic brain damage; myocardial infarction; post-surgical brain-damage; and
other destructive
processes related to stress or activation of the inflammatory system.
BACKGROUND OF THE INVENTION
Adiponectin (30 kDa) is a secreted protein expressed exclusively in
differentiated adipocyteS.
Primary sequence analysis reveals four main domains: a cleaved amino-terminal
signal sequence, a
region without homology to known proteins, a collagen-like region, and a
globular segment at the
carboxy terminus. The globular domain forms homotrimers, and additional
interactions between
adiponectin collagenous segments cause the protein to form higher order
structures. Adiponectin was
cloned in 1995/96 and is also known as AdipoQ and Acrp30, and its human
homologue has been
designated independently as apMl and GBP28.
Acrp30 protein shares sequence homology with a family of proteins showing a
modular 'design
containing a characteristic C-terminal complement factor C 1 q-like globular
domain. In addition to C 1 q,
members of this family include the human type VIII and X collagens,
precerebellin, and the hibernation-
3o regulated proteins hib 20, 25, and 27. Other than C 1 q, little is known
regarding the function of the C-
terminal globular regions of these proteins. In active and hibernating animals
members of the hib family
are differentially expressed in liver, suggesting a role in energy storage or
mobilization. A similar
function has been suggested for Acrp30 because the three-dimensional structure
of its C-terminal
globular domain is strikingly similar to that of tumor necrosis factor-a
(TNFa.), even though there is no
homology at the primary sequence level. Among its various biological effects
TNFcx (TNF-alpha)
regulates several aspects of energy homeostasis.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
2
A variety of factors has been shown to modulate the activity of components of
the insulin
signalling pathway, suggesting potential roles in the aetiology of insulin
resistance and type 2 diabetes.
TNF-alpha, for example, has been shown to inhibit the tyrosine kinase activity
of the insulin receptor in
adipocytes, reducing the phosphorylation and activation of IRS-1 and so
inhibiting the insulin signalling
pathway. Given that obesity is associated with over-expression of TNF-alpha
this suggests that TNF-
alpha impairment of IRS-mediated insulin signalling may be responsible, at
least in part, for obesity-
associated insulin resistance. Furthermore, insulin receptors and IRS-1 are
present in pancreatic beta
cells, and TNF-alpha and other cytokines have been shown to alter insulin
secretion. Thus, impairment
of insulin signalling by TNF-alpha and/or other pro-inflammatory cytokines may
be important
pathogenic mechanism linking obesity and type 2 diabetes.
T. Yokota et al (Blood, 2000; 96, 1723-1732) showed that human full-length
adiponectin
(produced in E.coli) specifically inhibits LPS-induced TNF-alpha production in
human macrophages,
indicating that adiponectin also may have anti-inflammatory activity.
PPARgamma agonists can suppress the activation of macrophages and so reduce
the production
of cytokines by these cells. For example, they have been shown to suppress the
LPS-induced TNF-alpha
synthesis by human peripheral mononuclear cells (C. Jiang et al, Nature, 1998;
391, 82-86).
BRIEF DISCLOSURE OF THE INVENTION
In the literature, both full-length adiponectin, (that is human adiponectin
produced from E. Coli,
and mouse adiponectin produced from E. Coli and mammalian cells), and globular
fragments of
adiponectin, (that is mouse adiponectin ACRP30 produced from E. Coli and
mammalian cells), have
been reported.
Common for the reported types of globular domains is that they are without a
larger part of the
collagenous domain that includes one to four lysines, thus, the known globular
fragments of adiponectin
do not include one or more lysines to be hydroxylated and glycosylated.
Moreover, these globular
fragments have been shown to be potent in muscle tissue, but they are not able
to show any effect on
insulin-reduced glucose output in hepatocytes. Furthermore,~no ieports of
globular fragments of
adiponectin in normalizing blood glucose levels have been made.
The reported full-length adiponectins are not as potent as globular fragments
of adiponectin in
3o muscle tissue, moreover, full-length adiponectin produced in E. Coli did
not show any effect on insulin-
reduced glucose output in hepatocytes. Full-length mouse adiponectin produced
in mammalian cells
showed effect on insulin-reduced glucose output in hepatocytes. Furthermore,
full-length mouse
adiponectin produced in mammalian cells have been able to reduce blood glucose
in a mouse model
(ob/ob) to near normal levels, when given in high dose.
Our medium sized fragments of adiponectin having a collagen domain (e.g. apM 1
(82-244))
produced in mammalian cells comprises at least one lysine which is
hydroxylated and glycosylated,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
3
moreover, they have been shown to transiently normalize blood glucose level in
a db/db mouse model in
a relatively low dose.
Without being bound by theory we believe that our medium sized fragments of
adiponectin are
more potent in the treatment of impaired glucose tolerance, and type 2
diabetes than the reported
globular forms due~to the hydroxy-glycosylation of one or more lysines in the
collagenous domain.
Since our fragments transiently normalize blood glucose level in a db/db mouse
model, this
indicates that the adiponectin polypeptide fragment should be administered
several times a day or more
conveniently should be conjugated to, for instance, a polymer, such as a PEG,
or a sugar moiety, to
thereby increase the half life, and reduce the frequency in administration.
Another approach to deal with
the transient normalization of blood glucose level would be to administer the
adiponectin polypeptide
fragment by gene therapy.
Accordingly, in one aspect the invention concerns an adiponectin polypeptide
fragment, such as
any one of seq id no 3, 4, 5, 10, 11, 12, or 13, as well as analogues thereof,
which fragment comprises a
globular domain, and a collagen domain, wherein at least one lysine in the
collagen domain is
15 hydroxylated and glycosylated.
Furthermore, we have analysed the structure of adiponectin, and located the
amino acids which
are surface exposed, and as such potential sites for introducing a non-
polypeptide moiety.
Thus, in a further aspect the invention concerns a conjugate comprising an
adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
2o wherein the adiponectin polypeptide comprises an amino acid residue having
an attachment group for
said first non-polypeptide moiety, wherein said amino acid residue is a
surface exposed amino acid
residue.
Since not all the surface exposed amino acids are desired for attaching a non-
polypeptide
moiety, it is a further aspect of the invention to introduce suitable amino
acids having an attachment
25 group for the non-polypeptide moiety into the position of a surface exposed
amino acid.
Thus, in a further aspect the invention concerns a conjugate comprising an
adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein said amino acid residue has been
introduced in a position that
3o in the parent adiponectin is occupied by a surface exposed amino acid
residue.
Furthermore, the wild type adiponectin has two conserved cysteine residues of
which Cys152
relative to seq id no 1, is non-surface exposed according to our analysis, and
as such not an obvious
choice when looking for a suitable attachment site for a non-polypeptide
moiety.
Thus, in a further aspect the invention concerns a conjugate comprising an
adiponeetin
35 polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein said amino acid residue is a
cysteine residue.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
4
Furthermore, the N-terminal of the adiponectin may also be suitable for
conjugation to a non-
polypeptide, provided that activity is not lost.
Thus, in a further aspect the invention concerns a conjugate comprising an
adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein the amino acid residue is the N-
terminal amino acid residue.
Moreover, we have discovered that calcium ions are crucial for the adiponectin
polypeptide to
form stable trimers and that removal of such calcium ions leads to
destabilization of the trimer structure.
Thus, in a further aspect the invention relates to an isolated complex
comprising a) an
1o adiponectin polypeptide or a conjugate comprising an adiponectin
polypeptide, and a first non-
polypeptide moiety covalently attached to the adiponectin polypeptide, and b)
calcium ions.
Furthermore, we have discovered that the introduction of a polymer, such as a
PEG, leads to
trimers having one, two, or three polymers attached to the adiponectin
polypeptide. Such trimers are
further stabilized with calcium ions.
15 Thus, in a further aspect the invention relates to an isolated complex
comprising
a) a conjugate comprising an adiponectin polypeptide trimer wherein the
adiponectin polypeptide trimer
contains three adiponectin polypeptide monomers, and one first polymer
covalently attached to any one
of the three monomers of the adiponectin polypeptide trimer in such a way that
the resulting trimer only
contains one polymer, and
2o b) calcium ions.
In a further aspect the invention concerns a nucleotide sequence encoding the
adiponectin
polypeptide part of the conjugate of the invention.
In a further aspect the invention concerns an expression vector comprising a
nucleotide
sequence of the invention.
25 In a further aspect the invention concerns a host cell comprising the
nucleotide sequence of the
invention.
In a further aspect the invention concerns a pharmaceutical composition
comprising the
conjugate of the invention and a pharmaceutically acceptable diluent, Garner
or adjuvant.
In a further aspect the invention concerns a pharmaceutical composition
comprising the
3o adiponectin polypeptide fragment of the invention and a pharmaceutically
acceptable diluent, Garner or
adjuvant.
In a further aspect the invention concerns use of a conjugate of the invention
for the
manufacture of a medicament for treatment of type 1 diabetes; impaired glucose
tolerance (herein after
referred to as IGT); type 2 diabetes; syndrome X; obesity; cardiovascular
disease, such as
35 atherosclerosis; dyslipidemia; or for lowering body weight without reducing
food intake; rheumatoid
arthritis; Crohn's disease; systemic lupus erythematosus; Sjogren's disease;
cachexia; septic shock;
myasthenia gravis; post-traumatic brain damage; myocardial infarction; post-
surgical brain-damage; and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
other destructive processes related to stress or activation of the
inflammatory system; in particular IGT,
type 2 diabetes, syndrome X, dyslipidemia, septic shock, or cardiovascular
disease, such as
atherosclerosis.
In a further aspect the invention concerns use of a conjugate or an
adiponectin polypeptide
fragment of the invention for preparing a medicament for treatment of a
disease, disorder, or condition
caused by expression or release of TNF-alpha in a human cell, wherein said
medicament inhibits
expression or release of TNF-alpha.
In a further aspect the invention concerns use of an adiponectin polypeptide
fragment of the
invention for the manufacture of a medicament for treatment of type 1
diabetes; impaired glucose
1 o tolerance (herein after referred to as IGT); type 2 diabetes; syndrome X;
obesity; cardiovascular disease,
such as atherosclerosis; dyslipidemia; or for lowering body weight without
reducing food intake;
rheumatoid arthritis; Crohn's disease; systemic lupus erythematosus; Sjogren's
disease; cachexia; septic
shock; myasthenia gravis; post-traumatic brain damage; myocardial infarction;
post-surgical brain-
damage; and other destructive processes related.to stress or activation of the
inflammatory system; in
15 particular IGT, type 2 diabetes, syndrome X, dyslipidemia, septic shock, or
cardiovascular disease, such
as atherosclerosis.
In a further aspect the invention concerns a method of treating a mammal with
type 1 diabetes;
impaired glucose tolerance (herein after referred to as IGT); type 2 diabetes;
syndrome X; obesity;
cardiovascular disease, such as atherosclerosis; dyslipidemia; or for lowering
body weight without
2o reducing food intake; rheumatoid arthritis; Crohn's disease; systemic lupus
erythematosus; Sjogren's
disease; cachexia; septic shock; myasthenia gravis; post-traumatic brain
damage; myocardial infarction;
post-surgical brain-damage; and other destructive processes related to stress
or activation of the
inflammatory system; in particular IGT, type 2 diabetes, syndrome X,
dyslipidemia, septic shock, or
cardiovascular disease, such as atherosclerosis, which method comprises
administering an effective
25 amount of a conjugate or an adiponectin polypeptide fragment of the
invention.
In a further aspect the present invention relates to a method of preparing an
adiponectin
polypeptide, comprising
a) preparing a nucleotide sequence encoding: a signal peptide and the
adiponectin polypeptide, wherein
the last three C-terminal amino acids of the signal peptide are HDG,
30 b) inserting the nucleotide sequence into a vector,
c) transfecting the vector into a mammalian cell,
d) expressing and optionally secreting the adiponectin polypeptide, and
e) optionally obtaining the adiponectin polypeptide.
Sequences of adiponectin polypeptides, fragments, and analogs, are listed in
the "sequence list".
DETAILED DISCLOSURE OF THE INVENTION
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
6
In the present application a number of references are referred to. They are
all intended to be
incorporated herein by reference.
In the context of the present application and invention the following
definitions apply:
The term "a" or "an", eg as used in "a non-polypeptide", "an amino acid
residue", "a
substitution", or "an attachment group", is intended to indicate one or more,
or at least one, eg a non
polypeptide means one or more non-polypeptides. "a" or "an" may be used
interchangeably with "one or
more" or "at least one" throughout the description.
The term "conjugate" (or interchangeably "conjugated polypeptide") is intended
to indicate a
heterogeneous (in the sense of composite or chimeric) molecule formed by the
covalent attachment of
t o one or more polypeptide(s) to one or more non-polypeptide moieties. The
term "covalent attachment"
means that the polypeptide and the non-polypeptide moiety are either directly
covalently joined to one
another, or else are indirectly covalently joined to one another through an
intervening moiety or
moieties, such as a bridge, spacer, or linkage moiety or moieties using an
attachment group present in
the polypeptide. Preferably, the conjugate is soluble at relevant
concentrations and conditions, i.e.
15 soluble in physiological fluids such as blood. Examples of conjugated
polypeptides of the invention
include glycosylated polypeptides, PEGylated polypeptides, glycosylated and
PEGylated polypeptides,
as well as glycosylated polypeptides having a PEG attached to the sugar
moiety. The term "non-
conjugated polypeptide" may be used about the polypeptide part of the
conjugate.
The term "non-polypeptide moiety" is intended to indicate a molecule that
20 is capable of conjugating to an attachment group of the polypeptide of the
invention. Preferred examples
of such molecule include polymer molecules, sugar moieties, lipophilic
compounds, or organic
derivatizing agents. When used in the context of a conjugate of the invention
it will be understood that
the non-polypeptide moiety is linked to the polypeptide part of the conjugate
through an attachment
group of the polypeptide.
25 The term "polymer molecule" is defined as a molecule formed by covalent
linkage of two or
more monomers, wherein none of the monomers is an amino acid residue. The term
"polymer" may be
used interchangeably with the term "polymer molecule". The term is intended to
cover carbohydrate
molecules attached by in vitro glycosylation, i.e. a synthetic glycosylation
performed in vitro normally
involving covalently linking a carbohydrate molecule to an attachment group of
the polypeptide,
30 optionally using a cross-linking agent. Carbohydrate molecules attached by
in vivo glycosylation, such
as N- or O-glycosylation (as further described below) are referred to herein
as "a sugar moiety". Except
where the number of non-polypeptide moieties, such as polymer molecules) or
sugar moieties in the
conjugate is expressly indicated every reference to "a non-polypeptide moiety"
contained in a conjugate
or otherwise used in the present invention shall be a reference to one or more
non-polypeptide moieties,
35 such as polymer molecules) or sugar moieties, in the conjugate.
The term "mono-pegylated" is intended to mean that the adiponectin polypeptide
has only one
polymer comprising a polyethylene glycol (PEG) covalently attached to it. Mono-
pegylation means that
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
7
the conjugate may be homogenous, eg. mono-pegylation of the N-terminal, or it
may be heterogenous,
eg. mono-pegylation of one lysine residue in each adiponectin molecule, for
instance, some of the
adiponectin molecules may be pegylated in position K134, and some of the
adiponectin molecules may
be pegylated in position K149 relative to seq id no 1 (these examples are
merely illustrative and are not
intended to limit the invention in any way).
The term "isolated" is intended to mean 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
to 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.
The term "attachment group" is intended to indicate an amino acid residue
group of the
polypeptide capable of coupling to the relevant non-polypeptide moiety. For
instance, for polymer, in
15 particular polyethylene glycol (PEG), conjugation a frequently used
attachment group is the s-amino
group of lysine or the N-terminal amino group. Other polymer attachment groups
include a free
carboxylic acid group (e.g. that of the C-terminal amino acid residue or of an
aspartic acid or glutamic
acid residue), suitably activated carbonyl groups, oxidized carbohydrate
moieties and mercapto groups
(eg. the sulthydryl group of cysteine).
20 For in vivo N-glycosylation, the term "attachment group" is used in an
unconventional way to
indicate the amino acid residues constituting an N-glycosylation site (with
the sequence N-X'-S/T/C-
X", wherein X' is any amino acid residue except proline, X" is any amino acid
residue that may or may
not be identical to X' and preferably is different from proline, N is
asparagine and S/T/C is either serine,
threonine or cysteine, preferably serine or threonine, and most preferably
threonine). Although the
25 asparagine residue of the N-glycosylation site is the one to which the
sugar moiety is attached during
glycosylation, such attachment cannot be achieved unless the other amino acid
residues of the N-
glycosylation site is present. Accordingly, when the non-polypeptide moiety is
an N-linked sugar
moiety, the term "amino acid residue having an attachment group for the first
non-polypeptide moiety"
as used in connection with alterations of the amino acid sequence of the
parent polypeptide is to be
3o understood as amino acid residues constituting an N-glycosylation site
is/are to be altered in such a
manner that a functional N-glycosylation site is introduced into the amino
acid sequence. For an "O-
glycosylation site" the attachment group is the OH-group of a serine or
threonine residue, and in that
respect the non-polypeptide moiety is an O-linked sugar moiety.
In the present application, amino acid names and atom names (e.g. CA, CB, CD,
CG, SG, NZ,
35 N, O, C, etc) are used as defined by the Protein DataBank (PDB)
(www.pdb.or~) which are based on the
IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and
Peptides (residue
names, atom names e.t.c.), Eur. J. Biochem., 138, 9-37 (1984) together with
their corrections in Eur. J.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
8
Biochem., 152, 1 (1985). CA is sometimes referred to as Ca, CB as C(3. The
term "amino acid residue"
is intended to indicate an amino acid residue contained in the group
consisting of alanine (Ala or A),
cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E),
phenylalanine (Phe or F), glycine
(Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),
leucine (Leu or L), methionine
(Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q),
arginine (Arg or R), serine
(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W),
and tyrosine (Tyr or Y)
residues. The terminology used for identifying amino acid
positions/substitutions is illustrated as
follows: C152 (indicates position #152 occupied by a cysteine residue in the
amino acid sequence e.g.
shown in SEQ ID NO 1). C152S indicates that the cysteine residue of position
152 has been replaced
1 o with a serine. The numbering of amino acid residues made herein is made
relative to the amino acid
sequence shown in SEQ ID NO 1. Multiple substitutions are indicated with a
"+", e.g. Fl 15N+V117T/S
means an amino acid sequence which comprises a substitution of the
phenylalanine residue in position
115 with an asparagine and a substitution of the valine residue in position
117 with a threonine or serine
residue, preferably a threonine residue. T/S as used about a given
substitution herein means either a T or
a S residue, preferably a T residue. As explained above the nomenclature X151Y
is intended to mean
that amino acid X in position 151 relative to human adiponectin has been
substituted with amino acid Y,
such as H151N.
The term "nucleotide sequence" is intended to indicate a consecutive stretch
of two or more
nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA,
semisynthetic,
2o synthetic origin, or any combinations thereof.
The term "polymerase chain reaction" or "PCR" generally refers to a method for
amplification
of a desired nucleotide sequence in vitro, as described, for example, in US
4,683,195. In general, the
PCR method involves repeated cycles of primer extension synthesis, using
oligonucleotide primers
capable of hybridising preferentially to a template nucleic acid.
"Cell", "host cell", "cell line" and "cell culture" are used interchangeably
herein and all such
terms should be understood to include progeny resulting from growth or
culturing of a cell.
"Transformation" and "transfection" are used interchangeably to refer to the
process of introducing
DNA into a cell.
"Operably linked" refers to the covalent joining of two or more nucleotide
sequences, by means
3o of enzymatic ligation or otherwise, in a configuration relative to one
another such that the normal
function of the sequences can be performed. For example, the nucleotide
sequence encoding a
presequence or secretory leader is operably linked to a nucleotide sequence
for a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide: a promoter or enhancer is
operably linked to a coding sequence if it affects the transcription of the
sequence; a ribosome binding
site is operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally,
"operably linked" means that the nucleotide sequences being linked are
contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is accomplished by
ligation at convenient
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
9
restriction sites. If such sites do not exist, then synthetic oligonucleotide
adaptors or linkers are used, in
conjunction with standard recombinant DNA methods.
The term "introduce" is primarily intended to mean substitution of an existing
amino acid
residue, but may also mean insertion of an additional amino acid residue. The
term "remove" is
primarily intended to mean substitution of the amino acid residue to be
removed by another amino acid
residue, but may also mean deletion (without substitution) of the amino acid
residue to be removed.
The term "immunogenicity" as used in connection with a given substance is
intended to indicate
the ability of the substance to induce a response from the immune system. The
immune response may be
a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology
(8"' Edition, Blackwell) for
t 0 further definition of immunogenicity). Immunogenicity may be determined by
use of any suitable
method known in the art, e.g. in vivo or in vitro. The term "reduced
immunogenicity" is intended to
indicate that the conjugate or polypeptide of the present invention gives rise
to a measurably lower
immune response than a reference molecule, such as wildtype human adiponectin
(apMl), or a variant
of wild-type human adiponectin, as determined under comparable conditions.
Normally, reduced
15 antibody reactivity is an indication of reduced immunogenicity.
The term "functional in vivo half life" is used in its normal meaning, i.e.
the time at which 50%
of a given functionality of the conjugate is retained (such as the time at
which 50% of the biological
activity of the conjugate is still present in the body/target organ, or the
time at which the activity of the
conjugate is 50% of the initial value). As an alternative to determining
functional in vivo half life,
2o "serum half life" may be determined, i.e. the time in which 50% of the
conjugate molecules circulate in
the plasma or bloodstream prior to being cleared. Determination of serum half
life is often more simple
than determining functional in vivo half life and the magnitude of serum half
life is usually a good
indication of the magnitude of functional in vivo half life. Alternative terms
to serum half life include
"plasma half life", "circulating half life", "serum clearance", "plasma
clearance" and "clearance half
25 life". The functionality to be retained is normally selected from
antiviral, antiproliferative,
immunomodulatory or receptor binding activity. Functional in vivo half life
and serum half life may be
determined by any suitable method known in the art.
The conjugate is normally cleared by the action of one or more of the
reticuloendothelial
systems (RES), kidney, spleen or liver, or by specific or unspecific
proteolysis. Clearance taking place
3o by the kidneys may also be referred to as "renal clearance" and is e.g.
accomplished by glomerular
filtration, tubular excretion or tubular elimination. Normally, clearance
depends on physical
characteristics of the conjugate, including molecular weight, size (diameter)
(relative to the cut-off for
glomerular filtration), charge, symmetry, shape/rigidity, attached
carbohydrate chains, and the presence
of cellular receptors for the protein. A molecular weight of about 67 kDa is
considered to be an
35 important cut-off value for renal clearance.
Reduced renal clearance may be established by any suitable assay, e.g. an
established in vivo
assay. Typically, the renal clearance is determined by administering a
labelled (e.g. radiolabelled or
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
fluorescence labelled) polypeptide conjugate to a patient and measuring the
label activity in urine
collected from the patient. Reduced renal clearance is determined relative to
the corresponding non-
conjugated polypeptide or the non-conjugated corresponding wild-type
polypeptide under comparable
conditions.
The term "increased" as used about the functional in vivo half life or serum
half life is used to
indicate that the relevant half life of the conjugate is statistically
significantly increased relative to that
of a reference molecule, such as an non-conjugated wildtype human adiponectin
or an non-conjugated
variant human adiponectin as determined under comparable conditions.
The term "reduced immunogenicity and/or increased functional in vivo half life
and/or increased
1 o serum half life" is to be understood as covering any one, two or all of
these properties. Preferably, a
conjugate of the invention has at least two or these properties, i.e. reduced
immunogenicity and
increased functional in vivo half life, reduced immunogenicity and increased
serum half life or increased
functional in vivo half life and increased serum half life. Most preferably,
the conjugate of the invention
has all properties.
t 5 The conjugates of the invention are useful as inter alia (ia) insulin
sensitizers based on their
ability to exhibit activity in the Test Assay (described in the experimental
section) by stimulating the
insulin-dependent reduction in glucose output in primary hepatocytes.
The term "one difference" or "differs from" as used in connection with
specific mutations is
intended to allow for additional differences being present apart from the
specified amino acid difference.
2o For instance, in addition to the removal and/or introduction of amino acid
residues comprising an
attachment group for the non-polypeptide moiety the adiponectin polypeptide
may comprise other
substitutions that are not related to introduction and/or removal of such
amino acid residues. The terms
"mutation" and "substitution" are used interchangeably herein.
The term "adiponectin polypeptide" is intended to indicate that the
polypeptide has a sequence
25 selected from any one of seq id no 1-8, 10-12, or 13, as well as
homologues, analogues, and fragments
thereof. Typically, the adiponectin polypeptide is selected from any one of
seq id no 1-8, 10-12, or 13,
as well as sequences that differs from any one of the specified sequences, in
one or more substitution(s),
preferably from one to eight, eg one to six. For convenience, the single
strands of the cDNA encoding
apMl(52-244), apMl(58-244), and apMl(82-244), are shown in seq id no 14-16,
respectively. The term
30 "homologue" is intended to indicate that a polypeptide has at least 50%
identity, such as at least 60%,
70%, 80%, 90%, or 95% identity, with any one of seq id no 1-8, 10-12, or 13.
The term "fragment" or
"adiponectin polypeptide fragment" is intended to indicate any one of seq id
nos 2-8, 10-12, or 13, as
well as homologues, analogues, and truncated versions thereof. Such truncation
may take place at the N-
or C-terminal end in accordance with known procedures, eg seq id no 5 may be C-
terminally truncated
35 by cleaving off two amino acids, thereby producing a sequence having amino
acid 101 to 242 of human
wild type adiponectin (human adiponectin (101-242), or apMl(101-242)). Another
couple of examples
of the nomenclature that has been used throughout this specification is apMl
(82-244) which means the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
11
sequence of human wild type adiponectin from amino acid 82 to 244; apMl(52-
244) which means the
sequence of human wild type adiponectin from amino acid 52 to 244; and apMl(58-
244) which means
the sequence of human wild type adiponectin from amino acid 58 to 244. A
further nomenclature that
has been used throughout this specification is, for instance, T121C-apMl (82-
244) which means the
sequence of human wild type adiponectin from amino acid 82 to 244, wherein Thr
in position 121 has
been substituted with Cys.
Typically, the term fragment means that any one of the seq id no 2-8, 10-12,
or 13, is truncated
N-terminally with 1, 2, 3, 4, 5, or 6 amino acid residues, or truncated C-
terminally with l, 2, 3, 4, 5, or 6
amino acid residues. In a non-limiting example, the fragment is truncated N-
terminally with 6 amino
1 o acid residues, and optionally truncated C-terminally with 2 amino acid
residues.
The percent identity as stated above can be determined conventionally using
known computer
programs. Typically, we are using the CLUSTALW program. (Thompson et al.,
1994, CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment through
sequence weighting,
position-specific gap penalties and weight matrix choice, Nucleic Acids
Research, 22:4673-4680).
15 Typically the adiponectin polypeptide exhibits activity in the Test Assay
(described in the
experimental section): Determination of adiponectin's effect on glucose uptake
in C2C12 cells.
The adiponectin polypeptide also exhibits activity in the Test Assay
(described in the
experimental section) by inhibiting LPS-induced TNF-alpha production in
monocytic cell line.
The adiponectin polypeptide also exhibits activity in the Test Assay
(described in the
2o experimental section) by enhancing the insulin mediated suppression of
glucose out-put in primary
hepatocytes.
The adiponectin polypeptide also exhibits activity in db/db mice (described in
the experimental
section) by lowering and normalizing blood glucose level.
Human wildtype adiponectin (or interchangeably "human adiponectin") (seq id no
1 ) consists of
25 244 amino acid residues, that is, a signal sequence from amino acid 1-17, a
non-homologous domain
from amino acid 18-41, a collagen domain from amino acid 42-107, and a
globular domain from amino
acid 108-244. The single strand of the cDNA encoding human adiponectin is
shown in seq id no 9.
The term "globular domain" is intended to indicate the sequence of human
adiponectin (108-
244) (shown in seq id no 6) and analogues thereof. Fragments are also intended
to be comprised, that is
3o both C-terminally truncated as well as N-terminally truncated. The globular
domain of human
adiponectin (apMl) is known to form trimers.
The term "trimer" as used in connection with an adiponectin polypeptide trimer
means that three
molecules of an adiponectin polypeptide monomer forms a trimer.
The term "homotrimer" means that the trimer consists of three identical
monomers.
35 The term "heterotrimer" means that the trimer consists of different
monomers, such as, two of
the monomers may be the same and the third may be different, or all three
monomers may be different.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
12
The difference being that one or two monomers) has/have an amino acid sequence
that differs from that
of the other monomer(s).
The term "collagen domain" is intended to indicate the sequence of human
adiponectin (42-107)
(as indicated in seq id no 1 ) and analogues thereof. Fragments are also
intended to be comprised, that is
both C-terminally truncated as well as N-terminally truncated. A collagen
domain is well known to have
repeating sequences of Gly-X-Y, wherein X and Y are the same or different and
selected from the amino
acids (one letter code): A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W,
Y, and V. An example of the
collagen domain is the amino acids from G1y99 to G1y107 (G99-G107). Another
example of the
collagen domain is the amino acids from G1u82 to G1y107 (E82-6107)
to The term "non-homologuous domain" is intended to indicate the sequence of
human adiponectin
(18-41 ) (as indicated in seq id no 1 ) and analogues thereof. Fragments are
also intended to be comprised,
that is both C-terminally truncated as well as N-terminally truncated.
The term "signal peptide" is intended to indicate the sequence of human
adiponectin (1-17) (as
indicated in seq id no 1 ) and analogues thereof. Fragments are also intended
to be comprised, that is both
IS C-terminally truncated as well as N-terminally truncated. An example of the
signal sequence is the
amino acids from Met 1 to Asp 17 (M 1-D 17).
The term "parent adiponectin" (or interchangeably "parent adiponectin
polypeptide") is intended
to indicate the starting molecule to be improved in accordance with the
present invention. While the
parent adiponectin may be of any origin, such as vertebrate or mammalian
origin (e.g. any of the origins
2o defined in WO 01/51645), or fragments thereof, the parent adiponectin is
typically wild-type human
adiponectin with SEQ ID NO l, or any of the fragments of seq id nos 2-8, 10-
12, or 13, or an analogue
thereof.
An "analogue" is a polypeptide, which differs in one or more amino acid
residues from a parent
polypeptide, normally in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues.
25 The term "functional site" as used about a polypeptide or conjugate of the
invention is intended
to indicate one or more amino acid residues which is/are essential for or
otherwise involved in the
function or performance of adiponectin, and thus "located at" the functional
site.
Characterisation of the apMl(82-244) prepared in example 2 revealed that
apMl(82-244)
produced in CHO cells is partially hydroxylated on the Pro-residues (P95 and
P104) and partially
3o hydroxylated and subsequently glycosylated on the Lys101-residue in the
collagen-like part of the
molecule (hereinafter also referred to as glyco-hydroxy-Lys). Thus, eucaryotic
cells, typically,
mammalian cells expressing, for instance, adiponectin polypeptide of seq id no
3, 10, 12, or 13,
produces sequences with four, one, four, and four glyco-hydroxy-Lys residues,
respectively. Whenever,
one, two, three or four Lys are present in the collagenous domain, and the
adiponectin polypeptide is
35 produced in mammalian cells, it comprises such post-translational
modifications. Moreover, if a more
optimized hydroxylation of the Pro-residues is desired, then Vitamine C should
be present during
expression of the polypeptide. Typically, the adiponectin polypeptide is
selected from any one of seq id
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
13
no 2, 3, 4, 5, 10, 11, 12, or 13, as well as sequences that differs from any
one of the specified sequences,
in one or more substitution(s), preferably from one to eight (in the situation
with seq id no 5, the
hydroxylated and glycosylated N-terminal lysine, may be prepared by
constructing a longer fragment,
such as apMl(82-244), and subsequently cutting with a suitable enzyme, such as
trypsin). If the
adiponectin polypeptide differs in one or more substitutions it means that one
or more amino acid
residues are introduced or removed, or some may be introduced and some may be
removed.
Adigonectin polypeptide fragmentl~s) of the invention
In a first aspect the invention concerns an adiponectin polypeptide fragment
comprising any one of seq
id no 2, 3, 4, 5, 10, 11, 12, or 13, as well as homologues, analogues, and
fragments thereof. Typically,
the adiponectin polypeptide fragment is selected from any one of seq id no 2,
3, 4, 5, 10, 11, 12, or 13,
as well as sequences that differs from any one of the specified sequences, in
one or more substitution(s),
preferably from one to eleven, such as from one to eight.
The one or more substitutions) (as explained above) in addition to the removal
and/or
15 introduction of amino acid residues comprising an attachment group for the
non-polypeptide moiety
may also comprise other substitutions that are not related to introduction
and/or removal of such amino
acid residues. However, the adiponectin polypeptide fragment as well as
sequences that differs in one or
more substitution(s), should have biological activity, such activity could be
tested in a relevant animal
model, such as mouse models of insulin resistance and diabetes, such as the
db/db mouse described in:
20 A. E. Halseth et al, Biochemical and Biophysical Research Communications
294 (2002) 798-805) mice;
or the ob/ob mouse described in: X. M. Song et al, Diabetologia 45 (2002) 56-
65; or rat models such as
zucker rats, or could be tested in a relevant in vitro assay, such as any one
of the Test Assays A, B, or C
described in the experimental section..
In a further embodiment the adiponectin polypeptide fragment is selected from
any one of seq id
25 no 3, 10, 12, or 13, as well as sequences that differs from any one of the
specified sequences in one or
more substitution(s), preferably from one to eleven, such as in one to eight
substitutions, eg. 1-6
substitutions.
In a further embodiment the adiponectin polypeptide fragment is selected from
any one of seq id
no 2, 3, 4, S, 10, 11, 12, or 13, preferably 3, 10, 12, or 13. A typical
adiponectin polypeptide fragment is
30 seq id no 10. Another typical adiponectin polypeptide fragment is seq id no
12. A further typical
adiponectin polypeptide fragment is seq id no 13.
In a alternative embodiment the adiponectin polypeptide fragment is selected
from sequences
that differs from any one of the seq id no 2, 3, 4, 5, 10, 11, 12, or 13,
preferably 3, 10, 12, or 13, in one
or more substitutions, preferably from one to eleven, such as in one to eight
substitutions, eg. 1-6
35 substitutions.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
14
In a further alternative embodiment the adiponectin polypeptide fragment is
selected from
sequences that differs from the seq id no 3 in one or more substitutions,
preferably from one to eleven,
such as in one to eight substitutions, eg. 1-6 substitutions.
In a further alternative embodiment the adiponectin polypeptide fragment is
selected from
sequences that differs from the seq id no 10 in one or more substitutions,
preferably from one to eleven,
such as in one to eight substitutions, eg. 1-6 substitutions.
In a further alternative embodiment the adiponectin polypeptide fragment is
selected from
sequences that differs from the seq id no 12 in one or more substitutions,
preferably from one to eleven,
such as in one to eight substitutions, eg. 1-6 substitutions.
1 o In a further alternative embodiment the adiponectin polypeptide fragment
is selected from
sequences that differs from the seq id no 13 in one or more substitutions,
preferably from one to eleven,
such as in one to eight substitutions, eg. 1-6 substitutions.
Typically, the adiponectin polypeptide fragment is produced in a mammalian
cell, eg a CHO,
BHK, HEK293 cell or an SF9 cell. The lysines in the collagenous domain are
hydroxylated and
15 glycosylated, when produced in a eucaryotic cell, such as a mammalian cell.
In a further embodiment the adiponectin polypeptide fragment comprises one to
four lysine
residues selected from any one of the positions K65, K68, K77, or K101. In a
further embodiment the
adiponectin polypeptide fragment comprises at least one lysine residue
selected from any one of the
positions K65, K68, K77, or K101. Preferably, the lysine residues are
hydroxylated and glycosylated. In
2o a further embodiment the adiponectin polypeptide fragment comprises one
lysine residue selected from
any one of the positions K65, K68, K77, or K101, preferably K101, and
preferably the position is
hydroxylated and glycosylated, such as glyco-hydroxy-K101. In a further
embodiment the adiponectin
polypeptide fragment comprises two lysine residues selected from any one of
the positions K65, K68,
K77, or K101, preferably K77 and K101, and preferably both of the positions
are hydroxylated and
25 glycosylated, such as glyco-hydroxy-K77 and glyco-hydroxy-K101. In a
further embodiment the
adiponectin polypeptide fragment comprises three lysine residues selected from
any one of the positions
K65, K68, K77, or K101, preferably K68, K77 and K101, and preferably all three
of the positions are
hydroxylated and glycosylated, such as glyco-hydroxy-K68, glyco-hydroxy-K77
and glyco-hydroxy-
K101. In a further embodiment the adiponectin polypeptide fragment comprises
four lysine residues
3o selected from positions K65, K68, K77, and K101, and preferably all four of
the positions are
hydroxylated and glycosylated. The N-terminal amino acid of the collagen
domain is typically not a
lysine, eg. K65, K68, or K77, since such a lysine will not be hydroxylated and
glycosylated upon
expression in a eucaryotic cell, such as a mammalian cell. However, as
explained above if the desired
adiponectin polypeptide fragment has the N-terminal amino acid, K101, then the
lysine may be
35 hydroxylated and glycosylated upon expression of a longer fragment in a
eucaryotic cell, and
subsequently cutting with a suitable enzyme, such as a trypsin.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
When the adiponectin polypeptide fragment comprises a collagen domain, such as
any one of
seq id no 3, 4, 5, 10, 11, 12, or 13, such collagen domain comprises lysines,
which when produced in a
eucaryotic cell are hydroxylated and glycosylated. If the adiponectin
polypeptide fragment only has 7
amino acids, or less, of the collagen domain, such as apMl(101-244) shown in
seq id no 5, then the
lysine will not be hydroxylated and glycosylated. However, the apMl(101-244)
could be constructed so
as to have a glyeo-hydroxy-K101 residue, since production of, eg. apMl(82-244)
in a CHO cell, and
subsequently cutting with an enzyme (that cuts between arginine and lysine),
such as trypsin, between
position 8100 and K101 would create the apMl(101-244) having the position K101
hydroxylated and
glycosylated.
10 Thus, in a certain aspect the invention concerns an adiponectin polypeptide
fragment comprising
a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence as indicated in
seq id no 1 from
position A108 to N244 as well as sequences that differs from the amino acid
sequence in one or more
substitution(s), and
15 wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as
indicated in seq id no 1 to 66 amino acids corresponding to position G42 as
indicated in seq id no 1, and
wherein the collagen domain comprises a lysine which is hydroxylated and
glycosylated.
The above adiponectin polypeptide fragment comprising a globular domain and a
collagen
domain wherein the collagen domain comprises a lysine which is hydroxylated
and glycosylated is
2o particularly preferred over adiponectin polypeptide fragments which do not
have a collagen domain or
which do not comprise a lysine which is hydroxylated and glycosylated. The
presence of a lysine which
is hydroxylated and glycosylated improves the overall performance of the
molecule as a therapeutic
agent useful for treating eg. impaired glucose tolerance, type 2 diabetes,
syndrome X, obesity, a
cardiovascular disease, such as atherosclerosis, or dyslipidemia. Moreover, we
have discovered that if
the adiponectin polypeptide fragment is to be expressed in acceptable yields
in a eucaryotic cell, such as
a mammalian cell, then the collagen domain should not comprise more than 56
amino acids, preferably
not more than 50 amino acids. However, it was possible to increase the
expression, with the aid of an
expression enhancer, such as UCOE, when the collagen domain comprises more
than 50 amino acids.
Typically, so-called UCOE's may be obtained from Cobra Therapeutics Limited,
or may be prepared,
3o for instance, as described in WO 00/05393.
Thus, the above adiponectin polypeptide fragment comprising a globular domain
and a collagen
domain is expressed in high yields from a eucaryotic, such as a mammalian
expression system so as to
be reproducible in large scale culturing.
Accordingly, a preferred aspect of the invention concerns an adiponectin
polypeptide fragment
comprising a globular domain and a collagen domain,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
16
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1 as well as sequences that differs from the amino acid
sequence in one or more
substitution(s), and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as
indicated in seq id no 1 to 56 amino acids corresponding to position A52 as
indicated in seq id no 1, and
wherein the collagen domain comprises a lysine which is hydroxylated and
glycosylated.
Typically, the globular domain should not contain too many amino acid changes
as this may
reduce the biological activity or lead to increased immunogenicity.
Accordingly, in a further embodiment of the adiponectin polypeptide fragment
comprising a
1 o globular domain and a collagen domain, the globular domain comprises an
amino acid sequence from
position 108 to 244 as indicated in seq id no 1 as well as sequences that
differs from the amino acid
sequence in up to eleven substitution(s).
In a further embodiment the globular domain comprises an amino acid sequence
from position
A108 to N244 as indicated in seq id no 1.
15 In the situation were it is desired to introduce glycosylation site(s), or
remove/introduce amino
acids) in the globular domain, the globular domain differs from the amino acid
sequence from position
A108 to N244 as indicated in seq id no 1 in one or more substitution(s).
Typically, the globular domain
differs from the amino acid sequence from position A108 to N244 as indicated
in seq id no 1 in one to
eleven (11) substitution(s), such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,
1-2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
20 or 11 substitution(s).
Furthermore, the adiponectin polypeptide fragment comprises any one of the
above
embodiments of the globular domain together with a collagen domain that
comprises from 7 amino acids
corresponding to position K101 as indicated in seq id no 1 to 66 amino acids
corresponding to position
G42 as indicated in seq id no l, and wherein the collagen domain comprises a
lysine which is
25 hydroxylated and glycosylated. In a further embodiment the collagen domain
comprises from 7 amino
acids corresponding to position K101 as indicated in seq id no 1 to 56 amino
acids corresponding to
position A52 as indicated in seq id no 1. Typically, the collagen domain
comprises from 7 amino acids
corresponding to position K101 as indicated in seq id no 1 to 50 amino acids
corresponding to position
R58 as indicated in seq id no 1, such as from 8 amino acids corresponding to
position 8100 as indicated
3o in seq id no 1 to SO amino acids corresponding to position R58 as indicated
in seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 47 amino
acids corresponding to
position T61 as indicated in seq id no 1, from 8 amino acids corresponding to
position 8100 as indicated
in seq id no 1 to 44 amino acids corresponding to position E64 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 41 amino
acids corresponding to
35 position E67 as indicated in seq id no 1, from 8 amino acids corresponding
to position 8100 as indicated
in seq id no 1 to 38 amino acids corresponding to position D70 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 35 amino
acids corresponding to
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
17
position L73 as indicated in seq id no 1, from 8 amino acids corresponding to
position 8100 as indicated
in seq id no 1 to 32 amino acids corresponding to position P76 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 29 amino
acids corresponding to
position D79 as indicated in seq id no 1, from 8 amino acids corresponding to
position 8100 as indicated
in seq id no 1 to 26 amino acids corresponding to position E82 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 23 amino
acids corresponding to
position V85 as indicated in seq id no 1, from 8 amino acids corresponding to
position 8100 as indicated
in seq id no 1 to 20 amino acids corresponding to position A88 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 17 amino
acids corresponding to
t o position P91 as indicated in seq id no 1, from 8 amino acids corresponding
to position 8100 as indicated
in seq id no 1 to 14 amino acids corresponding to position F94 as indicated in
seq id no 1, from 8 amino
acids corresponding to position 8100 as indicated in seq id no 1 to 11 amino
acids corresponding to
position I97 as indicated in seq id no 1, from 11 amino acids corresponding to
position I97 as indicated
in seq id no 1 to 50 amino acids corresponding to position R58 as indicated in
seq id no 1, from 14
amino acids corresponding to position F94 as indicated in seq id no 1 to 47
amino acids corresponding
to position T61 as indicated in seq id no 1, from 17 amino acids corresponding
to position P91 as
indicated in seq id no 1 to 44 amino acids corresponding to position E64 as
indicated in seq id no 1,
from 20 amino acids corresponding to position A88 as indicated in seq id no 1
to 41 amino acids
corresponding to position E67 as indicated in seq id no 1, from 23 amino acids
corresponding to position
V85 as indicated in seq id no 1 to 38 amino acids corresponding to position
D70 as indicated in seq id
no l, from 26 amino acids corresponding to position E82 as indicated in seq id
no 1 to 35 amino acids
corresponding to position L73 as indicated in seq id no 1, including the
collagen domain comprising 7
amino acids corresponding to position K101 as indicated in seq id no 1, 8
amino acids corresponding to
position 8100 as indicated in seq id no 1, 11 amino acids corresponding to
position I97 as indicated in
seq id no 1, 14 amino acids corresponding to position F94 as indicated in seq
id no 1, 17 amino acids
corresponding to position P91 as indicated in seq id no 1, 20 amino acids
corresponding to position A88
as indicated in seq id no 1, 23 amino acids corresponding to position V85 as
indicated in seq id no 1, 26
amino acids corresponding to position E82 as indicated in seq id no 1, 29
amino acids corresponding to
position D79 as indicated in seq id no 1, 32 amino acids corresponding to
position P76 as indicated in
3o seq id no 1, 35 amino acids corresponding to position L73 as indicated in
seq id no 1, 38 amino acids
corresponding to position D70 as indicated in seq id no 1, 41 amino acids
corresponding to position E67
as indicated in seq id no 1, 44 amino acids corresponding to position E64 as
indicated in seq id no 1, 47
amino acids corresponding to position T61 as indicated in seq id no 1, SO
amino acids corresponding to
position R58 as indicated in seq id no 1, 56 amino acids corresponding to
position A52 as indicated in
seq id no 1, 66 amino acids corresponding to position G42 as indicated in seq
id no 1. Any one of the
above collagen domains comprises a lysine which is hydroxylated and
glycosylated. Typically, as is the
case with the collagen domain of human adiponectin, the lysine to be
hydroxylated and glycosylated
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
18
should be N-terminally adjacent to a glycine, c~ also The Journal of
Biological Chemistry,
"Conformational Requirement for Lysine Hydroxylation in Collagen", Vol. 266,
No. 34, Issue of
December 5, pp. 22960-22967, 1991.
Depending on the length of the collagen domain it may comprise one to four
lysine(s), such as
1,2,3,or4.
Typically, the collagen domain of the adiponectin polypeptide fragment
comprises one to four lysine
residues selected from any one of the positions K65, K68, K77, or K101 as
indicated in seq id no 1. As
mentioned above (in connection with adiponectin polypeptide having seq id no
5, having an N-terminal
lysine at K101) a lysine in the collagen domain, which is the N-terminal
residue, will not be
hydroxylated and glycosylated upon expression of such adiponectin polypeptide
fragment in a
eucaryotic cell. If for instance an adiponectin polypeptide fragment having
four lysine residues in the
positions K65, K68, K77, and K101, wherein K65 (as indicated in seq id no 1)
is the N-terminal amino
acid, is desired, then expression of such fragment will lead to a fragment
having a collagen domain
wherein the three positions K68, K77, and K101, are hydroxylated and
glycosylated, and wherein K65 is
not. Another example is an adiponectin polypeptide fragment having three
lysine residues in the
positions K68, K77, and K101, wherein K68 (as indicated in seq id no 1) is the
N-terminal amino acid,
then expression of such fragment will lead to a fragment having a collagen
domain wherein the two
positions K77, and K101, are hydroxylated and glycosylated, and wherein K68 is
not. However, if the
desired adiponectin polypeptide fragment has the N-terminal amino acid, K68,
then the lysine may be
2o hydroxylated and glycosylated upon expression of a longer fragment in a
eucaryotic cell, and
subsequently cutting with a suitable protease, that specifically cleave
proteins following a glutamic acid
residue, such as the protease purified from Staphylococcus aureus V8, which is
comercially available.
Another example is an adiponectin polypeptide fragment having two lysine
residues in the positions
K77, and K101, wherein K77 (as indicated in seq id no 1) is the N-terminal
amino acid, then expression
of such fragment will lead to a fragment having a collagen domain wherein the
position K101, is
hydroxylated and glycosylated, and wherein K77 is not. However, if the desired
adiponectin polypeptide
fragment has the N-terminal amino acid, K77, then the lysine may be
hydroxylated and glycosylated
upon expression of a longer fragment in a eucaryotic cell, and subsequently
cutting with a suitable
Prolyl endoprotease, (in somecases also called prolyl oligopeptidases, which
are widely present in
3o microorganisms, plants and animals) which act as a post-proline cleaving
enzyme, such as the enzyme
from the microorganism Flavobacterium meningosepticum (which is commercially
available). In the
situation wherein the N-terminal amino acid is not a lysine, then an
adiponectin polypeptide fragment
comprising 1, 2, 3, or 4 lysine(s) will contain 1, 2, 3, or 4 lysine residues
that are hydroxylated and
glycosylated, respectively, upon expression in a eucaryotic cell. Thus, in a
particular embodiment the
adiponectin polypeptide fragment comprises one lysine residue which is
hydroxylated and glycosylated,
such as the position K101 as indicated in seq id no 1. In another particular
embodiment the adiponectin
polypeptide fragment comprises two lysine residues which are hydroxylated and
glycosylated, such as
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
19
the positions K77, and K101 as indicated in seq id no 1. In a further
particular embodiment the
adiponectin polypeptide fragment comprises three lysine residues which are
hydroxylated and
glycosylated, such as the positions K68, K77, and K101 as indicated in seq id
no 1. In a further
particular embodiment the adiponectin polypeptide fragment comprises four
lysine residues which are
hydroxylated and glycosylated, such as the positions K65, K68, K77, and K101
as indicated in seq id no
1.
Any one of the above adiponectin polypeptide fragments) of the invention may
be prepared
according to methods known in the art. Such method include recombinant DNA
techniques, preferably
the methods mentioned in the section "Methods of preparing an adiponectin
polypeptide for use in the
1 o invention" are used, and a particular suitable method of preparation, is
the method of preparing an
adiponectin polypeptide (including a fragment thereof), comprising
a) preparing a nucleotide sequence encoding: a signal peptide and the
adiponectin polypeptide, wherein
the last three C-terminal amino acids of the signal peptide are I~G,
b) inserting the nucleotide sequence into a vector,
15 c) transfecting the vector into a mammalian cell,
d) expressing and optionally secreting the adiponectin polypeptide, and
e) obtaining the adiponectin polypeptide.
Any one of the above adiponectin polypeptide fragments) comprising any one of
seq id no 3, 4, 5, 10,
11, 12, or 13, as well as homologues, analogues, and fragments thereof,
including any one of the
2o specified embodiments may be tested for biological activity in a suitable
animal model or in vitro assay
as mentioned above. Thus, in one embodiment the adiponectin polypeptide
fragment normalises blood
glucose concentration in a db/db mouse. In another embodiment the adiponectin
polypeptide fragment
enhances glucose uptake in muscle cells. A suitable in vitro assay for testing
glucose uptake is Test
Assay A. In a further embodiment the adiponectin polypeptide fragment inhibit
LPS-induced TNF-alpha
25 production in a monocytic cell line or in a macrophage. A suitable in vitro
assay for testing inhibition of
LPS-induced TNF-alpha production is Test Assay B. In a further embodiment the
adiponectin
polypeptide fragment enhances the insulin mediated suppression of glucose out-
put in primary
hepatocytes. A suitable in vitro assay for testing reduced glucose production
is Test Assay C.
Adiponectin polypeptide fragments which enhance glucose uptake in muscle cells
and inhibit LPS-
3o induced TNF-alpha production in a monocytic cell line or in a macrophage
are preferred. Other
preferred adiponectin polypeptide fragments are those which enhance glucose
uptake in muscle cells and
reduce glucose production in primary hepatocytes. It should be clear that in
all the test models/assays the
adiponectin polypeptide is tested and compared to a control group which did
not receive the adiponectin
polypeptide.
First group of conL ag te(s) of the invention
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
As stated above, in a further aspect the invention relates to a conjugate
comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, wherein the adiponectin polypeptide comprises an amino acid
residue having an attachment
group for said first non-polypeptide moiety, wherein said amino acid residue
is a surface exposed amino
acid residue.
In a second aspect the invention relates to a conjugate consisting essentially
of an adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein said amino acid residue is a
surface exposed amino acid
10 residue.
In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide
selected from seq id no 5 or 6, and one first non-polypeptide moiety
covalently attached to the
adiponectin polypeptide, wherein the adiponectin polypeptide comprises an
amino acid residue having
an attachment group for said first non-polypeptide moiety, wherein said amino
acid residue is a surface
15 exposed amino acid residue.
The amino acid residue having the attachment group for the first non-
polypeptide moiety is
located at the surface of the adiponectin polypeptide, and typically has more
than 25% of its side chain
exposed to the solvent, such as more than SO% of its side chain exposed to the
solvent. We believe that
such positions in the globular domain may be identified on the basis of an
analysis of the 3D structure of
2o the crystal structure of the globular domain of mouse ACRP30, cf Brief
Communication, "The crystal
structure of a complement-1 q family protein suggets an evolutionary link to
tumor necrosis factor",
Shapiro et al, pp 335-338. Typically, in the globular and collagen domains all
lysine residues are surface
exposed. The surface exposed amino acid residues have been identified as
outlined in the experimental
section herein.
By using a surface exposed amino acid residue which is already present in the
wildtype
molecule having an attachment group for a non-polypeptide moiety it will not
be necessary to make
mutations, however, this does not exclude that mutations can be made, provided
that the conjugate
maintain biological activity, and thereby its usefulness for treating eg.
impaired glucose tolerance, type 2
diabetes, syndrome X, obesity, a cardiovascular disease, such as
atherosclerosis, or dyslipidemia, such
activity could be tested in a relevant animal model, such as mouse models of
insulin resistance and
diabetes, such as db/db or ob/ob mice, or rat models such as tucker rats, or
could be tested in a relevant
in vitro assay, such as any one of the Test Assays A, B, or C described in the
experimental section.
In one embodiment the surface exposed amino acid residue is an amino acid
residue having at
least 25%, such as at least 50% of its side chain exposed to the surface. In a
particular embodiment the
surface exposed amino acid residue is an amino acid residue having 100% of its
side chain exposed to
the surface.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
21
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Y111, 8112, L119, E120, T121, Y122, V123, T124, I125, P126, N127, M128,
I130, 8131, T133,
K134, I135, F136, Y137, N138, Q139, Q140, N141, H142, D144, 6145, 5146, T147,
K149, H151,
N153, I154, P155, Y159, A161, H163, I164, T165, Y167, M168, K169, D170, V171,
K172, F176,
K177, K178, D179, K180, A181, M182, F184, T185, Y186, D187, Q188, Y189, Q190,
E191, N192,
N193, V194, D195, Q196, 5198, 6199, 5200, H204, E206, V207, 6208, D209, Q210,
W212, Q214,
V215, Y216, 6217, E218, 6219, E220, 8221, N222, 6223, L224, Y225, A226, D227,
N228, D229,
N230, D231, T233, F234, F237, L238, L239, Y240, H241, D242, T243, or N244 of
human adiponectin.
Each of these positions is considered an embodiment and may be made the
subject of a claim, moreover,
any one of these positions may be combined with any one of the embodiments
hereinafter.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Y111, 8112, E120, T121, Y122, V123, T124, I125, P126, N127, M128, 8131,
T133, K134, I135,
Q139, N141, D144, 6145, S146, T147, K149, H151, N153, P155, Y167, M168, K169,
D170, K178,
D179, K180, A181, F184, Y186, D187, Q188, Y189, Q190, E191, N192, N193, V194,
D195, H204,
E206, V207, 6208, Q210, V215, Y216, 6217, E218, 6219, E220, 8221, N222, 6223,
L224, Y225,
A226, D227, N228, D229, N230, H241, D242, T243, or N244 of human adiponectin.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Y111, E120, T121, Y122, V123, T124, I125, P126, N127, M128, 8131, Q139,
N141, D144,
6145, S146, N153, Y167, M168, K169, K178, D179, K180, A181, Y186, D187, Q188,
Y189, Q190,
2o E191, N192, N193, V194, D195, E206, V207, 6208, V215, Y216, 6217, E218,
6219, E220, 8221,
N222, 6223, L224, Y225, A226, D227, N228, D229, N230, H241, T243, or N244 of
human
adiponectin.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
E120, T121, Y122, V123, T124, I125, P126, N127, Y167, M168, K169, A181, Y186,
D187, Q188,
Y189, Q190, E191, N192, N193, V194, D195, V215, Y216, 6217, E218, 6219, E220,
8221, N222,
6223, L224, Y225, A226, D227, N228, D229, N230, T243, or N244 of human
adiponectin.
The identification of surface exposed amino acids in the globular domain of
human adiponectin
has made it possible to select the desired target for attaching a non-
polypeptide moiety. Such a non-
polypeptide moiety is typically selected from a polymer molecule, a lipophilic
compound, or an organic
3o derivatizing agent. Suitable methods for attaching a non-polypeptide moiety
to any one of the surface
exposed amino acids in the globular domain of human adiponectin are well known
to the skilled person.
The preferred methods of attaching a non-polypeptide moiety selected from a
polymer molecule, a
lipophilic compound, or an organic derivatizing agent are described in more
detail in the section
"Methods of preparing a conjugate of the invention" hereinafter.
The adiponectin polypeptide should have a globular domain, such as indicated
in the sequence
of human adiponectin (108-244) (shown in seq id no 6). The adiponectin
polypeptide part of the
conjugate comprises the globular domain having the amino acid sequence shown
in seq id no 6 as well
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
22
as analogues thereof, including fragments. As mentioned also analogues are
comprised, in particular
analogues that differs in l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues relative to
the amino acid sequence shown in seq id no 6.
Thus, in a further embodiment the adiponectin polypeptide part of the
conjugate comprises a
globular domain, preferably a collagen and a globular domain. In a still
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 10. In
a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 11. In
a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 12. In
a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 13. In
a further embodiment the
to adiponectin polypeptide comprises the amino acid sequence of seq id no 6.
In a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 5. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 4. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 3. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 2. In a
further embodiment the
15 adiponectin polypeptide is consisting essentially of a globular domain. In
a further embodiment the
adiponectin polypeptide is consisting essentially of a collagen and a globular
domain. In a further
embodiment the adiponectin polypeptide is consisting essentially of the amino
acid sequence of seq id
no 10. In a further embodiment the adiponectin polypeptide is consisting
essentially of the amino acid
sequence of seq id no 11. In a further embodiment the adiponectin polypeptide
is consisting essentially
20 of the amino acid sequence of seq id no 12. In a further embodiment the
adiponectin polypeptide is
consisting essentially of the amino acid sequence of seq id no 13. In a
further embodiment the
adiponectin polypeptide is consisting essentially of the amino acid sequence
of seq id no 6. In a further
embodiment the adiponectin polypeptide is consisting essentially of the amino
acid sequence of seq id
no S. In a further embodiment the adiponectin polypeptide is consisting
essentially of the amino acid
25 sequence of seq id no 4. In a further embodiment the adiponectin
polypeptide is consisting essentially of
the amino acid sequence of seq id no 3. In a further embodiment the
adiponectin polypeptide is
consisting essentially of the amino acid sequence of seq id no 2.
Typically, the adiponectin polypeptide is selected from any one of seq id no
2, 3, 4, 5, 10, 1 l,
12, or 13, as well as sequences that differs from any one of the specified
sequences, in one or more .
3o substitution(s), preferably from one to eleven, more preferably from one to
eight. In one embodiment the
adiponectin polypeptide is selected from any one of seq id no 3, 10, 12, or
13, as well as sequences that
differs from any one of the specified sequences in one to eleven
substitutions. In another embodiment
the adiponectin polypeptide is selected from any one of seq id no 3, 10, 12,
or 13, as well as sequences
that differs from any one of the specified sequences in one to eight
substitutions, such as 1-6
35 substitutions.
In a particular embodiment the adiponectin polypeptide is selected from any
one of seq id no 2,
3, 4, 5, 10, 1 l, 12, or 13, as well as sequences that differs from any one of
the specified sequences, in
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
23
one or more substitutions, and comprises one to four lysine residues selected
from any one of the
positions K65, K68, K77, or K101. In a further embodiment the adiponectin
polypeptide is selected
from any one of seq id no 2, 3, 4, 5, 10, 11, 12, or 13, preferably 3, 10, 12,
or 13. In a alternative
embodiment the adiponectin polypeptide is selected from sequences that differs
from any one of the seq
id no 2, 3, 4, 5, 10, 11, 12, or 13, preferably 3, 10, 12, or 13, in one or
more substitutions, preferably
from one to eleven, more preferably from one to eight, such as 1-6. In a
further embodiment the
adiponectin polypeptide comprises at least one lysine residue selected from
any one of the positions
K65, K68, K77, or K101. As mentioned above when produced in a eucaryotic cell,
such as a mammalian
cell, lysine residues in the collagen domain are hydroxylated and
glycosylated. Typically, the lysine
1o residues are hydroxylated and glycosylated. In a further embodiment the
adiponectin polypeptide
comprises one lysine residue selected from any one of the positions K65, K68,
K77, or K101, preferably
K101, and preferably the position is hydroxylated and glycosylated, such as
glyco-hydroxy-K101. In a
further embodiment the adiponectin polypeptide comprises two lysine residues
selected from any one of
the positions K65, K68, K77, or K101, preferably K77 and K101, and preferably
both of the positions
t5 are hydroxylated and glycosylated, such as glyco-hydroxy-K77 and glyco-
hydroxy-K101. In a further
embodiment the adiponectin polypeptide comprises three lysine residues
selected from any one of the
positions K65, K68, K77, or K101, preferably K68, K77 and K101, and preferably
all three of the
positions are hydroxylated and glycosylated, such as glyco-hydroxy-K68, glyco-
hydroxy-K77 and
glyco-hydroxy-K101. In a further embodiment the adiponectin polypeptide
comprises four lysine
2o residues selected from positions K65, K68, K77, and K101, and preferably
all four of the positions are
hydroxylated and glycosylated.
In a still further embodiment the adiponectin polypeptide is selected from any
one of the
adiponectin polypeptide fragments described in the above section "Adiponectin
polypeptide fragments)
of the invention". Each of the described adiponectin polypeptide fragments is
considered an embodiment
25 suitable as the adiponectin polypeptide part of the conjugate.
Accordingly, one example of a preferred aspect of the conjugate is a conjugate
comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1 as well as sequences that differs from the amino acid
sequence in one or more
3o substitution(s), and wherein the collagen domain comprises from 7 amino
acids corresponding to
position K101 as indicated in seq id no 1 to 56 amino acids corresponding to
position A52 as indicated
in seq id no 1, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated, and
a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide fragment,
35 wherein the adiponectin polypeptide fragment comprises an amino acid
residue having an
attachment group for said first non-polypeptide moiety, wherein said amino
acid residue is a surface
exposed amino acid residue. As mentioned above the surface exposed amino acid
residue is selected
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
24
from any one of E82, T83, G84, V85, P86, A88, E89, P91, R92, F94, P95, I97,
Q98, 8100, K101, E103,
P104, 6105, E106, 6107, A108, Y109, V110, Y111, 8112, E120, T121, Y122, V123,
T124, I125,
P126, N127, M128, 8131, T133, K134, I135, Q139, N141, D144, 6145, 5146, T147,
K149, H151,
N153, P155, Y167, M168, K169, D170, K178, D179, K180, A181, F184, Y186, D187,
Q188, Y189,
Q190, E191, N192, N193, V194, D195, H204, E206, V207, 6208, Q210, V215, Y216,
6217, E218,
6219, E220, 8221, N222, 6223, L224, Y225, A226, D227, N228, D229, N230, H241,
D242, T243, or
N244 relative to human adiponectin, preferably any one of A108, Y109, V110, Yl
11, 8112, E120,
T121, Y122, V123, T124, I125, P126, N127, M128, 8131, T133, K134, I135, Q139,
N141, D144,
6145, 5146, T147, K149, H151, N153, P155, Y167, M168, K169, D170, K178, D179,
K180, A181,
1o F184, Y186, Q188, Y189, Q190, E191, N192, N193, V194, H204, E206, V207,
6208, Q210, V215,
Y216, 6217, E218, 6219, E220, 8221, N222, L224, Y225, D227, N228, D229, N230,
H241, D242,
T243, or N244. However, particular a preferred amino acid residue having an
attachment group for said
first non-polypeptide moiety is selected from a lysine, aspartic acid, or
glutamic acid. In this respect the
surface exposed amino acid residue may be selected from any one of E120, K134,
D144, K149, K169,
D170, K172, K177, K178, D179, K180, E191, E206, D209, E218, E220, D227, D229,
D231, or D242,
such as from any one of E120, E191, E206, E218, or E220, or from any one of
K134, K149, K169,
K172, K177, K178, or K180, or from any one of D144, D170, D179, D209, D227,
D229, D231, or
D242.
It should be clear that the surface exposed amino acid residue having an
attachment group for
the first non-polypeptide moiety may either be located in the globular domain
or in the collagen domain,
or in case of more than one non-polypeptide moiety being attached they may be
located in the globular
domain or in the collagen domain, or in both the globular domain and the
collagen domain.
Accordingly in a further embodiment the attachment group is located in the
globular domain. In
a further embodiment the adiponectin polypeptide further comprises a collagen
domain. In one
embodiment the attachment group is located in the collagen domain. If only one
non-polypeptide is
attached then it may be in the globular domain or in the collagen domain. If
more than one, such as two
non-polypeptides, are attached then one may be located in the collagen domain
and one in the globular
domain, or both may be in the collagen domain, or both may be in the globular
domain.
In a further embodiment the adiponectin polypeptide comprises a non-homologous
domain.
In a further embodiment the adiponectin polypeptide comprises a signal
peptide.
In a further embodiment the adiponectin polypeptide is isolated.
In a further embodiment only one first non-polypeptide moiety is attached to
the adiponectin
polypeptide.
In a further embodiment the conjugate of the invention is mono pegylated.
In a further embodiment the first non-polypeptide moiety is selected from a
polymer molecule, a
lipophilic compound, and an organic derivatizing agent.
In a further embodiment the first non-polypeptide moiety is selected from a
polymer molecule.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
In a further embodiment the amino acid residue having the attachment group for
said first non-
polypeptide moiety is selected from a lysine, aspartic acid, or glutamic acid.
In this respect the surface
exposed amino acid residue may be selected from any one of K65, K68, K77,
K101, E120, K134, D144,
K149, K169, D170, K172, K177, K178, D179, K180, D187, E191, D195, E206, D209,
E218, E220,
5 D227, D229, D231, or D242. Typically, the surface exposed amino acid residue
may be selected from
any one of E120, K134, D144, K149, K169, D170, K172, K177, K178, D179, K180,
E191, E206, D209,
E218, E220, D227, D229, D231, or D242, preferably from any one of E120, K134,
D144, K149, K169,
D170, K178, D179, K180, E191, E206, E218, E220, D227, D229, or D242, more
preferably from any
one of E120, D144, K169, K178, D179, K180, E191, E206, E218, E220, D227, or
D229, in particular
to from any one of E120, K169, E191, E218, E220, D227, or D229.
In a further embodiment the first non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol. Such polymers are available from Shearwater,
SunBio, Pierce, or Enzon.
In a further embodiment the polymer has a molecular weight of from lkDa to
200kDa (kDa is a
well known abbreviation and means kilo Dalton). In a still further embodiment
the polymer has a
t 5 molecular weight of from 2kDa to 95kDa. In a still further embodiment the
polymer has a molecular
weight of from SkDa to 80kDa. In a still further embodiment the polymer has a
molecular weight of
from l2kDa to 60kDa, such as 5-20 kDa, 12-40 kDa, 20-40 kDa, 5 kDa, 10 kDa, 12
kDa, or 20 kDa.
In a further embodiment the amino acid residue having the attachment group is
a lysine residue.
Such lysine residue may be present in the non-homologous, collagen or globular
domain, depending on
2o the length of the adiponectin polypeptide. Typically, a part of the
collagen domain linked to the globular
domain will contain one to four lysine residues, that is positions K65, K68,
K77, or K101. For instance,
the sequence of seq id no 3 has four lysines in the collagen domain, the
sequence of seq id no 4 has one
lysine in the collagen domain, the sequence of seq id no 5 has one lysine in
the collagen domain, the
sequence of seq id no 10 has one lysine in the collagen domain, the sequence
of seq id no 11 has one
25 lysine in the collagen domain, the sequence of seq id no 12 has four
lysines in the collagen domain, and
the sequence of seq id no 13 has four lysines in the collagen domain.
When a lysine intended as the amino acid residue having the attachment group
is located in the
collagen domain of the adiponectin polypeptide then the lysine may be
hydroxylated and glycosylated if
produced in eg. a mammalian cell or may be free of any such glyco-hydroxy
groups. If the lysine is
3o hydroxylated and glycosylated then it is not preferred as an attachment
group, although such glyco-
hydroxy group could be attached to a polymer such as a PEG, eg. by using a
mPEG-AMINE, c~ also the
section "Conjugate of the invention comprising a second non-polypeptide
moiety". Thus, if it is intended
that a lysine located in the collagen domain of the adiponectin polypeptide
should be conjugated to a
non-polypeptide, then such adiponectin polypeptide should be expressed in a
bacterial cell, such as E.
Coli.
In a further embodiment the lysine is selected from any one of the positions
K65, K68, K77, or
K101 of the collagen domain of human adiponectin.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
26
In a further embodiment the lysine is selected from any one of the positions
K134, K149, K169,
K172, K177, K178, or K180 of the globular domain of human adiponectin,
preferably any one of the
positions K134, K149, K169, K178, or K180.
In a further embodiment the lysine is selected from any one of the positions
K65, K68, K77,
K101, K134, K149, K169, K172, K177, K178, or K180 of human adiponectin,
preferably any one of the
positions K134, K149, K169, K178, or K180.
Typically, the lysine is selected from any one of the positions K68, K77,
K101, K134, K149,
Kl 69, K172, K177, K178, or K180 of human adiponectin, however, depending on
the length of the
adiponectin polypeptide, the skilled person will recognize that the lysine
residues may also be selected
to from any one of the positions K77, K101, K134, K149, K169, K172, K177,
K178, or K180 of human
adiponectin, in particular from any one of the positions K101, K134, K149,
K169, K172, K177, K178,
or K180 of human adiponectin, preferably any one of the positions K134, K149,
K169, K178, or Kl 80.
In a further embodiment the polymer molecule is selected from the group
consisting of SS-PEG,
NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SBA, PEG-SCM, mPEG-BTC (All available
from
Shearwater), and SC-PEG (available from Enzon).
In a further embodiment the polymer molecule is selected from the group
consisting of Sk-PEG-
SCM, 12k-PEG-SCM, 20k-PEG-SCM, Sk-PEG-SPA, 12k-PEG-SPA, 20k-PEG-SPA. (All
available
from Shearwater).
In a further embodiment the conjugate further comprises a second non-
polypeptide moiety
2o selected from the group consisting of a polymer molecule, a lipophilic
compound, a sugar moiety and an
organic derivatizing agent. The second non-polypeptide moiety is different
from the first non-
polypeptide.
In a further embodiment the second non-polypeptide moiety is selected from a
polymer
molecule.
In a further embodiment the amino acid residue having the attachment group for
said second
non-polypeptide moiety is selected from a lysine, aspartic acid, glutamic acid
or cysteine residue. In this
respect the surface exposed amino acid residue may be selected from any one of
K65, K68, K77, K101,
E120, K134, D144, K149, K169, D170, K172, K177, K178, D179, K180, D187, E191,
D195, E206,
D209, E218, E220, D227, D229, D231, or D242. Typically, the surface exposed
amino acid residue may
3o be selected from any one of E120, K134, D144, K149, K169, D170, K172, K177,
K178, D179, K180,
E191, E206, D209, E218, E220, D227, D229, D231, or D242, preferably from any
one of E120, K134,
D144, K149, K169, D170, K178, D179, K180, E191, E206, E218, E220, D227, D229,
or D242, more
preferably from any one of E120, D144, K169, K178, D179, K180, E191, E206,
E218, E220, D227, or
D229, in particular from any one of E120, K169, E191, E218, E220, D227, or
D229.
In a further embodiment the second non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
27
In a further embodiment the amino acid sequence of the adiponectin polypeptide
further
comprises at least one removed lysine residue.
In a further embodiment one to four lysine residues selected from any one of
the positions K65,
K68, K77, or K101 of the collagen domain of human adiponectin is/are removed.
In a further embodiment one to six lysine residues selected from any one of
the positions K134,
K149, K169, K172, K177, K178, or K180 of the globular domain of wild-type
human adiponectin is/are
removed.
Such lysine residues may be removed from the collagen and/or globular domain,
depending on
the length of the adiponectin polypeptide. The skilled person will understand
that the group of lysines to
1o select from will depend on whether the full collagen domain or only a
fragment thereof is present in the
adiponectin polypeptide, and thus whether the group of lysine residues are the
positions K65, K68, K77,
or K101 of the collagen domain of human adiponectin and positions K134, K149,
K169, K172, K177,
K178, or K180 of the globular domain of human adiponectin, or a smaller group,
such as K77, or K101
of the collagen domain and positions K134, K149, K169, K172, K177, K178, or Kl
80 of the globular
15 domain, or even a smaller group, such as K101 of the collagen domain and
positions K134, K149, K169,
K172, K177, K178, or K180 of the globular domain. Obviously, at least one
lysine should be present in
the adiponectin polypeptide in order to make possible the conjugation to a
lysine.
Second group of con'u ate s) of the invention
20 In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide,
and a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, wherein the
adiponectin polypeptide comprises an amino acid residue having an attachment
group for said first non-
polypeptide moiety, wherein said amino acid residue is a cysteine residue.
In a further aspect the invention relates to a conjugate consisting
essentially of an adiponectin
25 polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein said amino acid residue is a
cysteine residue.
In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide
selected from seq id no 5 or 6, and one first non-polypeptide moiety
covalently attached to the
3o adiponectin polypeptide, wherein the adiponectin polypeptide comprises an
amino acid residue having
an attachment group for said first non-polypeptide moiety, wherein said amino
acid residue is a cysteine
residue.
By using a cysteine residue which is already present in the wildtype molecule
having a
sulfliydryl attachment group for a non-polypeptide moiety it will not be
necessary to make mutations,
35 however, this does not exclude that mutations can be made, provided that
the conjugate maintain
biological activity, and thereby its usefulness for treating eg. impaired
glucose tolerance, type 2 diabetes,
syndrome X, obesity, a cardiovascular disease, such as atherosclerosis, or
dyslipidemia, such activity
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
28
could be tested in a relevant animal model, such as mouse models of insulin
resistance and diabetes,
such as db/db or ob/ob mice, or rat models such as zucker rats, or could be
tested in a relevant in vitro
assay, such as any one of the Test Assays A, B, or C described in the
experimental section. The wildtype
adiponectin polypeptide has two cysteine residues, that is, position C36 and
C152 relative to seq id no 1.
The use of C152 relative to seq id no 1 in the globular domain of human
adiponectin for
conjugation to a non-polypeptide moiety is not an obvious choice, since this
cysteine does not have its
sulfllydryl group (-SH) exposed to the surface of human adiponectin, c~ the
experimental section under
"Surface exposure". Such a non-polypeptide moiety is typically selected from a
polymer molecule, a
lipophilic compound, or an organic derivatizing agent. Suitable methods for
attaching a non-polypeptide
1 o moiety to a cysteine residue in the globular domain of human adiponectin
are well known to the skilled
person. The preferred methods of attaching a non-polypeptide moiety selected
from a polymer molecule,
a lipophilic compound, or an organic derivatizing agent are described in more
detail in the section
"Methods of preparing a conjugate of the invention" hereinafter.
T'he adiponectin polypeptide should have a globular domain, such as indicated
in the sequence
15 of human adiponectin (108-244) (shown in seq id no 6). The adiponectin
polypeptide part of the
conjugate comprises the globular domain having the amino acid sequence shown
in seq id no 6 as well
as analogues thereof, including fragments. As mentioned also analogues are
comprised, in particular
analogues that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues relative to
the amino acid sequence shown in seq id no 6.
20 Thus, in a further embodiment the adiponectin polypeptide part of the
conjugate comprises a
globular domain, preferably a collagen and a globular domain. In a still
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 10. In
a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 11. In
a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 12. In
a further embodiment the
25 adiponectin polypeptide comprises the amino acid sequence of seq id no 13.
In a further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 6. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 5. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 4. In a
further embodiment the
adiponectin polypeptide comprises the amino acid sequence of seq id no 3. In a
further embodiment the
3o adiponectin polypeptide comprises the amino acid sequence of seq id no 2.
In a further embodiment the
adiponectin polypeptide is consisting essentially of a globular domain. In a
further embodiment the
adiponectin polypeptide is consisting essentially of a collagen and a globular
domain. In a further
embodiment the adiponectin polypeptide is consisting essentially of the amino
acid sequence of seq id
no 10. In a further embodiment the adiponectin polypeptide is consisting
essentially of the amino acid
35 sequence of seq id no 11. In a further embodiment the adiponectin
polypeptide is consisting essentially
of the amino acid sequence of seq id no 12. In a further embodiment the
adiponectin polypeptide is
consisting essentially of the amino acid sequence of seq id no 13. In a
further embodiment the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
29
adiponectin polypeptide is consisting essentially of the amino acid sequence
of seq id no 6. In a further
embodiment the adiponectin polypeptide is consisting essentially of the amino
acid sequence of seq id
no 5. In a further embodiment the adiponectin polypeptide is consisting
essentially of the amino acid
sequence of seq id no 4. In a further embodiment the adiponectin polypeptide
is consisting essentially of
the amino acid sequence of seq id no 3. In a further embodiment the
adiponectin polypeptide is
consisting essentially of the amino acid sequence of seq id no 2.
Typically, the adiponectin polypeptide is selected from any one of seq id no
3, 4, 5, 10, 11, 12,
or 13, as well as sequences that differs from any one of the specified
sequences, in one or more
substitution(s), preferably from one to eleven, such as from one to eight. In
one embodiment the
t o adiponectin polypeptide is selected from any one of seq id no 3, 10, 12,
or 13, as well as sequences that
differs from any one of the specified sequences in one to eleven
substitutions, such as one to eight
substitutions, eg. 1-6 substitutions.
In a particular embodiment the adiponectin polypeptide is selected from any
one of seq id no 3,
4, 5, 10, 11, 12, or 13, as well as sequences that differs from any one of the
specified sequences, in one
15 or more substitutions, and comprises one to four lysine residues selected
from any one of the positions
K65, K68, K77, or K101. In a further embodiment the adiponectin polypeptide is
selected from any one
of seq id no 3, 4, 5, 10, 11, 12, or 13, preferably 3, 10, 12, or 13. In a
alternative embodiment the
adiponectin polypeptide is selected from sequences that differs from any one
of the seq id no 3, 4, 5, 10,
11, 12, or 13, preferably 3, 10, 12, or 13, in one or more substitutions,
preferably from one to eleven
2o substitutions, such as one to eight substitutions, eg. 1-6 substitutions .
In a further embodiment the
adiponectin polypeptide comprises at least one lysine residue selected from
any one of the positions
K65, K68, K77, or K101. As mentioned above when produced in a eucaryotic cell,
such as a mammalian
cell, lysine residues in the collagen domain are hydroxylated and
glycosylated. Typically, the lysine
residues are hydroxylated and glycosylated. In a further embodiment the
adiponectin polypeptide
25 comprises one lysine residue selected from any one of the positions K65,
K68, K77, or K101, preferably
K101, and preferably the position is hydroxylated and glycosylated, such as
glyco-hydroxy-K101. In a
further embodiment the adiponectin polypeptide comprises two lysine residues
selected from any one of
the positions K65, K68, K77, or K101, preferably K77 and K101, and preferably
both of the positions
are hydroxylated and glycosylated, such as glyco-hydroxy-K77 and glyco-hydroxy-
K101. In a further
3o embodiment the adiponectin polypeptide comprises three lysine residues
selected from any one of the
positions K65, K68, K77, or K101, preferably K68, K77 and K101, and preferably
all three of the
positions are hydroxylated and glycosylated, such as glyco-hydroxy-K68, glyco-
hydroxy-K77 and
glyco-hydroxy-K101. In a further embodiment the adiponectin polypeptide
comprises four lysine
residues selected from positions K65, K68, K77, and K101, and preferably all
four of the positions are
3s hydroxylated and glycosylated.
In a still further embodiment the adiponectin polypeptide is selected from any
one of the
adiponectin polypeptide fragments described in the above section "Adiponectin
polypeptide fragments)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
of the invention". Each of the described adiponectin polypeptide fragments is
considered an embodiment
suitable as the adiponectin polypeptide part of the conjugate.
Accordingly, one example of a preferred aspect of the conjugate is a conjugate
comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1 as well as sequences that differs from the amino acid
sequence in one or more
substitution(s), and wherein the collagen domain comprises from 7 amino acids
corresponding to
position K101 as indicated in seq id no 1 to 56 amino acids corresponding to
position A52 as indicated
in seq id no 1, and wherein the collagen domain comprises a lysine which is
hydroxylated and
to glycosylated, and
a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide fragment,
wherein the adiponectin polypeptide fragment comprises an amino acid residue
having an
attachment group for said first non-polypeptide moiety, wherein said amino
acid residue is a cysteine
residue.
15 In a further embodiment the cysteine is Cys 152 in the globular domain of
human adiponectin.
In a further embodiment the adiponectin polypeptide comprises a collagen
domain.
In a further embodiment the adiponectin polypeptide comprises a non-homologous
domain. In a
further embodiment the cysteine is Cys36 in the non-homologous domain of human
adiponectin.
In a further embodiment the adiponectin polypeptide comprises a signal
peptide.
2o In a further embodiment the adiponectin polypeptide is isolated.
In a further embodiment only one first non-polypeptide moiety is attached to
the adiponectin
polypeptide.
In a further embodiment the conjugate of the invention is mono pegylated.
In a further embodiment the first non-polypeptide moiety is. selected from a
polymer molecule, a
25 lipophilic compound, and an organic derivatizing agent.
In a further embodiment the first non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
In a further embodiment the polymer has a molecular weight of from lkDa to
200kDa (kDa is a
well known abbreviation and means kilo Dalton). In a still further embodiment
the polymer has a
3o molecular weight of from 2kDa to 95kDa. In a still further embodiment the
polymer has a molecular
weight of from SkDa to 80kDa. In a still further embodiment the polymer has a
molecular weight of
from l2kDa to 60kDa, such as 12-40 kDa, 20-40 kDa, 5 kDa, 12 kDa, or 20 kDa.
In a further embodiment the polymer molecule is selected from the group
consisting of
mPEG(MAL), mPEG2(MAL), mPEG-OPSS, PEG-vinylsulphone, OPSS-PEG-hydrazide in
combination with mPEG-ALD. In a further embodiment the polymer molecule is
selected from the
group consisting of 5k-mPEG(MAL), 20k-mPEG(MAL), 40k-mPEG2(MAL), 5k-mPEG-OPSS,
lOk-
mPEG-OPSS, 20k-mPEG-OPSS, OPSS-PEGZk-hydrazide in combination with mPEG3ok~-
ALD.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
31
In a further embodiment the conjugate further comprises a second non-
polypeptide moiety
selected from the group consisting of a polymer molecule, a lipophilic
compound, and an organic
derivatizing agent. The second non-polypeptide moiety is different from the
first non-polypeptide.
In a further embodiment the second non-polypeptide moiety is selected from a
polymer
molecule.
In a further embodiment the amino acid residue having the attachment group for
said second
non-polypeptide moiety is selected from a lysine, aspartic acid, glutamic acid
or cysteine residue.
In a further embodiment the second non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
t o In a further embodiment the amino acid sequence of the adiponectin
polypeptide further
comprises at least one removed lysine residue.
In a further embodiment one to four lysine residues selected from any one of
the positions K65,
K68, K77, or K101 of the collagen domain of human adiponectin is/are removed.
In a further embodiment one to six lysine residues selected from any one of
the positions Kl 34,
15 K149, K169, K172, K177, K178, or Kl 80 of the globular domain of wild-type
human adiponectin is/are
removed.
Such lysine residues may be removed from the collagen and/or globular domain,
depending on
the length of the adiponectin polypeptide. The skilled person will understand
that the group of lysines to
select from will depend on whether the full collagen domain or only a fragment
thereof is present in the
2o adiponectin polypeptide, and thus whether the group of lysine residues are
the positions K65, K68, K77,
or K101 of the collagen domain of human adiponectin and positions K134, K149,
K169, K172, K177,
K178, or Kl 80 of the globular domain of human adiponectin, or a smaller
group, such as K77, or K1 O1
of the collagen domain and positions K134, K149, K169, K172, K177, K178, or
K180 of the globular
domain, or even a smaller group, such as K101 of the collagen domain and
positions K134, K149, K169,
25 K172, K177, K178, or K180 of the globular domain. If desired to introduce a
second non-polypeptide
moiety by conjugating it to a lysine, then obviously, at least one lysine
should be present in the
adiponectin polypeptide in order to make possible the conjugation to a lysine.
Third g_rou~ of coniu~ate(s) of the invention
30 In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide,
and a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, wherein the
adiponectin polypeptide comprises an amino acid residue having an attachment
group for said first non-
polypeptide moiety, wherein the amino acid residue is the N-terminal amino
acid residue.
In a further aspect the invention relates to a conjugate consisting
essentially of an adiponectin
35 polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
said first non-polypeptide moiety, wherein the amino acid residue is the N-
terminal amino acid residue.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
32
By using the N-terminal amino acid residue which is already present in the
wildtype molecule
having an attachment group for a non-polypeptide moiety it will not be
necessary to make mutations,
however, this does not exclude that mutations can be made, provided that the
conjugate maintain
biological activity, and thereby its usefulness for treating eg. impaired
glucose tolerance, type 2 diabetes,
syndrome X, obesity, a cardiovascular disease, such as atherosclerosis, or
dyslipidemia, such activity
could be tested in a relevant animal model, such as mouse models of insulin
resistance and diabetes,
such as db/db or ob/ob mice, or rat models such as zucker rats, or could be
tested in a relevant in vitro
assay, such as any one of the Test Assays A, B, or C described in the
experimental section.
Such a non-polypeptide moiety is typically selected from a polymer molecule, a
lipophilic
t o compound, or an organic derivatizing agent. Suitable methods for attaching
a non-polypeptide moiety to
the N-terminal amino acid residue in the adiponectin polypeptide are well
known to the skilled person.
The preferred methods of attaching a non-polypeptide moiety selected from a
polymer molecule, a
lipophilic compound, or an organic derivatizing agent are described in more
detail in the section
"Methods of preparing a conjugate of the invention" hereinafter.
15 The adiponectin polypeptide should have a globular domain, such as
indicated in the sequence
of human adiponectin (108-244) (shown in seq id no 6). The adiponectin
polypeptide part of the
conjugate comprises the globular domain having the amino acid sequence shown
in seq id no 6 as well
as analogues thereof, including fragments. As mentioned also analogues are
comprised, in particular
analogues that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid residues relative to
2o the amino acid sequence shown in seq id no 6.
Thus, in a further embodiment the adiponectin polypeptide comprises a globular
domain,
preferably a collagen and a globular domain. In a still further embodiment the
adiponectin polypeptide
comprises the amino acid sequence of seq id no 10. In a further embodiment the
adiponectin polypeptide
comprises the amino acid sequence of seq id no 11. In a further embodiment the
adiponectin polypeptide
25 comprises the amino acid sequence of seq id no 12. In a further embodiment
the adiponectin polypeptide
comprises the amino acid sequence of seq id no 13. In a further embodiment the
adiponectin polypeptide
comprises the amino acid sequence of seq id no 6. In a further embodiment the
adiponectin polypeptide
comprises the amino acid sequence of seq id no 5. In a further embodiment the
adiponectin polypeptide
comprises the amino acid sequence of seq id no 4. In a further embodiment the
adiponectin polypeptide
3o comprises the amino acid sequence of seq id no 3. In a further embodiment
the adiponectin polypeptide
comprises the amino acid sequence of seq id no 2. In a further embodiment the
adiponectin polypeptide
is consisting essentially of a globular domain. In a further embodiment the
adiponectin polypeptide is
consisting essentially of a collagen and a globular domain. In a further
embodiment the adiponectin
polypeptide is consisting essentially of the amino acid sequence of seq id no
10. In a further embodiment
35 the adiponectin polypeptide is consisting essentially of the amino acid
sequence of seq id no 11. In a
further embodiment the adiponectin polypeptide is consisting essentially of
the amino acid sequence of
seq id no 12. In a further embodiment the adiponectin polypeptide is
consisting essentially of the amino
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
33
acid sequence of seq id no 13. In a further embodiment the adiponectin
polypeptide is consisting
essentially of the amino acid sequence of seq id no 6. In a further embodiment
the adiponectin
polypeptide is consisting essentially of the amino acid sequence of seq id no
5. In a further embodiment
the adiponectin polypeptide is consisting essentially of the amino acid
sequence of seq id no 4. In a
further embodiment the adiponectin polypeptide is consisting essentially of
the amino acid sequence of
seq id no 3. In a further embodiment the adiponectin polypeptide is consisting
essentially of the amino
acid sequence of seq id no 2.
Typically, the adiponectin polypeptide is selected from any one of seq id no
2, 3, 4, 5, 10, 11,
12, or 13, as well as sequences that differs from any one of the specified
sequences, in one or more
t0 substitution(s), preferably from one to eleven, such as one to eight. In
one embodiment the adiponectin
polypeptide is selected from any one of seq id no 3, 10, 12, or 13, as well as
sequences that differs from
any one of the specified sequences in one to eleven, such as one to eight
substitutions, eg. 1-6
substitutions.
In a particular embodiment the adiponectin polypeptide is selected from any
one of seq id no 3,
15 4, 5, 10, 11, 12, or 13, as well as sequences that differs from any one of
the specified sequences, in one
or more substitutions, and comprises one to four lysine residues selected from
any one of the positions
K65, K68, K77, or K101. In a further embodiment the adiponectin polypeptide is
selected from any one
of seq id no 3, 4, 5, 10, 1 l, 12, or 13, preferably 3, 10, 12, or 13. In a
alternative embodiment the
adiponectin polypeptide is selected from sequences that differs from any one
of the seq id no 3, 4, 5, 10,
20 11, 12, or 13, preferably 3, 10, 12, or 13, in one or more substitutions,
preferably from one to eleven,
such as one to eight, eg. 1-6. In a further embodiment the adiponectin
polypeptide comprises at least one
lysine residue selected from any one of the positions K65, K68, K77, or K101.
As mentioned above
when produced in a eucaryotic cell, such as a mammalian cell, lysine residues
in the collagen domain
are hydroxylated and glycosylated. Typically the lysine residues are
hydroxylated and glycosylated. In a
25 further embodiment the adiponectin polypeptide comprises one lysine residue
selected from any one of
the positions K65, K68, K77, or K101, preferably K101, and preferably the
position is hydroxylated and
glycosylated, such as glyco-hydroxy-K101. In a further embodiment the
adiponectin polypeptide
comprises two lysine residues selected from any one of the positions K65, K68,
K77, or K101,
preferably K77 and K101, and preferably both of the positions are hydroxylated
and glycosylated, such
3o as glyco-hydroxy-K77 and glyco-hydroxy-K101. In a further embodiment the
adiponectin polypeptide
comprises three lysine residues selected from any one of the positions K65,
K68, K77, or K101,
preferably K68, K77 and K101, and preferably all three of the positions are
hydroxylated and
glycosylated, such as glyco-hydroxy-K68, glyco-hydroxy-K77 and glyco-hydroxy-
K101. In a further
embodiment the adiponectin polypeptide comprises four lysine residues selected
from positions K65,
35 K68, K77, and K101, and preferably all four of the positions are
hydroxylated and glycosylated.
In a still further embodiment the adiponectin polypeptide is selected from any
one of the
adiponectin polypeptide fragments described in the above section "Adiponectin
polypeptide fragments)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
34
of the invention". Each of the described adiponectin polypeptide fragments is
considered an embodiment
suitable as the adiponectin polypeptide part of the conjugate.
Accordingly, one example of a preferred aspect of the conjugate is a conjugate
comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1 as well as sequences that differs from the amino acid
sequence in one or more
substitution(s), and wherein the collagen domain comprises from 7 amino acids
corresponding to
position K101 as indicated in seq id no 1 to 56 amino acids corresponding to
position A52 as indicated
in seq id no 1, and wherein the collagen domain comprises a lysine which is
hydroxylated and
1o glycosylated, and
a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide fragment,
wherein the adiponectin polypeptide fragment comprises an amino acid residue
having an
attachment group for said first non-polypeptide moiety, wherein the amino acid
residue is the N-terminal
amino acid residue.
15 In a further embodiment the adiponectin polypeptide comprises a collagen
domain.
In a further embodiment the adiponectin polypeptide comprises a non-homologous
domain.
In a further embodiment the adiponectin polypeptide comprises a signal
peptide.
In a further embodiment the adiponectin polypeptide is isolated.
In a further embodiment only one first non-polypeptide moiety is attached to
the adiponectin
2o polypeptide.
In a further embodiment the conjugate of the invention is mono pegylated.
In a further embodiment the first non-polypeptide moiety is selected from a
polymer molecule, a
lipophilic compound, and an organic derivatizing agent.
In a further embodiment the first non-polypeptide moiety is a polymer,
typically a linear or
25 branched polyethylene glycol.
In a further embodiment the polymer has a molecular weight of from lkDa to
200kDa (kDa is a
well known abbreviation and means kilo Dalton). In a still further embodiment
the polymer has a
molecular weight of from 2kDa to 95kDa. In a still further embodiment the
polymer has a molecular
weight of from SkDa to 80kDa. In a still further embodiment the polymer has a
molecular weight of
3o from l2kDa to 60kDa, such as 5-20 kDa, 12-40 kDa, 20-40 kDa, 5 kDa, 12 kDa,
or 20 kDa.
In a further embodiment the polymer molecule is selected from the group
consisting of SS-PEG,
NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SBA, PEG-SCM, mPEG-OPSS, mPEG-BTC (All
available from Shearwater), and SC-PEG.
In a further embodiment the polymer molecule is selected from the group
consisting of Sk-PEG-
35 SCM, Sk-mPEG-OPSS, lOk-mPEG-OPSS, 20k-mPEG-OPSS, 12k-PEG-SCM, 20k-PEG-SCM,
Sk-
mPEG-ALD, 20k-mPEG-ALD, 30k-mPEG-ALD, and 40k-mPEG2-ALD. (All available from
Shearwater)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
In a further embodiment the conjugate further comprises a second non-
polypeptide moiety
selected from the group consisting of a polymer molecule, a lipophilic
compound, and an organic
derivatizing agent. The second non-polypeptide moiety is different from the
first non-polypeptide.
In a further embodiment the second non-polypeptide moiety is selected from a
polymer
molecule.
In a further embodiment the amino acid residue having the attachment group for
said second
non-polypeptide moiety is selected from a lysine, aspartic acid, glutamic acid
or cysteine residue.
In a further embodiment the second non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
1 o In a further embodiment the amino acid sequence of the adiponectin
polypeptide further
comprises at least one removed lysine residue.
In a further embodiment one to four lysine residues selected from any one of
the positions K65,
K68, K77, or K101 of the collagen domain of human adiponectin is/are removed.
In a further embodiment one to six lysine residues selected from any one of
the positions K134,
~s K149, K169, K172, K177, K178, or K180 of the globular domain of wild-type
human adiponectin is/are
removed.
Such lysine residues may be removed from the collagen and/or globular domain,
depending on
the length of the adiponectin polypeptide. The skilled person will understand
that the group of lysines to
select from will depend on whether the full collagen domain or only a fragment
thereof is present in the
2o adiponectin polypeptide, and thus whether the group of lysine residues are
the positions K65, K68, K77,
or K101 of the collagen domain of human adiponectin and positions K134, K149,
K169, K172, K177,
K178, or K180 of the globular domain of human adiponectin, or a smaller group,
such as K77, or K101
of the collagen domain and positions K134, K149, K169, K172, K177, K178, or
K180 of the globular
domain, or even a smaller group, such as K101 of the collagen domain and
positions K134, K149, K169,
25 K172, K177, K178, or K180 of the globular domain. If desired to introduce a
second non-polypeptide
moiety by conjugating it to a lysine, then obviously, at least one lysine
should be present in the
adiponectin polypeptide in order to make possible the conjugation to a lysine.
Fourth group of conju~ate(s) of the invention
3o In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide,
and a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, wherein the
adiponectin polypeptide comprises an amino acid residue having an attachment
group for said first non-
polypeptide moiety, wherein said amino acid residue has been introduced in a
position that in the parent
adiponectin is occupied by a surface exposed amino acid residue.
35 In a further aspect the invention relates to a conjugate consisting
essentially of an adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
wherein the adiponectin polypeptide comprises an amino acid residue having an
attachment group for
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
36
said first non-polypeptide moiety, wherein said amino acid residue has been
introduced in a position that
in the parent adiponectin is occupied by a surface exposed amino acid residue.
In a further aspect the invention relates to a conjugate comprising an
adiponectin polypeptide,
and one first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, wherein the
adiponectin polypeptide comprises an amino acid residue having an attachment
group for said first non-
polypeptide moiety, wherein said amino acid residue has been introduced in a
position that in the parent
adiponectin selected from seq id no 5 or 6 is occupied by a surface exposed
amino acid residue.
It is clear that the introduction of an amino acid residue in a position that
in the parent
adiponectin is occupied by a surface exposed amino acid residue will lead to a
novel adiponectin
to polypeptide. Such novel adiponectin polypeptide is also intended to be
comprised within the scope of
the present invention.
Thus, in a further aspect the invention relates to an adiponectin polypeptide
comprising an
amino acid residue having an attachment group for a first non-polypeptide
moiety, wherein said amino
acid residue has been introduced in a position that in the parent adiponectin
is occupied by a surface
15 exposed amino acid residue.
The amino acid residue having the attachment group for the first non-
polypeptide moiety is
located at the surface of the adiponectin polypeptide, and typically has more
than 25% of its side chain
exposed to the solvent, such as more than 50% of its side chain exposed to the
solvent. We believe that
such positions in the globular domain may be identified on the basis of an
analysis of the 3D structure of
2o the crystal structure of the globular domain of mouse ACRP30, cf Brief
Communication, "The crystal
structure of a complement-lq family protein suggets an evolutionary link to
tumor necrosis factor",
Shapiro et al, pp 335-338. Typically, in the globular and collagen domains all
lysine residues are surface
exposed. The surface exposed amino acid residues have been identified as
outlined in the experimental
section herein.
25 By introducing an amino acid residue having an attachment group for a non-
polypeptide moiety
in a position that in the parent adiponectin polypeptide is occupied by a
surface exposed amino acid
residue a novel molecule is created. Such novel adiponectin polypeptide may or
may not comprise
further mutations, however, this does not exclude that mutations can be made,
provided that the
adiponectin polypeptide or the conjugate maintain biological activity, and
thereby its usefulness for
3o treating eg. impaired glucose tolerance, type 2 diabetes, syndrome X,
obesity, a cardiovascular disease,
such as atherosclerosis, or dyslipidemia, such activity could be tested in a
relevant animal model, such
as mouse models of insulin resistance and diabetes, such as db/db or ob/ob
mice, or rat models such as
zucker rats, or could be tested in a relevant in vitro assay, such as any one
of the Test Assays A, B, or C
described in the experimental section.
35 In one embodiment the surface exposed amino acid residue is an amino acid
residue having at
least 25%, such as at least 50% of its side chain exposed to the surface. In a
particular embodiment the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
37
surface exposed amino acid residue is an amino acid residue having 100% of its
side chain exposed to
the surface.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Y111, 8112, L119, E120, T121, Y122, V123, T124, I125, P126, N127, M128,
I130, 8131, T133,
s K134, I135, F136, Y137, N138, Q139, Q140, N141, H142, D144, 6145, S146,
T147, K149, H151,
N153, I154, P155, Y159, A161, H163, I164, T165, Y167, M168, K169, D170, V171,
K172, F176,
K177, K178, D179, K180, A181, M182, F184, T185, Y186, D187, Q188, Y189, Q190,
E191, N192,
N193, V194, D195, Q196, 5198, 6199, S200, H204, E206, V207, 6208, D209, Q210,
W212, Q214,
V215, Y216, 6217, E218, 6219, E220, 8221, N222, 6223, L224, Y225, A226, D227,
N228, D229,
1o N230, D231, T233, F234, F237, H241, D242, T243, or N244 of human
adiponectin.
In a fi~rther embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Y111, 8112, E120, T121, Y122, V123, T124, I125, P126, N127, M128, 8131,
T133, K134, I135,
Q139, N141, D144, 6145, 5146, T147, K149, H151, N153, P155, Y167, M168, K169,
D170, K178,
D179, K180, A181, F184, Y186, D187, Q188, Y189, Q190, E191, N192, N193, V194,
D195, H204,
15 E206, V207, 6208, Q210, V215, Y216, 6217, E218, 6219, E220, 8221, N222,
6223, L224, Y225,
A226, D227, N228, D229, N230, H241, D242, T243, or N244 of human adiponectin.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
V110, Yl l l, E120, T121, Y122, V123, T124, I125, P126, N127, M128, 8131,
Q139, N141, D144,
6145, S146, N153, Y167, M168, K169, K178, D179, K180, A181, Y186, D187, Q188,
Y189, Q190,
2o E191, N192, N193, V194, D195, E206, V207, 6208, V215, Y216, 6217, E218,
6219, E220, 8221,
N222, 6223, L224, Y225, A226, D227, N228, D229, N230, H241, T243, or N244 of
human
adiponectin.
In a further embodiment the surface exposed amino acid residue is selected
from A108, Y109,
E120, T121, Y122, V123, T124, I125, P126, N127, Y167, M168, K169, A181, Y186,
D187, Q188,
25 Y189, Q190, E191, N192, N193, V194, D195, V215, Y216, 6217, E218, 6219,
E220, 8221, N222,
6223, L224, Y225, A226, D227, N228, D229, N230, T243, or N244 of human
adiponectin.
Any one of the above positions which have been identified as surface exposed
amino acid
residues may be substituted with an amino acid residue having an attachment
group for the first non-
polypeptide moiety, and such amino acid residue is typically selected from a
lysine, aspartic acid,
30 glutamic acid or cysteine residue. Each of these positions is considered an
embodiment and may be
made the subject of a claim, moreover, any one of these positions may be
combined with any one of the
embodiments hereinafter.
The identification of surface exposed amino acids in the globular domain of
human adiponectin
has made it possible to select the desired target for introducing an amino
acid residue having an
35 attachment group for a first non-polypeptide moiety and subsequently
attaching the first non-polypeptide
moiety. Such a non-polypeptide moiety is typically selected from a polymer
molecule, a lipophilic
compound, or an organic derivatizing agent. Suitable methods for attaching a
non-polypeptide moiety to
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
38
any one of the surface exposed amino acids in the globular domain of human
adiponectin are well
known to the skilled person. The preferred methods of attaching a non-
polypeptide moiety selected from
a polymer molecule, a lipophilic compound, or an organic derivatizing agent
are described in more
detail in the section "Methods of preparing a conjugate of the invention"
hereinafter.
The adiponectin polypeptide should have a globular domain, such as indicated
in the sequence
of human adiponectin (108-244) (shown in seq id no 6). The adiponectin
polypeptide part of the
conjugate comprises the globular domain having the amino acid sequence shown
in seq id no 6 as well
as analogues thereof, including fragments. As mentioned also analogues are
comprised, in particular
analogues that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 l, 12, 13, 14, or
15 amino acid residues relative to
1o the amino acid sequence shown in seq id no 6.
Thus, in a further embodiment the parent adiponectin polypeptide comprises a
globular domain,
preferably a collagen and a globular domain. In a still further embodiment the
parent adiponectin
comprises the amino acid sequence of seq id no 10. In a further embodiment the
parent adiponectin
comprises the amino acid sequence of seq id no 11. In a further embodiment the
adiponectin polypeptide
15 comprises the amino acid sequence of seq id no 12. In a further embodiment
the adiponectin polypeptide
comprises the amino acid sequence of seq id no 13. In a further embodiment the
parent adiponectin
comprises the amino acid sequence of seq id no 6. In a further embodiment the
parent adiponectin
comprises the amino acid sequence of seq id no 5. In a further embodiment the
parent adiponectin
comprises the amino acid sequence of seq id no 4. In a further embodiment the
parent adiponectin
2o comprises the amino acid sequence of seq id no 3. In a further embodiment
the parent adiponectin
comprises the amino acid sequence of seq id no 2. In a further embodiment the
parent adiponectin
consist essentially of a globular domain. In a further embodiment the parent
adiponectin consist
essentially of a collagen and a globular domain. In a further embodiment the
parent adiponectin consist
essentially of the amino acid sequence of seq id no 10. In a further
embodiment the parent adiponectin
25 consist essentially of the amino acid sequence of seq id no 11. In a
further embodiment the parent
adiponectin consist essentially of the amino acid sequence of seq id no 12. In
a further embodiment the
parent adiponectin consist essentially of the amino acid sequence of seq id no
13. In a further
embodiment the parent adiponectin consist essentially of the amino acid
sequence of seq id no 6. In a
further embodiment the parent adiponectin consist essentially of the amino
acid sequence of seq id no S.
3o In a further embodiment the parent adiponectin consist essentially of the
amino acid sequence of seq id
no 4. In a further embodiment the parent adiponectin consist essentially of
the amino acid sequence of
seq id no 3. In a further embodiment the parent adiponectin consist
essentially of the amino acid
sequence of seq id no 2.
Typically, the parent adiponectin is selected from any one of seq id no 2, 3,
4, 5, 10, 11, 12, or
35 13, as well as sequences that differs from any one of the specified
sequences, in one or more
substitution(s), preferably from one to eleven, such as one to eight. In one
embodiment the adiponectin
polypeptide is selected from any one of seq id no 3, 10, 12, or 13, as well as
sequences that differs from
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
39
any one of the specified sequences in one to eleven substitutions, such as one
to eight substitutions, eg.
1-6 substitutions.
In a particular embodiment the parent adiponectin is selected from any one of
seq id no 3, 4, 5,
10, 11, 12, or 13, as well as sequences that differs from any one of the
specified sequences, in one or
more substitutions, and comprises one to four lysine residues selected from
any one of the positions
K65, K68, K77, or K101. In a further embodiment the parent adiponectin is
selected from any one of seq
id no 3, 4, 5, 10, 11, 12, or 13, preferably 3, 10, 12, or 13. In a
alternative embodiment the parent
adiponectin is selected from sequences that differs from any one of the seq id
no 3, 4, 5, 10, 11, 12, or
13, preferably 3, 10, 12, or 13, in one or more substitutions, preferably from
one to eleven substitutions,
1o such as one to eight substitutions, eg. 1-6 substitutions. In a further
embodiment the parent adiponectin
comprises at least one lysine residue selected from any one of the positions
K65, K68, K77, or K101. As
mentioned above when produced in a eucaryotic cell, such as a mammalian cell,
lysine residues in the
collagen domain are hydroxylated and glycosylated. Typically the lysine
residues are hydroxylated and
glycosylated. In a further embodiment the parent adiponectin comprises one
lysine residue selected from
15 any one of the positions K65, K68, K77, or K101, preferably K101, and
preferably the position is
hydroxylated and glycosylated, such as glyco-hydroxy-K101. In a further
embodiment the parent
adiponectin comprises two lysine residues selected from any one of the
positions K65, K68, K77, or
K101, preferably K77 and K101, and preferably both of the positions are
hydroxylated and glycosylated,
such as glyco-hydroxy-K77 and glyco-hydroxy-K101. In a further embodiment the
parent adiponectin
20 comprises three lysine residues selected from any one of the positions K65,
K68, K77, or K101,
preferably K68, K77 and K101, and preferably all three of the positions are
hydroxylated and
glycosylated, such as glyco-hydroxy-K68, glyco-hydroxy-K77 and glyco-hydroxy-
K101. In a further
embodiment the parent adiponectin comprises four lysine residues selected from
positions K65, K68,
K77, and K101, and preferably all four of the positions are hydroxylated and
glycosylated.
25 In a still further embodiment the parent adiponectin polypeptide is
selected from any one of the
adiponectin polypeptide fragments described in the above section "Adiponectin
polypeptide fragments)
of the invention". Each of the described adiponectin polypeptide fragments is
considered an embodiment
suitable as the parent adiponectin polypeptide in either the adiponectin
polypeptide or the conjugate.
Accordingly, one example of a preferred aspect of the adiponectin polypeptide
relates to an
3o adiponectin polypeptide fragment comprising an amino acid residue having an
attachment group for a
first non-polypeptide moiety, wherein said amino acid residue has been
introduced in a position that in
the parent adiponectin is occupied by a surface exposed amino acid residue. In
one embodiment the
parent adiponectin is selected from an adiponectin polypeptide fragment
comprising a globular domain
and a collagen domain, wherein the globular domain comprises an amino acid
sequence from position
35 A108 to N244 as indicated in seq id no 1 as well as sequences that differs
from the amino acid sequence
in one or more substitution(s), and wherein the collagen domain comprises from
7 amino acids
corresponding to position K101 as indicated in seq id no 1 to 56 amino acids
corresponding to position
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
A52 as indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is
hydroxylated and glycosylated.
Furthermore, one example of a preferred aspect of the conjugate relates to a
conjugate
comprising an adiponectin polypeptide fragment, and a first non-polypeptide
moiety covalently attached
to the adiponectin polypeptide fragment, wherein the adiponectin polypeptide
fragment comprises an
amino acid residue having an attachment group for said first non-polypeptide
moiety, wherein said
amino acid residue has been introduced in a position that in the parent
adiponectin is occupied by a
surface exposed amino acid residue. In one embodiment the parent adiponectin
is selected from an
adiponectin polypeptide fragment comprising a globular domain and a collagen
domain, wherein the
1o globular domain comprises an amino acid sequence from position A108 to N244
as indicated in seq id
no 1 as well as sequences that differs from the amino acid sequence in one or
more substitution(s), and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no 1, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated.
t5 It should be clear that the surface exposed amino acid residue having an
attachment group for
the first non-polypeptide moiety may either be introduced in the globular
domain or in the collagen
domain, or in case of more than one non-polypeptide moiety being attached they
may be introduced in
the globular domain or in the collagen domain, or in both the globular domain
and the collagen domain.
Accordingly, in a further embodiment of the conjugate or the adiponectin
polypeptide or the
2o adiponectin polypeptide fragment the surface exposed amino acid residue
having the attachment group
is introduced in the globular domain.
In a further embodiment the adiponectin polypeptide comprises a collagen
domain.
In a further embodiment the surface exposed amino acid residue having the
attachment group is
introduced in the collagen domain. If only one first non-polypeptide is
attached then it may be in the
25 globular domain or in the collagen domain. If more than one, such as two
non-polypeptides, are attached
then one may be located in the collagen domain and one in the globular domain,
or both may be in the
collagen domain, or both may be in the globular domain.
In a further embodiment the adiponectin polypeptide comprises a non-homologous
domain.
In a fiirther embodiment the adiponectin polypeptide comprises a signal
peptide.
3o In a further embodiment the adiponectin polypeptide is isolated.
In a further embodiment only one first non-polypeptide moiety is attached to
the adiponectin
polypeptide.
In a further embodiment the conjugate of the invention is mono pegylated.
In a further embodiment the amino acid residue having the attachment group for
said first non-
35 polypeptide moiety is selected from a lysine, aspartic acid, glutamic acid
or cysteine residue.
In a further embodiment the amino acid residue having the attachment group for
said first non-
polypeptide moiety is an glutamic acid residue. Preferred Glu mutations are
made in the globular
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
41
domain and may be selected from any one of A108E, Y109E, V110E, Y111E, R112E,
T121E, Y122E,
V123E, T124E, I125E, P126E, N127E, M128E, R131E, T133E, K134E, I135E, Q139E,
N141E,
D144E, G145E, S146E, T147E, K149E, H151E, N153E, P155E, Y167E, M168E, K169E,
D170E,
K178E, D179E, K180E, A181E, F184E, Y186E, Q188E, Y189E, Q190E, N192E, N193E,
V194E,
H204E, E206E, V207E, G208E, Q210E, V215E, Y216E, G217E, G219E, R221E, N222E,
L224E,
Y225E, D227E, N228E, D229E, N230E, H241E, D242E, T243E, or N244E.
In a further embodiment the amino acid residue having the attachment group for
said first non-
polypeptide moiety is an aspartic acid residue. Preferred Asp mutations are
made in the globular domain
and may be selected from any one of A108D, Y109D, V110D, Y111D, Rl 12D, E120D,
T121D,
1o Y122D, V123D, T124D, I125D, P126D, N127D, M128D, R131D, T133D, K134D,
I135D, Q139D,
N141D, G145D, S146D, T147D, K149D, H151D, N153D, P155D, Y167D, M168D, K169D,
K178D,
K180D, A181D, F184D, Y186D, Q188D, Y189D, Q190D, E191D, N192D, N193D, V194D,
H204D,
E206D, V207D, G208D, Q210D, V215D, Y216D, G217D, E218D, G219D, E220D, R221D,
N222D,
L224D, Y225D, N228D, N230D, H241D, T243D, or N244D.
~ 5 In a further embodiment the amino acid residue having the attachment group
for said first non-
polypeptide moiety is a lysine residue. Preferred Lys mutations are made in
the globular domain and
may be selected from any one of A108K, Y109K, V110K, Y111K, Rl 12K, E120K,
T121K, Y122K,
V123K, T124K, I125K, P126K, N127K, M128K, R131K, T133K, I135K, Q139K, N141K,
D144K,
G145K, S146K, T147K, H151K, N153K, P155K, Y167K, M168K, D170K, D179K, A181K,
F184K,
2o Y186K, Q188K, Y189K, Q190K, E191K, N192K, N193K, V194K, H204K, E206K,
V207K, G208K,
Q210K, V215K, Y216K, G217K, E218K, G219K, E220K, R221K, N222K, IJ224K, Y225K,
D227K,
N228K, D229K, N230K, H241K, D242K, T243K, or N244K.
In a further embodiment the amino acid residue having the attachment group for
said first non-
polypeptide moiety is a cysteine residue. Preferred Cys mutations are made in
the globular domain and
25 may be selected from any one of A108C, Y109C, V110C, Y111C, R112C, E120C,
T121C, Y122C,
V123C, T124C, I125C, P126C, N127C, M128C, R131C, T133C, K134C, I135C, Q139C,
N141C,
D144C, G145C, S146C, T147C, K149C, H151C, N153C, P155C, Y167C, M168C, K169C,
D170C,
K178C, D179C, K180C, A181C, F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C,
N193C,
V194C, H204C, E206C, V207C, G208C, Q210C, V215C, Y216C, G217C, E218C, G219C,
E220C,
30 8221 C, N222C, L224C, Y225C, D227C, N228C, D229C, N230C, H241 C, D242C,
T243C, or N244C,
such as T121C, S146C, or T243C. Cys152 (which is not surface exposed) relative
to human adiponectin
is preferably maintained so that the adiponectin polypeptide contains two
cysteins in the globular
domain.
The above Lys, Glu, Asp, or Cys mutations may be introduced in any one of the
parent
35 adiponectin polypeptides as part of the conjugate or as the adiponectin
polypeptide, including fragments
thereof, such as any one of the sequences seq id no 3, 4, 5, 6, 10, 1 l, 12,
or 13, or the adiponectin
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
42
polypeptide fragments selected from any one of the adiponectin polypeptide
fragments described in the
above section "Adiponectin polypeptide fragments) of the invention".
To illustrate the non-conjugated polypeptide part of the invention some
embodiments have been
outlined hereafter.
Typical, embodiments of the invention relates to an adiponectin polypeptide
comprising a
mutation selected from any one of A108C, Y109C, V110C, Y111C, R112C, E120C,
T121C, Y122C,
V123C, T124C, I125C, P126C, N127C, M128C, R131C, T133C, K134C, I135C, Q139C,
N141C,
D144C, G145C, S146C, T147C, K149C, H151C, N153C, P155C, Y167C, M168C, K169C,
D170C,
K178C, D179C, K180C, A181C, F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C,
N193C,
to V194C, H204C, E206C, V207C, G208C, Q210C, V215C, Y216C, G217C, E218C,
G219C, E220C,
R221C, N222C, L224C, Y225C, D227C, N228C, D229C, N230C, H241C, D242C, T243C,
or N244C,
such as T121C, S146C, or T243C. Preferably, the adiponectin polypeptide
contains only one of these
cysteine mutations, since two or more may lead to loss of product upon
expression, for instance, due to
inter and/or intra molecular sulphurbridges being formed. Cys152 (which is not
surface exposed)
~5 relative to human adiponectin is preferably maintained so that the
adiponectin polypeptide contains two
or more cysteins in the globular domain, that is Cys152 and one or more
introduced cysteins, preferably
one introduced cystein and the conserved Cys152.
Typically, the adiponectin polypeptide is selected from any one of the
sequences seq id no 3, 4,
5, 6, 10, 11, 12, or 13, or any one of the adiponectin polypeptide fragments
described in the above
2o section "Adiponectin polypeptide fragments) of the invention". Typical
embodiments of the
adiponectin polypeptide are selected from any one of the sequences seq id no
17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51,
or 52.
Accordingly, the present invention relates to an adiponectin polypeptide
having an amino acid
25 sequence selected from any one of the seq id no 3, 4, 5, 6, 10, 1 l, 12, or
13, wherein the adiponectin
polypeptide comprises a mutation selected from any one of A108C, Y109C, V1
IOC, Y111C, R112C,
E120C, T121C, Y122C, V123C, T124C, I125C, P126C, N127C, M128C, R131C, T133C,
K134C,
I135C, Q139C, N141C, D144C, G145C, S146C, T147C, K149C, H151C, N153C, P155C,
Y167C,
M168C, K169C, D170C, K178C, D179C, K180C, A181C, F184C, Y186C, Q188C, Y189C,
Q190C,
3o E191C, N192C, N193C, V194C, H204C, E206C, V207C, G208C, Q210C, V215C,
Y216C, G217C,
E218C, G219C, E220C, R221C, N222C, L224C, Y225C, D227C, N228C, D229C, N230C,
H241C,
D242C, T243C, or N244C. Thus, each of these amino acid sequences in
combination with one of the
specified mutations constitutes embodiments of the invention and may be made
the subject of one or
more claims. Typically, the adiponectin polypeptide comprising a mutation is
produced in a eucaryotic
35 cell, such as a mammalian cell, and thus, any one of the sequences seq id
no 3, 4, 5, 10, 11, 12, or 13
comprises a lysine in the collagen domain which is hydroxylated and
glycosylated. Alternatively, any
one of the sequences seq id no 3, 4, 5, 10, 11, 12, or 13 is produced in a
bacterial cell, such as E. Coli,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
43
and thus, is not hydroxylated and glycosylated. For instance, in one example
the present invention
relates to an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
wherein the adiponectin polypeptide comprises the mutation T121C, such as the
adiponectin polypeptide
having the amino acid sequence of seq id no 17; in another example the present
invention relates to an
adiponectin polypeptide having an amino acid sequence selected from the seq id
no 10, wherein the
adiponectin polypeptide comprises the mutation S 146C, such as the adiponectin
polypeptide having the
amino acid sequence of seq id no 18; in a further example the present
invention relates to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 10,
wherein the adiponectin
polypeptide comprises the mutation T243C, such as the adiponectin polypeptide
having the amino acid
to sequence of seq id no 19; in a further example the present invention
relates to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 10,
wherein the adiponectin
polypeptide comprises the mutation N127C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 35; in a further example the present invention relates
to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 10,
wherein the adiponectin
15 polypeptide comprises the mutation N141C, such as the adiponectin
polypeptide having the amino acid
sequence of seq id no 36; in a further example the present invention relates
to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 10,
wherein the adiponectin
polypeptide comprises the mutation N228C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 37;in a further example the present invention relates to
an adiponectin polypeptide
2o having an amino acid sequence selected from the seq id no 5, wherein the
adiponectin polypeptide
comprises the mutation T121C, such as the adiponectin polypeptide having the
amino acid sequence of
seq id no 23; in a further example the present invention relates to an
adiponectin polypeptide having an
amino acid sequence selected from the seq id no 5, wherein the adiponectin
polypeptide comprises the
mutation S 146C, such as the adiponectin polypeptide having the amino acid
sequence of seq id no 24; in
25 a further example the present invention relates to an adiponectin
polypeptide having an amino acid
sequence selected from the seq id no 5, wherein the adiponectin polypeptide
comprises the mutation
T243C, such as the adiponectin polypeptide having the amino acid sequence of
seq id no 25; in a further
example the present invention relates to an adiponectin polypeptide having an
amino acid sequence
selected from the seq id no 5, wherein the adiponectin polypeptide comprises
the mutation N127C, such
3o as the adiponectin polypeptide having the amino acid sequence of seq id no
41; in a further example the
present invention relates to an adiponectin polypeptide having an amino acid
sequence selected from the
seq id no 5, wherein the adiponectin polypeptide comprises the mutation N141
C, such as the adiponectin
polypeptide having the amino acid sequence of seq id no 42; in a further
example the present invention
relates to an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
35 wherein the adiponectin polypeptide comprises the mutation N228C, such as
the adiponectin
polypeptide having the amino acid sequence of seq id no 43; in a further
example the present invention
relates to an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 13,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
44
wherein the adiponectin polypeptide comprises the mutation T121 C, such as the
adiponectin polypeptide
having the amino acid sequence of seq id no 32; in a further example the
present invention relates to an
adiponectin polypeptide having an amino acid sequence selected from the seq id
no 13, wherein the
adiponectin polypeptide comprises the mutation S146C, such as the adiponectin
polypeptide having the
amino acid sequence of seq id no 33; in a further example the present
invention relates to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 13,
wherein the adiponectin
polypeptide comprises the mutation T243C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 34; in a further example the present invention relates
to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 13,
wherein the adiponectin
1o polypeptide comprises the mutation N127C, such as the adiponectin
polypeptide having the amino acid
sequence of seq id no 50; in a further example the present invention relates
to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 13,
wherein the adiponectin
polypeptide comprises the mutation N141C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 51; in a further example the present invention relates
to an adiponectin
polypeptide having an amino acid sequence selected from the seq id no 13,
wherein the adiponectin
polypeptide comprises the mutation N228C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 52; and so forth. Preferably, any one of the above
adiponectin polypeptides
comprising a cystein introduced in the globular domain is produced in a
eucaryotic cell, such as a
mammalian cell.
2o Furthermore, the present invention relates to an adiponectin polypeptide
fragment comprising a
globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence as indicated in
seq id no 1 from
position A108 to N244, and wherein the collagen domain comprises from 7 amino
acids corresponding
to position K101 as indicated in seq id no 1 to 66 amino acids corresponding
to position G42 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated,
wherein the adiponectin polypeptide fragment comprises a mutation selected
from any one of
A108C, Y109C, V110C, Y111C, R112C, E120C, T121C, Y122C, V123C, T124C, I125C,
P126C,
N127C, M128C, R131C, T133C, K134C, I135C, Q139C, N141C, D144C, G145C, S146C,
T147C,
3o K149C, H151C, N153C, P155C, Y167C, M168C, K169C, D170C, K178C, D179C,
K180C, A181C,
F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C, N193C, V194C, H204C, E206C,
V207C,
G208C, Q210C, V215C, Y216C, G217C, E218C, G219C, E220C, R221C, N222C, L224C,
Y225C,
D227C, N228C, D229C, N230C, H241C, D242C, T243C, or N244C. Thus, each of these
amino acid
sequences, including the embodiments described in the above section
"Adiponectin polypeptide
fragments) of the invention", in combination with one of the specified
mutations constitutes
embodiments of the invention and may be made the subject of one or more
claims. As mentioned above
it is preferred that the adiponectin polypeptide fragment only contains one
introduced Cys. However, the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
adiponectin polypeptide fragment may contain other mutations as long as the
biological activity is
maintained (as mentioned above), which means that preferably up to eleven (11)
substitutions may be
made in the globular domain. For instance, in one example the present
invention relates to an
adiponectin polypeptide fragment comprising a globular domain and a collagen
domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T121C. Examples
t o of such adiponectin polypeptide fragments are any one of the sequences seq
id no 17, 23, 26, 29, or 32.
In another example the present invention relates to an adiponectin polypeptide
fragment comprising a
globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
15 to position 8100 as indicated in seq id no 1 to 50 amino acids
corresponding to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation S
146C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 18,
27, or 33. In a further
example the present invention relates to an adiponectin polypeptide fragment
comprising a globular
20 domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
25 glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 19,
or 28. In a further example
the present invention relates to an adiponectin polypeptide fragment
comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
3o indicated in seq id no l, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N127C. Examples
of such adiponectin polypeptide fragments are any one of the sequences seq id
no 35, 41, 44, 47, or 50.
35 In a further example the present invention relates to an adiponectin
polypeptide fragment comprising a
globular domain and a collagen domain,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
46
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation N141
C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 36,
45, or 51. In a further
example the present invention relates to an adiponectin polypeptide fragment
comprising a globular
domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
1o indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 37,
or 46. And so forth.
t 5 Also, as described above the above Lys, Glu, Asp, or Cys mutations may be
introduced in any
one of the parent adiponectin polypeptides as part of the conjugate, including
fragments thereof, such as
any one of the sequences seq id no 3, 4, 5, 6, 10, 11, 12, or 13, or the
adiponectin polypeptide fragments
selected from any one of the adiponectin polypeptide fragments described in
the above section
"Adiponectin polypeptide fragments) of the invention", in which respect a
first non-polypeptide moiety
2o is attached to the introduced amino acid residue having an attachment group
for said first non-
polypeptide moiety.
To illustrate this conjugate part of the invention some embodiments have been
outlined
hereafter. Typical, embodiments of the invention relates to a conjugate
comprising an adiponectin
polypeptide comprising a mutation selected from any one of A108C, Y109C, V1
lOC, Y111C, R112C,
25 E120C, T121C, Y122C, V123C, T124C, I125C, P126C, N127C, M128C, R131C,
T133C, K134C,
I135C, Q139C, N141C, D144C, G145C, S146C, T147C, K149C, H151C, N153C, P155C,
Y167C,
M168C, K169C, D170C, K178C, D179C, K180C, A181C, F184C, Y186C, Q188C, Y189C,
Q190C,
E191C, N192C, N193C, V194C, H204C, E206C, V207C, G208C, Q210C, V215C, Y216C,
G217C,
E218C, G219C, E220C, R221C, N222C, L224C, Y225C, D227C, N228C, D229C, N230C,
H241C,
30 D242C, T243C, or N244C, such as T121C, S146C, or T243C, and a first non-
polypeptide moiety
covalently attached to the introduced cystein residue. Preferably, the
adiponectin polypeptide of the
conjugate contains only one of these cysteine mutations, since two or more may
lead to loss of product
upon expression, for instance, due to inter and/or intra molecular
sulphurbridges being formed.
Typically, the adiponectin polypeptide of the conjugate is selected from any
one of the sequences seq id
35 no 3, 4, 5, 6, 10, 11, 12, or 13, or any one of the adiponectin polypeptide
fragments described in the
above section "Adiponectin polypeptide fragments) of the invention".
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
47
Accordingly, the present invention relates to a conjugate comprising an
adiponectin polypeptide
having an amino acid sequence selected from any one of the seq id no 3, 4, 5,
6, 10, 11, 12, or 13,
wherein the adiponectin polypeptide comprises a mutation selected from any one
of A108E, Y109E,
V110E, Y111E, R112E, T121E, Y122E, V123E, T124E, I125E, P126E, N127E, M128E,
R131E,
T133E, K134E, I135E, Q139E, N141E, D144E, G145E, S146E, T147E, K149E, H151E,
N153E,
P155E, Y167E, M168E, K169E, D170E, K178E, D179E, K180E, A181E, F184E, Y186E,
Q188E,
Y189E, Q190E, N192E, N193E, V194E, H204E, E206E, V207E, G208E, Q210E, V215E,
Y216E,
G217E, G219E, R221E, N222E, L224E, Y225E, D227E, N228E, D229E, N230E, H241E,
D242E,
T243E, or N244E, and a first non-polypeptide moiety covalently attached to the
introduced Glu residue.
Thus, each of these amino acid sequences in combination with one of the
specified mutations constitutes
embodiments of the invention and may be made the subject of one or more
claims.
Furthermore, the present invention relates to a conjugate comprising an
adiponectin polypeptide
having an amino acid sequence selected from any one of the seq id no 3, 4, 5,
6, 10, 11, 12, or 13,
wherein the adiponectin polypeptide comprises a mutation selected from any one
of A108D, Y109D,
V110D, Y111D, R112D, E120D, T121D, Y122D, V123D, T124D, I125D, P126D, N127D,
M128D,
R131D, T133D, K134D, I135D, Q139D, N141D, G145D, S146D, T147D, K149D, H151D,
N153D,
P155D, Y167D, M168D, K169D, K178D, K180D, A181D, F184D, Y186D, Q188D, Y189D,
Q190D,
E191D, N192D, N193D, V194D, H204D, E206D, V207D, G208D, Q210D, V215D, Y216D,
G217D,
E218D, G219D, E220D, R221D, N222D, L224D, Y225D, N228D, N230D, H241D, T243D,
or N244D,
2o and a first non-polypeptide moiety covalently attached to the introduced
Asp residue. Thus, each of
these amino acid sequences in combination with one of the specified mutations
constitutes embodiments
of the invention and may be made the subject of one or more claims.
Furthermore, the present invention relates to a conjugate comprising an
adiponectin polypeptide
having an amino acid sequence selected from any one of the seq id no 3, 4, 5,
6, 10, 11, 12, or 13,
wherein the adiponectin polypeptide comprises a mutation selected from any one
of A108K, Y109K,
V110K, Y111K, R112K, E120K, T121K, Y122K, V123K, T124K, I125K, P126K, N127K,
M128K,
R131K, T133K, I135K, Q139K, N141K, D144K, G145K, S146K, T147K, H151K, N153K,
P155K,
Y167K, M168K, D170K, D179K, A181K, F184K, Y186K, Q188K, Y189K, Q190K, E191K,
N192K,
N193K, V194K, H204K, E206K, V207K, G208K, Q210K, V215K, Y216K, G217K, E218K,
G219K,
3o E220K, R221K, N222K, L224K, Y225K, D227K, N228K, D229K, N230K, H241K,
D242K, T243K, or
N244K, and a first non-polypeptide moiety covalently attached to the
introduced Lys residue. Thus,
each of these amino acid sequences in combination with one of the specified
mutations constitutes
embodiments of the invention and may be made the subject of one or more
claims.
Furthermore, the present invention relates to a conjugate comprising an
adiponectin polypeptide
having an amino acid sequence selected from any one of the seq id no 3, 4, 5,
6, 10, 11, 12, or 13,
wherein the adiponectin polypeptide comprises a mutation selected from any one
of A108C, Y109C,
V110C, Y111C, R112C, E120C, T121C, Y122C, V123C, T124C, I125C, P126C, N127C,
M128C,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
48
R131C, T133C, K134C, I135C, Q139C, N141C, D144C, G145C, S146C, T147C, K149C,
H151C,
N153C, P155C, Y167C, M168C, K169C, D170C, K178C, D179C, K180C, A181C, F184C,
Y186C,
Q188C, Y189C, Q190C, E191C, N192C, N193C, V194C, H204C, E206C, V207C, G208C,
Q210C,
V215C, Y216C, G217C, E218C, G219C, E220C, 8221 C, N222C, L224C, Y225C, D227C,
N228C,
D229C, N230C, H241C, D242C, T243C, or N244C, and a first non-polypeptide
moiety covalently
attached to the introduced cystein residue. Thus, each of these amino acid
sequence sequences in
combination with one of the specified mutations constitutes embodiments of the
invention and may be
made the subject of one or more claims. Typical embodiments of the adiponectin
polypeptide part of the
conjugate are any one of the sequences seq id no 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31,
to 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, or 52. Preferably, any one of
the above adiponectin polypeptides comprising a cystein introduced in the
globular domain is produced
in a eucaryotic cell, such as a mammalian cell, e.g. a CHO, BHK, HEK293 cell
or an SF9 cell.
For instance, in one example the present invention relates to a conjugate
comprising an
adiponectin polypeptide having an amino acid sequence selected from the seq id
no 10, wherein the
15 adiponectin polypeptide comprises the mutation T121C, and a first non-
polypeptide moiety covalently
attached to the introduced cystein residue. Such as a conjugate comprising an
adiponectin polypeptide
having the sequence seq id no 17, and a first non-polypeptide moiety
covalently attached to the
introduced cystein residue T121 C. In another example the present invention
relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
2o wherein the adiponectin polypeptide comprises the mutation S 146C, and a
first non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 18, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue S 146C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
25 wherein the adiponectin polypeptide comprises the mutation T243C, and a
first non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 19, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue T243C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
3o wherein the adiponectin polypeptide comprises the mutation N127C, and a
first non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 35, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N127C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
35 wherein the adiponectin polypeptide comprises the mutation N141 C, and a
first non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 36, and a first non-polypeptide
moiety covalently attached to
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
49
the introduced cystein residue N141C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 10,
wherein the adiponectin polypeptide comprises the mutation N228C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 37, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N228C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation T121C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 23, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue T121C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation S146C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
t5 polypeptide having the sequence seq id no 24, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue S146C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation T243C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 25, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue T243C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation N127C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 41, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N127C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation N141C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
3o polypeptide having the sequence seq id no 42, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N141C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 5,
wherein the adiponectin polypeptide comprises the mutation N228C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 43, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N228C. In a further example the present
invention relates to a conjugate
comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 13,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
wherein the adiponectin polypeptide comprises the mutation T121C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 32, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue T121C. In a further example the present
invention relates to a conjugate
5 comprising an adiponectin polypeptide having an amino acid sequence selected
from the seq id no 13,
wherein the adiponectin polypeptide comprises the mutation S 146C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 33, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue S 146C. In a further example the present
invention relates to a conjugate
10 comprising an adiponectin polypeptide having an amino acid sequence
selected from the seq id no 13,
wherein the adiponectin polypeptide comprises the mutation T243C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 34, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue T243C. In a further example the present
invention relates to a conjugate
~ 5 comprising an adiponectin polypeptide having an amino acid sequence
selected from the seq id no 13,
wherein the adiponectin polypeptide comprises the mutation N127C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 50, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N127C. In a further example the present
invention relates to a conjugate
2o comprising an adiponectin polypeptide having an amino acid sequence
selected from the seq id no 13,
wherein the adiponectin polypeptide comprises the mutation N141 C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 51, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N141 C. In a further example the present
invention relates to a conjugate
25 comprising an adiponectin polypeptide having an amino acid sequence
selected from the seq id no 13,
wherein the adiponectin polypeptide comprises the mutation N228C, and a first
non-polypeptide moiety
covalently attached to the introduced cystein residue. Such as a conjugate
comprising an adiponectin
polypeptide having the sequence seq id no 52, and a first non-polypeptide
moiety covalently attached to
the introduced cystein residue N228C. And so forth. Preferably, any one of the
above adiponectin
3o polypeptides comprising a mutation, such as cystein, introduced in the
globular domain is produced in a
eucaryotic cell, such as a mammalian cell, e.g. a CHO, BHK, HEK293 cell or an
SF9 cell.
Furthermore, the present invention relates to a conjugate comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence as indicated in
seq id no 1 from position
35 A108 to N244, and wherein the collagen domain comprises from 7 amino acids
corresponding to
position K101 as 'indicated in seq id no 1 to 66 amino acids corresponding to
position G42 as indicated
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
51
in seq id no l, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated,
wherein the adiponectin polypeptide fragment comprises a mutation selected
from any one of
A108C, Y109C, V110C, Y111C, R112C, E120C, T121C, Y122C, V123C, T124C, I125C,
P126C,
s N127C, M128C, R131C, T133C, K134C, I135C, Q139C, N141C, D144C, G145C, S146C,
T147C,
K149C, H151C, N153C, P155C, Y167C, M168C, K169C, D170C, K178C, D179C, K180C,
A181C,
F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C, N193C, V194C, H204C, E206C,
V207C,
G208C, Q210C, V215C, Y216C, G217C, E218C, G219C, E220C, R221C, N222C, L224C,
Y225C,
D227C, N228C, D229C, N230C, H241C, D242C, T243C, or N244C; and
1 o a first non-polypeptide moiety covalently attached to the introduced
cystein residue. Thus, each
of these amino acid sequences as part of the conjugate, including the
embodiments described in the
above section "Adiponectin polypeptide fragments) of the invention", in
combination with one of the
specified mutations constitutes embodiments of the invention and may be made
the subject of one or
more claims. As mentioned above it is preferred that the adiponectin
polypeptide fragment only contains
15 one introduced Cys, and that Cys 152 is maintained. However, the
adiponectin polypeptide fragment may
contain other mutations as long as the biological activity is maintained (as
mentioned above), which
means that preferably up to eleven (11) substitutions may be made in the
globular domain. Typical
embodiments of the adiponectin polypeptide fragment part of the conjugate are
any one of the sequences
seq id no 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41,
20 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52.
For instance, in one example the present invention relates to a conjugate
comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 7
amino acids corresponding
25 to position K101 as indicated in seq id no 1 to 56 amino acids
corresponding to position A52 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T121C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In another example the present invention relates to a conjugate comprising an
adiponectin
3o polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
35 glycosylated, wherein the adiponectin polypeptide comprises the mutation
T121 C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
52
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 17,
23, 26, 29, or 32, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue T121C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
to indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T121C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 17,
~5 or 26, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue T121C.
In a further example the present invention relates to a conjugate comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
20 indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N127C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
25 In a further example the present invention relates to a conjugate
comprising an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
3o indicated in seq id no l, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N127C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 35,
35 41, 44, 47, or 50, and wherein the collagen domain comprises a lysine which
is hydroxylated and
glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue N127C.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
53
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N127C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
to an adiponectin polypeptide fragment having a sequence selected from any one
of seq id no 35,
or 44, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue N127C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular
15 domain comprises an amino acid sequence from position A108 to N244 as
indicated in seq id no 1, and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no 1, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated,
wherein the adiponectin
polypeptide fragment comprises the mutation S 146C; and
20 a first non-polypeptide moiety covalently attached to the introduced
cystein residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
25 to position 8100 as indicated in seq id no 1 to 50 amino acids
corresponding to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
S146C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
3o an adiponectin polypeptide fragment having a sequence selected from any one
of seq id no 18,
27, or 33, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated;
and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue S146C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
35 polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
54
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation S
146C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 18,
or 27, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue S 146C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
l0 polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular
domain comprises an amino acid sequence from position A108 to N244 as
indicated in seq id no 1, and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no 1, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated,
wherein the adiponectin
15 polypeptide fragment comprises the mutation N141C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
2o indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N141C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
25 conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 36,
45, or 51, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated;
and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue N141 C.
30 In a further example the present invention relates to a conjugate
comprising an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
35 indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N141C; and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 36,
or 45, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue N141C.
In a further example the present invention relates to a conjugate comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
1o to position K101 as indicated in seq id no 1 to 56 amino acids
corresponding to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T243C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
15 polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
2o glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
25 indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Such as a
30 conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 19,
or 28, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue T243C.
In a further example the present invention relates to a conjugate comprising
35 an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
56
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N228C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
1o indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
15 wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C; and
2o a first non-polypeptide moiety covalently attached to the introduced
cystein residue. Such as a
conjugate comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 37,
or 46, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue N228C.
25 And so forth.
'The first non-polypeptide moiety to be attached to the introduced amino acid
residue having the
attachment group for said first non-polypeptide moiety, such as a cysteine,
lysine, aspartic acid, or
glutamic acid may be introduced by methods known to the person skilled in the
art, or as suggested in
the section "Methods of preparing a conjugate of the invention" herein. Thus,
when a conjugate is to be
3o prepared, a further embodiment of the first non-polypeptide moiety is
selected from a polymer, a
lipophilic compound, and an organic derivatizing agent.
In a further embodiment the first non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol. Such polymers are available from Shearwater.
In a specific aspect the present invention relates to a conjugate comprising
35 an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
57
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T121C; and
a first polymer covalently attached to the introduced cystein residue.
In another example the present invention relates to a conjugate comprising an
adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
1o indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T121C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 17,
15 23, 26, 29, or 32, and wherein the collagen domain comprises a lysine which
is hydroxylated and
glycosylated; and
a first polymer covalently attached to the introduced cystein residue T121C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
2o wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T121C; and
25 a first polymer covalently attached to the introduced cystein residue. Such
as a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 17,
or 26, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first polymer covalently attached to the introduced cystein residue T121 C.
30 In a further example the present invention relates to a conjugate
comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
35 indicated in seq id no l, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N127C; and
a first polymer covalently attached to the introduced cystein residue.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
$8
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N127C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 35,
41, 44, 47, or 50, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated; and
a first polymer covalently attached to the introduced cystein residue N127C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
z0 glycosylated, wherein the adiponectin polypeptide comprises the mutation
N127C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 35,
or 44, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first polymer covalently attached to the introduced cystein residue N127C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular
domain comprises an amino acid sequence from position A108 to N244 as
indicated in seq id no l, and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no 1, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated,
wherein the adiponectin
polypeptide fragment comprises the mutation S 146C; and
a first polymer covalently attached to the introduced cystein residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
59
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
S146C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 18,
27, or 33, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated;
and
a first polymer covalently attached to the introduced cystein residue S 146C.
1o In a further example the present invention relates to a conjugate
comprising an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
15 indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
S146C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 18,
20 or 27, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first polymer covalently attached to the introduced cystein residue S 146C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular
domain comprises an amino acid sequence from position A108 to N244 as
indicated in seq id no 1, and
25 wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no l, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated,
wherein the adiponectin
polypeptide fragment comprises the mutation N141C; and
a first polymer covalently attached to the introduced cystein residue.
30 In a further example the present invention relates to a conjugate
comprising an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
35 indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N141C; and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 36,
45, or 51, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated;
and
a first polymer covalently attached to the introduced cystein residue N141C.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position RI00 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N141C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
15 comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 36,
or 45, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first polymer covalently attached to the introduced cystein residue N141 C.
In a further example the present invention relates to a conjugate comprising
2o an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
25 glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T243C; and
a first polymer covalently attached to the introduced cystein residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
30 indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position RI00 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C; and
a first polymer covalently attached to the introduced cystein residue.
35 In a further example the present invention relates to a conjugate
comprising an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
6I
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 19,
or 28, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
a first polymer covalently attached to the introduced cystein residue T243C.
In a further example the present invention relates to a conjugate comprising
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no I, and wherein the collagen domain comprises from 7
amino acids corresponding
15 to position K101 as indicated in seq id no 1 to 56 amino acids
corresponding to position A52 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N228C; and
a first polymer covalently attached to the introduced cystein residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
zo polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
25 glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C; and
a first polymer covalently attached to the introduced cystein residue.
In a further example the present invention relates to a conjugate comprising
an adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
3o indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C; and
a first polymer covalently attached to the introduced cystein residue. Such as
a conjugate
35 comprising
an adiponectin polypeptide fragment having a sequence selected from any one of
seq id no 37,
or 46, and wherein the collagen domain comprises a lysine which is
hydroxylated and glycosylated; and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
62
a first polymer covalently attached to the introduced cystein residue N228C.
In a further embodiment the polymer or first polymer has a molecular weight of
from lkDa to
200kDa (kDa is a well known abbreviation and means kilo Dalton). In a still
further embodiment the
polymer has a molecular weight of from 2kDa to 95kDa. In a still further
embodiment the polymer has a
molecular weight of from SkDa to 80kDa. In a still further embodiment the
polymer has a molecular
weight of from 5 or l2kDa to 60kDa, such as 5-20 kDa, 12-40 kDa, 20-40 kDa, 5
kDa, 12 kDa, or 20
kDa. In a further embodiment the polymer or first polymer molecule is selected
from the group
consisting of mPEG(MAL), mPEG2(MAL), PEG-vinylsulphone, mPEG-OPSS, OPSS-PEG-
hydrazide
in combination with mPEG-ALD.
l0 To attach for instance, a polymer, such as a PEG to an introduced Cys the
OPSS and VS
chemistries, e.g. as described in the examples, are suitable. Suitable methods
of preparing the conjugate,
such as attaching a polymer, is described in the section "Methods of preparing
a conjugate of the
invention".
In a further embodiment the polymer or first polymer molecule is selected from
the group
IS consisting of 5k-mPEG(MAL), Sk-mPEG-OPSS, lOk-mPEG-OPSS, 20k-mPEG-OPSS, 20k-
mPEG(MAL), 40k-mPEG2(MAL), OPSS-PEGZk-hydrazide in combination with mPEG3o,~-
ALD.
In a further embodiment the polymer molecule is selected from the group
consisting of SS-PEG,
NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SBA, PEG-SCM, mPEG-BTC (All available
from
Shearwater), and SC-PEG.
2o In a further embodiment the polymer molecule is selected from the group
consisting of Sk-PEG-
SCM, 12k-PEG-SCM, 20k-PEG-SCM, Sk-PEG-SPA, 12k-PEG-SPA, 20k-PEG-SPA. (All
available
from Shearwater).
In the situation where it is decided to introduce a glycosylation site in the
adiponectin
polypeptide, in order to attach a sugar moiety, such sugar moiety is comprised
within the term first non-
25 polypeptide moiety. Accordingly, such particular aspect of the invention
relates to a conjugate
comprising an adiponectin polypeptide, and a sugar moiety covalently attached
to the adiponectin
polypeptide, wherein the adiponectin polypeptide comprises an amino acid
residue having an attachment
group for said sugar moiety, wherein said amino acid residue has been
introduced in a position that in
the parent adiponectin is occupied by a surface exposed amino acid residue.
Moreover, since the
30 introduction of the amino acid residue in a position that in the parent
adiponectin is occupied by a
surface exposed amino acid residue will lead to a novel polypeptide, then a
still further aspect of the
invention relates to an adiponectin polypeptide comprising an amino acid
residue having an attachment
group for a sugar moiety, wherein said amino acid residue has been introduced
in a position that in the
parent adiponectin is occupied by a surface exposed amino acid residue.
Moreover, the introduction of a
35 glycosylation site is preferably done in the globular domain in order not
to disturb the collagen structure.
Typically, the surface exposed amino acid residue is selected from A108, Y109,
V110, Y111, 8112,
L119, E120, T121, Y122, V123, T124, I125, P126, N127, M128, I130, 8131, T133,
K134, I135, F136,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
63
Y137, N138, Q139, Q140, N141, H142, D144, 6145, S146, T147, K149, H151, N153,
I154, P155,
Y159, A161, I164, T165, Y167, M168, K169, D170, V171, K172, F176, K177, K178,
D179, K180,
A181, M182, F184, T185, Y186, Q188, Y189, Q190, E191, N192, N193, V194, Q196,
5198, 6199,
5200, H204, E206, V207, 6208, D209, Q210, W212, Q214, V215, Y216, 6217, E218,
6219, E220,
8221, N222, L224, Y225, D227, N228, D229, N230, D231, T233, H241, D242, T243,
or N244 relative
to seq id no 1.
Any one of the above positions which have been identified as surface exposed
amino acid
residues may be substituted with an amino acid residue having an attachment
group for the sugar
moiety. The attachment group for the sugar moiety is selected from an N- or O-
glycosylation site. As
1 o described above the N-glycosylation site must have the pattern N-X'-S/T/C-
X", wherein X' and X" are
as defined above.
Thus, in one embodiment the attachment group is selected from an O-
glycosylation site. In
particular, the adiponectin polypeptide comprises a mutation selected from any
one of A108T/S,
Y109T/S, V110T/S, Y111T/S, R112T/S, L119T/S, E120T/S, T121S, Y122T/S, V123T/S,
T124S,
I125T/S, P126T/S, N127T/S, M128T/S, I130T/S, R131T/S, T133S, K134T/S, I135T/S,
F136T/S,
Y137T/S, N138T/S, Q139T/S, Q140T/S, N141T/S, H142T/S, D144T/S, G145T/S, S146T,
T147S,
K149T/S, H151T/S, N153T/S, I154T/S, P155T/S, Y159T/S, A161T/S, H163T/S,
I164T/S, T165S,
Y167T/S, M168T/S, K169T/S, D170T/S, V171T/S, K172T/S, F176T/S, K177T/S,
K178T/S, D179T/S,
K180T/S, A181T/S, M182T/S, F184T/S, T185S, Y186T/S, D187T/S, Q188T/S, Y189T/S,
Q190T/S,
2o E191T/S, N192T/S, N193T/S, V194T/S, D195T/S, Q196T/S, S198T, G199T/S,
S200T, H204T/S,
E206T/S, V207T/S, G208T/S, D209T/S, Q210T/S, W212T/S, Q214T/S, V215T/S,
Y216T/S, G217T/S,
E218T/S, G219T/S, E220T/S, R221T/S, N222T/S, G223T/S, L224T/S, Y225T/S,
A226T/S, D227T/S,
N228T/S, D229T/S, N230T/S, D231T/S, T233S, F234T/S, F237T/S, H241T/S, D242T/S,
T243S, or
N244T/S relative to seq id no l, preferably A108T/S, Y109T/S, V110T/S,
Y111T/S, R112T/S, L119T/S,
E120T/S, T121S, Y122T/S, V123T/S, T124S, I125T/S, P126T/S, N127T/S, M128T/S,
I130T/S,
R131T/S, T133S, K134T/S, I135T/S, F136T/S, Y137T/S, N138T/S, Q139T/S, Q140T/S,
N141T/S,
H142T/S, D144T/S, G145T/S, S146T, T147S, K149T/S, H151T/S, N153T/S, I154T/S,
P155T/S,
Y159T/S, A161T/S, I164T/S, T165S, Y167T/S, M168T/S, K169T/S, D170T/S, V171T/S,
K172T/S,
F176T/S, K177T/S, K178T/S, D179T/S, K180T/S, A181T/S, M182T/S, F184T/S, T185S,
Y186T/S,
3o Q188T/S, Y189T/S, Q190T/S, E191T/S, N192T/S, N193T/S, V194T/S, Q196T/S,
G199T/S, S200T,
H204T/S, E206T/S, V207T/S, G208T/S, D209T/S, Q210T/S, W212T/S, Q214T/S,
V215T/S, Y216T/S,
G217T/S, E218T/S, G219T/S, E220T/S, R221T/S, N222T/S, L224T/S, Y225T/S,
D227T/S, N228T/S,
D229T/S, N230T/S, D231T/S, T233S, H241T/S, D242T/S, T243S, or N244T/S. T/S
means either T or
S, T is preferred, eg. D242T/S means D242T or D242S, where D242T is preferred.
Each of these
mutations constitutes an individual embodiment and may be the subject of a
claim in combination with
any one of the above adiponectin polypeptides, such as any one of the
sequences seq id no 3, 4, 5, 6, 10,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
64
11, 12, or 13, or any one of the adiponectin polypeptide fragments described
in the above section
"Adiponectin polypeptide fragments) of the invention".
Further, in a particular embodiment the invention relates to a conjugate
comprising an
adiponectin polypeptide, wherein the adiponectin polypeptide comprises a
mutation selected from any
one of A108T/S, Y109T/S, V110T/S, Y111T/S, R112T/S, L119T/S, E120T/S, T121S,
Y122T/S,
V123T/S, T124S, I125T/S, P126T/S, N127T/S, M128T/S, I130T/S, R131T/S, T133S,
K134T/S,
I135T/S, F136T/S, Y137T/S, N138T/S, Q139T/S, Q140T/S, N141T/S, H142T/S,
D144T/S, G145T/S,
S146T, T147S, K149T/S, H151T/S, N153T/S, I154T/S, P155T/S, Y159T/S, A161T/S,
I164T/S, T165S,
Y167T/S, M168T/S, K169T/S, D170T/S, V171T/S, K172T/S, F176T/S, K177T/S,
K178T/S, D179T/S,
1o K180T/S, A181T/S, M182T/S, F184T/S, T185S, Y186T/S, Q188T/S, Y189T/S,
Q190T/S, E191T/S,
N192T/S, N193T/S, V194T/S, Q196T/S, G199T/S, S200T, H204T/S, E206T/S, V207T/S,
G208T/S,
D209T/S, Q210T/S, W212T/S, Q214T/S, V215T/S, Y216T/S, G217T/S, E218T/S,
G219T/S, E220T/S,
R221T/S, N222T/S, L224T/S, Y225T/S, D227T/S, N228T/S, D229T/S, N230T/S,
D231T/S, T233S,
H241T/S, D242T/S, T243S, or N244T/S relative to seq id no 1; and a sugar
moiety covalently attached
to the introduced O-glycosylation site. Moreover, since the introduction of
the amino acid residue in a
position that in the parent adiponectin is occupied by a surface exposed amino
acid residue will lead to a
novel polypeptide, then in a still further embodiment the invention relates to
an adiponectin polypeptide
comprising a mutation selected from any one of A108T/S, Y109T/S, V110T/S,
Y111T/S, R112T/S,
L119T/S, E120T/S, T121S, Y122T/S, V123T/S, T124S, I125T/S, P126T/S, N127T/S,
M128T/S,
2o I130T/S, R131T/S, T133S, K134T/S, I135T/S, F136T/S, Y137T/S, N138T/S,
Q139T/S, Q140T/S,
N141T/S, H142T/S, D144T/S, G145T/S, S146T, T147S, K149T/S, H151T/S, N153T/S,
I154T/S,
P155T/S, Y159T/S, A161T/S, I164T/S, T165S, Y167T/S, M168T/S, K169T/S, D170T/S,
V171T/S,
K172T/S, F176T/S, K177T/S, K178T/S, D179T/S, K180T/S, A181T/S, M182T/S;
F184T/S, T185S,
Y186T/S, Q188T/S, Y189T/S, Q190T/S, E191T/S, N192T/S, N193T/S, V194T/S,
Q196T/S, G199T/S,
S200T, H204T/S, E206T/S, V207T/S, G208T/S, D209T/S, Q210T/S, W212T/S, Q214T/S,
V215T/S,
Y216T/S, G217T/S, E218T/S, G219T/S, E220T/S, R221T/S, N222T/S, L224T/S,
Y225T/S, D227T/S,
N228T/S, D229T/S, N230T/S, D231T/S, T233S, H241T/S, D242T/S, T243S, or N244T/S
relative to seq
id no 1, T is preferred.
Thus, in another embodiment the attachment group is selected from an N-
glycosylation site. In
particular, the adiponectin polypeptide comprises a mutation selected from any
one of
A108N+V110T/S, Y109N+y111T/S, V110N+R112T/S, Y111N, Y111N+S113T,
R112N+A114T/S,
L119N+T121S, L119N, E120N+Y122T/S, T121N+V123T/S, Y122N, Y122N+T124S,
V123N+I125T/S, T124N+p126T/S, P126N+M128T/S, P129T/S, M128N+I130T/S,
I130N+F132T/S,
R131N, R131N+T133S, T133N+I135T/S, K134N+F136T/S, I135N+y137T/S,
F136N+N138T/S,
Y137N+Q139T/S, Q140T/S, Q139N+N141T/S, Q140N+H142T/S, Y143T/S, H142N+D144T/S,
D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T/S, T147N+K149T/S,
K149N+H151T/S, H151N+N153T/S, P155T/S, P155N+L157T/S, Y159N+A161T/S,
A161N+H163T/S,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
H163N, H163N+T165S, I164N+V166T/S, T165N+Y167T/S, Y167N+K169T/S,
M168N+D170T/S,
K169N+V171T/S, D170N+K172T/S, V171N+V173T/S, K172N, K172N+S174T,
F176N+K178T/S,
K177N+D179T/S, K178N+K180T/S, D179N+A181T/S, K180N+M182T/S, A181N+L183T/S,
M182N+F184T/S, F184N+y186T/S, T185N+D187T/S, Y186N+Q188T/S, D187N+Y189T/S,
5 Q188N+Q190T/S, Y189N+E191T/S, Q190N+N192T/S, E191N+N193T/S, V194T/S,
D195T/S,
V194N+Q196T/S, D195N+A197T/S, Q196N, Q196N+S198T, S198N, S198N+S200T,
G199N+V201T/S, S200N+L202T/S, H204N+E206T/S, E206N+G208T/S, V207N+D209T/S,
G208N+Q210T/S, D209N+V211T/S, Q210N+W212T/S, W212N+Q214T/S, Q214N+Y216T/S,
V215N+G217T/S, Y216N+E218T/S, G217N+G219T/S, E218N+E220T/S, G219N+R221T/S,
1o E220N+N222T/S, R221N+G223T/S, L224T/S, G223N+Y225T/S, L224N+A226T/S,
Y225N+D227T/S,
A226N+N228T/S, D227N+D229T/S, N230T/S, D229N+D231T/S, S232T, D231N,
D231N+T233S,
T233N, T233N+T235S, F234N+G236T/S, F237N+L239T/S, H241N, H241N+T243S, or
D242N+N244T/S relative to seq id no 1, such as A108N+V110T/S, Y109N+Y111T/S,
V110N+R112T/S, Y111N, Y111N+S113T, R112N+A114T/S, L119N+T121S, L119N,
15 E120N+y122T/S, T121N+V123T/S, Y122N, Y122N+T124S, T124N+p126T/S,
P126N+M128T/S,
P129T/S, M128N+I130T/S, I130N+F132T/S, R131N, R131N+T133S, T133N+I135T/S,
K134N+F136T/S, I135N+y137T/S, F136N+N138T/S, Y137N+Q139T/S, Q140T/S,
Q139N+N141T/S,
Q140N+H142T/S, Y143T/S, H142N+D144T/S, D144N, D144N+S146T, G145N, G145N+T147S,
S146N+G148T/S, T147N+K149T/S, K149N+H151T/S, H151N+N153T/S, P155T/S,
P155N+L157T/S,
2o Y159N+A161T/S, I164N+V166T/S, T165N+Y167T/S, Y167N+K169T/S, M168N+D170T/S,
K169N+V171T/S, D170N+K172T/S, V171N+V173T/S, K172N, K172N+S174T,
F176N+K178T/S,
K177N+D179T/S, K178N+K180T/S, D179N+A181T/S, K180N+M182T/S, A181N+L183T/S,
M182N+F184T/S, F184N+y186T/S, Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S,
Q190N+N192T/S, E191N+N193T/S, V194T/S, V194N+Q196T/S, Q196N, Q196N+S198T,
25 G199N+V201T/S, S200N+L202T/S, H204N+E206T/S, E206N+G208T/S, V207N+D209T/S,
G208N+Q210T/S, D209N+V211T/S, Q210N+W212T/S, W212N+Q214T/S, Q214N+Y216T/S,
V215N+G217T/S, Y216N+E218T/S, G217N+G219T/S, E218N+E220T/S, G219N+R221T/S, or
E220N+N222T/S, e.g. A108N+V110T, Y109N+Y111T, V110N+R112T, Y111N, Y111N+S113T,
R112N+A114T, L119N+T121S, L119N, E120N+y122T, T121N+V123T, Y122N, Y122N+T124S,
3o T124N+P126T, P126N+M128T, P129T, M128N+I130T, I130N+F132T, R131N,
R131N+T133S,
T133N+I135T, K134N+F136T, I135N+Y137T, F136N+N138T, Y137N+Q139T, Q140T,
Q139N+N141T, Q140N+H142T, Y143T, H142N+D144T, D144N, D144N+S146T, G145N,
G145N+T147S, S146N+G148T, T147N+K149T, K149N+H151T, H151N+N153T, P155T,
P155N+L157T, Y159N+A161T, I164N+V166T, T165N+y167T, Y167N+K169T, M168N+D170T,
35 K169N+V171T, D170N+K172T, V171N+V173T, K172N, K172N+S174T, F176N+K178T,
K177N+D179T, K178N+K180T, D179N+A181T, K180N+M182T, A181N+L183T, M182N+F184T,
F184N+y186T, Y186N+Q188T, Q188N+Q190T, Y189N+E191T, Q190N+N192T, E191N+N193T,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
66
V 194T, V 194N+Q 196T, Q 196N, Q 196N+S 198T, G 199N+V201 T, S200N+L202T,
H204N+E206T,
E206N+G208T, V207N+D209T, G208N+Q210T, D209N+V211T, Q210N+W212T, W212N+Q214T,
Q214N+Y216T, V215N+G217T, Y216N+E218T, G217N+G219T, E218N+E220T, G219N+R221T,
or
E220N+N222T. Each of these mutations constitutes an individual embodiment and
may be the subject
of a claim in combination with any one of the above adiponectin polypeptides,
such as any one of the
sequences seq id no 3, 4, 5, 6, 10, 11, 12, or 13, or any one of the
adiponectin polypeptide fragments
described in the above section "Adiponectin polypeptide fragments) of the
invention".
Further, in a particular embodiment the invention relates to a conjugate
comprising an
adiponectin polypeptide, wherein the adiponectin polypeptide comprises a
mutation selected from any
one of A108N+V110T/S, Y109N+Y111T/S, V110N+R112T/S, Y111N, Y111N+S113T,
R112N+A114T/S, L119N+T121S, L119N, E120N+Y122T/S, T121N+V123T/S, Y122N,
Y122N+T124S, T124N+P126T/S, P126N+M128T/S, P129T/S, M128N+I130T/S,
I130N+F132T/S,
R131N, R131N+T133S, T133N+I135T/S, K134N+F136T/S, I135N+Y137T/S,
F136N+N138T/S,
Y137N+Q139T/S, Q140T/S, Q139N+N141T/S, Q140N+H142T/S, Y143T/S, H142N+D144T/S,
D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T/S, T147N+K149T/S,
K149N+H151T/S, H151N+N153T/S, P155T/S, P155N+L157T/S, Y159N+A161T/S,
I164N+V166T/S,
T165N+Y167T/S, Y167N+K169T/S, M168N+D170T/S, K169N+V171T/S, D170N+K172T/S,
V171N+V173T/S, K172N, K172N+S174T, F176N+K178T/S, K177N+D179T/S,
K178N+K180T/S,
D179N+A181T/S, K180N+M182T/S, A181N+L183T/S, M182N+F184T/S, F184N+Y186T/S,
2o Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S, Q190N+N192T/S, E191N+N193T/S,
V194T/S, V194N+Q196T/S, Q196N, Q196N+S198T, G199N+V201T/S, S200N+L202T/S,
H204N+E206T/S, E206N+G208T/S, V207N+D209T/S, G208N+Q210T/S, D209N+V211T/S,
Q210N+W212T/S, W212N+Q214T/S, Q214N+Y216T/S, V215N+G217T/S, Y216N+E218T/S,
G217N+G219T/S, E218N+E220T/S, G219N+R221T/S, or E220N+N222T/S relative to seq
id no 1; and
a sugar moiety covalently attached to the introduced N-glycosylation site.
Moreover, since the
introduction of the N-glycosylation site in a position that in the parent
adiponectin is occupied by a
surface exposed amino acid residue will lead to a novel polypeptide, then in a
still further embodiment
the invention relates to an adiponectin polypeptide comprising a mutation
selected from any one of
A108N+V110T/S, Y109N+Y111T/S, V110N+R112T/S, Y111N, Y111N+S113T,
R112N+A114T/S,
3o L119N+T121S, L119N, E120N+Y122T/S, T121N+V123T/S, Y122N, Y122N+T124S,
T124N+p126T/S, P126N+M128T/S, P129T/S, M128N+I130T/S, I130N+F132T/S, R131N,
R131N+T133S, T133N+I135T/S, K134N+F136T/S, I135N+Y137T/S, F136N+N138T/S,
Y137N+Q139T/S, Q140T/S, Q139N+N141T/S, Q140N+H142T/S, Y143T/S, H142N+D144T/S,
D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T/S, T147N+K149T/S,
K149N+H151T/S, H151N+N153T/S, P155T/S, P155N+L157T/S, Y159N+A161T/S,
I164N+V166T/S,
T165N+y167T/S, Y167N+K169T/S, M168N+D170T/S, K169N+V171T/S, D170N+K172T/S,
V171N+V173T/S, K172N, K172N+S174T, F176N+K178T/S, K177N+D179T/S,
K178N+K180T/S,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
67
D179N+A181T/S, K180N+M182T/S, A181N+L183T/S, M182N+F184T/S, F184N+Y186T/S,
Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S, Q190N+N192T/S, E191N+N193T/S,
V194T/S, V194N+Q196T/S, Q196N, Q196N+S198T, G199N+V201T/S, S200N+L202T/S,
H204N+E206T/S, E206N+G208T/S, V207N+D209T/S, G208N+Q210T/S, D209N+V211T/S,
Q210N+W212T/S, W212N+Q214T/S, Q214N+Y216T/S, V215N+G217T/S, Y216N+E218T/S,
G217N+G219T/S, E218N+E220T/S, G219N+R221 T/S, or E220N+N222T/S relative to seq
id no 1.
Typically, the adiponectin polypeptide wherein a glycosylation site is
introduced is selected
from any one of the sequences seq id no 3, 4, 5, 6, 10, 11, 12, or 13, or any
one of the adiponectin
polypeptide fragments described in the above section "Adiponectin polypeptide
fragments) of the
t o invention", such adiponectin polypeptide is preferably expressed in a
eucaryotic cell, such as a
mammalian cell, and in this respect will be conjugated to a sugar moiety. To
illustrate this a few
embodiments are outlined hereafter.
Accordingly, the invention relates to an adiponectin polypeptide selected from
any one of the
sequences seq id no 3, 4, 5, 6, 10, 11, 12, or 13, such as seq id no 10, or
seq id no 11, comprising a
mutation selected from any one of A108N+V110T/S, Y109N+Y111T/S, V110N+R112T/S,
Y111N,
Y111N+S113T, R112N+A114T/S, L119N+T121S, L119N, E120N+Y122T/S, T121N+V123T/S,
Y122N, Y122N+T124S, T124N+P126T/S, P126N+M128T/S, P129T/S, M128N+I130T/S,
I130N+F132T/S, R131N, R131N+T133S, T133N+I135T/S, K134N+F136T/S,
I135N+Y137T/S,
F136N+N138T/S, Y137N+Q139T/S, Q140T/S, Q139N+N141T/S, Q140N+H142T/S, Y143T/S,
2o H142N+D144T/S, D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T/S,
T147N+K149T/S, K149N+H151T/S, H151N+N153T/S, P155T/S, P155N+L157T/S,
Y159N+A161T/S,
I164N+V166T/S, T165N+Y167T/S, Y167N+K169T/S, M168N+D170T/S, K169N+V171T/S,
D170N+K172T/S, V171N+V173T/S, K172N, K172N+S174T, F176N+K178T/S,
K177N+D179T/S,
K178N+K180T/S, D179N+A181T/S, K180N+M182T/S, A181N+L183T/S, M182N+F184T/S,
F184N+Y186T/S, Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S, Q190N+N192T/S,
E 191N+N193T/S, V 194T/S, V 194N+Q 196T/S, Q 196N, Q 196N+S 198T, G 199N+V201
T/S,
S200N+L202T/S, H204N+E206T/S, E206N+G208T/S, V207N+D209T/S, G208N+Q210T/S,
D209N+V211T/S, Q210N+W212T/S, W212N+Q214T/S, Q214N+y216T/S, V215N+G217T/S,
Y216N+E218T/S, G217N+G219T/S, E218N+E220T/S, G219N+R221 T/S, or E220N+N222T/S
relative
3o to seq id no l, such as a mutation selected from any one of A108N+V110T/S,
Y109N+Y111T/S,
V110N+R112T/S, Y111N, Y111N+S113T, R112N+A114T/S, L119N+T121S, L119N,
E120N+Y122T/S, T121N+V123T/S, Y122N, Y122N+T124S, T124N+P126T/S,
P126N+M128T/S,
P129T/S, M128N+I130T/S, I130N+F132T/S, R131N, R131N+T133S, T133N+I135T/S,
K134N+F136T/S, I135N+Y137T/S, F136N+N138T/S, Y137N+Q139T/S, Q140T/S,
Q139N+N141T/S,
Q140N+H142T/S, Y143T/S, H142N+D144T/S, D144N, D144N+S146T, G145N, G145N+T147S,
S146N+G148T/S, T147N+K149T/S, K149N+H151T/S, H151N+N153T/S, P155T/S,
P155N+L157T/S,
Y159N+A161T/S, I164N+V166T/S, T165N+Y167T/S, Y167N+K169T/S, M168N+D170T/S,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
68
K169N+V171T/S, D170N+K172T/S, V171N+V173T/S, K172N, K172N+S174T,
F176N+K178T/S,
K177N+D179T/S, K178N+K180T/S, D179N+A181T/S, K180N+M182T/S, A181N+L183T/S,
M182N+F184T/S, F184N+Y186T/S, Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S,
Q190N+N192T/S, E191N+N193T/S, V194T/S, V194N+Q196T/S, Q196N, Q196N+S198T,
s G199N+V201 T/S, S200N+L202T/S, or H204N+E206T/S, in particular a mutation
selected from any
one ofA108N+V110T/S, Y109N+y111T/S, V110N+R112T/S, Y111N, Y111N+S113T,
R112N+A114T/S, L119N+T121S, L119N, E120N+y122T/S, T121N+V123T/S, Y122N,
Y122N+T124S, T124N+P126T/S, P126N+M128T/S, P129T/S, M128N+I130T/S,
I130N+F132T/S,
R131N, R131N+T133S, T133N+I135T/S, K134N+F136T/S, I135N+Y137T/S,
F136N+N138T/S,
Y137N+Q139T/S, Q140T/S, Q139N+N141T/S, Q140N+H142T/S, Y143T/S, H142N+D144T/S,
D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T/S, T147N+K149T/S,
K149N+H151T/S, H151N+N153T/S, P155T/S, P155N+L157T/S, Y159N+A161T/S,
I164N+V166T/S,
T165N+y167T/S, Y167N+K169T/S, M168N+D170T/S, K169N+V171T/S, D170N+K172T/S,
V171N+V173T/S, K172N, K172N+S174T, F176N+K178T/S, K177N+D179T/S,
K178N+K180T/S,
D179N+A181T/S, K180N+M182T/S, A181N+L183T/S, M182N+F184T/S, F184N+y186T/S,
Y186N+Q188T/S, Q188N+Q190T/S, Y189N+E191T/S, Q190N+N192T/S, E191N+N193T/S,
V 194T/S, or V 194N+Q 196T/S, preferably a mutation selected from any one of
A108N+V 1 l OT,
Y109N+Y111T, V110N+R112T, Y111N, Y111N+S113T, R112N+A114T, L119N+T121S, L119N,
E120N+y122T, T121N+V123T, Y122N, Y122N+T124S, T124N+P126T, P126N+M128T, P129T,
2o M128N+I130T, I130N+F132T, R131N, R131N+T133S, T133N+I135T, K134N+F136T,
I135N+Y137T, F136N+N138T, Y137N+Q139T, Q140T, Q139N+N141T, Q140N+H142T, Y143T,
H142N+D144T, D144N, D144N+S146T, G145N, G145N+T147S, S146N+G148T, T147N+K149T,
K149N+H151T, H151N+N153T, P155T, P155N+L157T, Y159N+A161T, I164N+V166T,
T165N+Y167T, Y167N+K169T, M168N+D170T, K169N+V171T, D170N+K172T, V171N+V173T,
K172N, K172N+S174T, F176N+K178T, K177N+D179T, K178N+K180T, D179N+A181T,
K180N+M182T, A181N+L183T, M182N+F184T, F184N+y186T, Y186N+Q188T, Q188N+Q190T,
Y189N+E191T, Q190N+N192T, E191N+N193T, V194T, or V194N+Q196T. In a further
embodiment
the invention relates to an adiponectin polypeptide selected from the sequence
seq id no 10 comprising a
mutation selected from any one of Y111N, Y122N, P129T, R131N, D144N+S146T,
G145N,
3o H151N+N153T, P155T, K178N+K180T, such as Y111N, Y122N, R131N, D144N+S146T,
H151N+N153T, K178N+K180T. In a further embodiment the invention relates to an
adiponectin
polypeptide selected from the sequence seq id no 10 comprising a mutation
selected from Y111N, such
as the adiponectin polypeptide having the seq id no 53. In a further
embodiment the invention relates to
an adiponectin polypeptide selected from the sequence seq id no 10 comprising
a mutation selected from
Y122N, such as the adiponectin polypeptide having the seq id no 54. In a
further embodiment the
invention relates to an adiponectin polypeptide selected from the sequence seq
id no 10 comprising a
mutation selected from R131N, such as the adiponectin polypeptide having the
seq id no 55. In a further
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
69
embodiment the invention relates to an adiponectin polypeptide selected from
the sequence seq id no 10
comprising a mutation selected from D144N+S146T, such as the adiponectin
polypeptide having the seq
id no 56. In a further embodiment the invention relates to an adiponectin
polypeptide selected from the
sequence seq id no 10 comprising a mutation selected from H151N+N153T, such as
the adiponectin
polypeptide having the seq id no 57. In a further embodiment the invention
relates to an adiponectin
polypeptide selected from the sequence seq id no 10 comprising a mutation
selected from
K178N+K180T, such as the adiponectin polypeptide having the seq id no 58. In a
further embodiment
the invention relates to an adiponectin polypeptide selected from the sequence
seq id no 10 comprising a
mutation selected from P129T, such as the adiponectin polypeptide having the
seq id no 59. In a fiu-ther
1 o embodiment the invention relates to an adiponectin polypeptide selected
from the sequence seq id no 10
comprising a mutation selected from G145N, such as the adiponectin polypeptide
having the seq id no
60. In a further embodiment the invention relates to an adiponectin
polypeptide selected from the
sequence seq id no 10 comprising a mutation selected from P155T, such as the
adiponectin polypeptide
having the seq id no 61. In a further embodiment the invention relates to an
adiponectin polypeptide
15 selected from the sequence seq id no 11 comprising a mutation selected from
any one of Y111N,
Y122N, P129T, R131N, D144N+S146T, G145N, H151N+N153T, P155T, K178N+K180T, such
as
Y111N, Y122N, R131N, D144N+S146T, H151N+N153T, K178N+K180T. In a further
embodiment the
invention relates to an adiponectin polypeptide selected from the sequence seq
id no 11 comprising a
mutation selected from Y111N. In a further embodiment the invention relates to
an adiponectin
2o polypeptide selected from the sequence seq id no 11 comprising a mutation
selected from Y122N. In a
further embodiment the invention relates to an adiponectin polypeptide
selected from the sequence seq
id no 11 comprising a mutation selected from R131N. In a further embodiment
the invention relates to
an adiponectin polypeptide selected from the sequence seq id no 11 comprising
a mutation selected from
D 144N+S 146T. In a further embodiment the invention relates to an adiponectin
polypeptide selected
25 from the sequence seq id no 11 comprising a mutation selected from
H151N+N153T. In a further
embodiment the invention relates to an adiponectin polypeptide selected from
the sequence seq id no 11
comprising a mutation selected from K178N+K180T.
Hereafter, in connection with the introduction of an N-glycosylation site in
the adiponectin
polypeptide either conjugated to a sugar moiety or non-conjugated, the
mutation may be selected from
3o any one of the above mentioned N-glycosylation sites, and the adiponectin
polypeptide may be selected
from any one of the above mentioned adiponectin polypeptide fragments,
however, for illustrative
purposes only a small group of adiponectin polypeptide fragments and of N-
glycosylation sites will be
indicated.
Thus, the invention relates to an adiponectin polypeptide selected from an
adiponectin
35 polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular
domain comprises an amino acid sequence from position A108 to N244 as
indicated in seq id no 1, and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as indicated
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
in seq id no 1 to 56 amino acids corresponding to position A52 as indicated in
seq id no 1, and wherein
the collagen domain comprises a lysine which is hydroxylated and glycosylated,
the adiponectin polypeptide fragment comprising a mutation selected from any
one of Y111N,
Y122N, P129T, R131N, D144N+S146T, G145N, H151N+N153T, P155T, K178N+K180T. In a
further
embodiment the adiponectin polypeptide fragment comprises a globular domain
and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no l, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no l, and wherein the collagen domain comprises a lysine
which is hydroxylated and
to glycosylated.
Furthermore, the invention relates to a conjugate comprising an adiponectin
polypeptide,
wherein the adiponectin polypeptide has the sequence seq id no 10 comprising a
mutation selected from
any one of Y111N, Y122N, P129T, R131N, D144N+S146T, G145N, H151N+N153T, P155T,
or
K178N+K180T relative to seq id no 1; and a sugar moiety covalently attached to
the introduced N-
15 glycosylation site. In a further embodiment the invention relates to a
conjugate comprising an
adiponectin polypeptide, wherein the adiponectin polypeptide has the sequence
seq id no 11 comprising
a mutation selected from any one of Y111N, Y122N, P129T, R131N, D144N+S146T,
G145N,
H151N+N153T, P155T, or K178N+K180T relative to seq id no l; and a sugar moiety
covalently
attached to the introduced N-glycosylation site.
20 Moreover, the invention relates to a conjugate comprising
an adiponectin polypeptide fragment which comprises a globular domain and a
collagen
domain, wherein the globular domain comprises an amino acid sequence from
position A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
25 indicated in seq id no 1, and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, the adiponectin polypeptide fragment comprising a mutation
selected from any one of
Y111N, Y122N, P129T, R131N, D144N+S146T, G145N, H151N+N153T, P155T,
K178N+K180T;
and a sugar moiety covalently attached to the introduced N-glycosylation site.
The sugar moiety may be introduced by methods known to the person skilled in
the art, or as
3o suggested in the section "Methods of preparing a conjugate of the
invention" herein. Preferably, a
mammalian cell line is used to express the adiponectin polypeptide, such as a
CHO cell, BHK cell, or
HEK cell.
When the collagen domain comprises a lysine which is hydroxylated and
glycosylated, it may
comprise l, 2, 3, or 4 lysine(s), as explained in detail above.
35 Although, the adiponectin polypeptide may be modified to comprise more than
one introduced
glycosylation site in the globular domain it is preferred that no more than
four glycosylation sites are
introduced, that is, one to four N-glycosylation sites) or one to four O-
glycosylation sites) or mixtures
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
71
thereof, provided that no more than four glycosylation sites are introduced,
such as one N-glycosylation
site, two N-glycosylation sites, three N-glycosylation sites, four N-
glycosylation sites, three O-
glycosylation sites, four O-glycosylation sites, or one N-glycosylation site
and one O-glycosylation site.
In a more preferred embodiment the adiponectin polypeptide comprises at least
one introduced N-
glycosylation site, such as one introduced N-glycosylation site. The
adiponectin polypeptide may be
non-conjugated, or preferably, conjugated to a sugar moiety attached to the
introduced glycosylation
site, such as an N-glycosylation site.
In addition to the first non-polypeptide moiety, which is typically selected
from a polymer
attached to an amino acid residue such as a lysine, aspartic acid, glutamic
acid or cysteine residue, or a
1 o sugar moiety attached to an introduced N-glycosylation site, the
adiponectin polypeptide may optionally
also comprise a second non-polypeptide moiety which is different from the
first non-polypeptide moiety.
Thus, if for instance, the first non-polypeptide moiety is a polymer, then the
second non-polypeptide
moiety is typically a sugar moiety, or if the first non-polypeptide moiety is
a sugar moiety, then the
second non-polypeptide moiety is typically a polymer.
15 Accordingly, in a further embodiment the conjugate further comprises a
second non-polypeptide
moiety selected from the group consisting of a polymer molecule, a lipophilic
compound, a sugar moiety
and an organic derivatizing agent. The second non-polypeptide moiety is
different from the first non-
polypeptide moiety, however, the above embodiments described in connection
with the first non-
polypeptide moiety are also considered embodiments for the second non-
polypeptide moiety.
2o In a further embodiment the second non-polypeptide moiety is selected from
a polymer
molecule.
In a further embodiment the amino acid residue having the attachment group for
said second
non-polypeptide moiety is selected from a lysine, aspartic acid, glutamic acid
or cysteine residue, such
as a cysteine residue.
25 In a further embodiment the second non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
In a further embodiment the second non-polypeptide moiety is a polymer
molecule having a
sugar moiety as an attachment group.
In a further embodiment the polymer molecule is selected from the group
consisting of mPEG-
3o AMINE. (Available from Shearwater).
In a further embodiment the polymer molecule is selected from the group
consisting of Sk-
mPEG-AMINE. (Available from Shearwater)
In a further embodiment the amino acid sequence of the adiponectin polypeptide
further
comprises at least one removed lysine residue.
35 In a further embodiment one to four lysine residues selected from any one
of the positions K65,
K68, K77, or K101 of the collagen domain of human adiponectin is/are removed.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
72
In a further embodiment one to six lysine residues selected from any one of
the positions K134,
K149, K169, K172, K177, K178, or K180 of the globular domain of wild-type
human adiponectin is/are
removed.
Such lysine residues may be removed from the collagen and/or globular domain,
depending on
the length of the adiponectin polypeptide. The skilled person will understand
that the group of lysines to
select from will depend on whether the full collagen domain or only a fragment
thereof is present in the
adiponectin polypeptide, and thus whether the group of lysine residues are the
positions K65, K68, K77,
or K101 of the collagen domain of human adiponectin and positions K134, K149,
K169, K172, K177,
K178, or Kl 80 of the globular domain of human adiponectin, or a smaller
group, such as K77, or K101
t o of the collagen domain and positions K134, K149, K169, K172, K177, K178,
or K180 of the globular
domain, or even a smaller group, such as K101 of the collagen domain and
positions K134, K149, K169,
K172, K177, K178, or K180 of the globular domain. If desired to introduce a
second non-polypeptide
moiety by conjugating it to a lysine, then obviously, at least one lysine
should be present in the
adiponectin polypeptide in order to make possible the conjugation to a lysine.
Calcium composition aspects
We have shown that calcium ions are crucial for the adiponectin polypeptide to
form stable
trimers and that removal of such calcium ions leads to destabilization of the
trimer structure. No effect
could be seen with other divalent cations such as magnesium and zinc ions. The
destabilization of the
trimer structure leads to a heterogenous composition as shown in native gels.
The addition of calcium
ions to a liquid solution of adiponectin which had a destabilized trimer
structure lead to recovery of the
stable trimer structure. In particular we have shown that lowering pH in the
absence of calcium ions
destabilize the trimer structure, and that adding calcium ions leads to a
stable trimer. The stable trimer
structure has biological activity which may be tested in various in vitro or
in vivo models, such in vivo
models may be one of the recognized mouse models for testing insulin
sensitivity, or obesity. From our
experimental analysis of the structure of a human adiponectin fragment
(apMl(82-244) it has become
clear that D187, and D195, releative to seq id no 1 in the globular domain of
human adiponectin are
involved in the binding of calcium ions, and that mutation in one or both of
these positions results in
reduced affinity to calcium ions. Furthermore H163 is also believed to be
important for calcium binding.
'Thus, in order to maintain the calcium binding it is preferred to maintain
D187, and D195 releative to
seq id no 1, and more preferably D187, D195, and H163 should be maintained.
A typical way of testing biological activity is in the test Assay A, B, or C,
described in the
experimental section. The adiponectin polypeptide trimer will usually consist
of three identical
monomers, however, the trimer may also be heterogenous, for instance, two of
the monomers may be
the same and the third may be different, or all three monomers may be
different. The difference being
that one or two monomers) has/have an amino acid sequence that differs from
that of the other
monomer(s). Another difference could be in a sugar moiety, eg. in different
hydroxy-glycosylations in
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
73
the collagenous domain on each adiponectin polypeptide monomer. In case the
individual monomers are
identical but have different sugar moieties attached, this is intended to be
comprised within the term
"homotrimer". In case the adiponectin polypeptide trimer consist of three
identical monomers, that is
three identical amino acid sequences, it is referred to as a homotrimer. In a
further embodiment the
trimer is a heterotrimer. In a further embodiment the trimer is a homotrimer.
Thus, calcium ions stabilize the adiponectin polypeptide trimer and this
intimate assembly is
referred to herein as a complex.
Accordingly, in a broad aspect the present invention relates to an isolated
complex comprising
a) an adiponectin polypeptide or a conjugate comprising an adiponectin
polypeptide, and a first non-
1 o polypeptide moiety covalently attached to the adiponectin polypeptide, and
b) calcium ions.
In a further aspect the present invention relates to an isolated complex
comprising a) an
adiponectin polypeptide, and b) calcium ions.
In a further aspect the present invention relates to an isolated complex
comprising a) a conjugate
comprising an adiponectin polypeptide, and a first non-polypeptide moiety
covalently attached to the
15 adiponectin polypeptide, and b) calcium ions.
In an embodiment of the above aspects the adiponectin polypeptide is expressed
and recovered
from mammalian host cells. Preferred host cells are CHO, BHK, or HEK cells, in
particular CHO-Kl
and HEK293 cells.
In a further embodiment of the above aspects the adiponectin polypeptide is
expressed and
2o recovered from yeast cells.
In an alternative embodiment of the above aspects the adiponectin polypeptide
is expressed and
recovered from bacterial cells. Examples of bacterial host cells include
grampositive bacteria such as
strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or
S"treptomyces, or gramnegative bacteria,
such as strains of E. coli. A typically, embodiment is an E. Coli host cell.
25 In a further aspect the present invention relates to an isolated complex
comprising a) an
adiponectin polypeptide or a conjugate comprising an adiponectin polypeptide,
and a first non-
polypeptide moiety covalently attached to the adiponectin polypeptide, and b)
calcium ions, provided
that the adiponectin polypeptide is expressed and recovered from mammalian
host cells.
In a further aspect the present invention relates to an isolated complex
comprising a) an
30 adiponectin polypeptide or a conjugate comprising an adiponectin
polypeptide, and a first non-
polypeptide moiety covalently attached to the adiponectin polypeptide, and b)
calcium ions, provided
that the adiponectin polypeptide is expressed and recovered from bacterial
host cells.
Thus, the group under a) may be selected from an adiponectin polypeptide in
one embodiment
or from a conjugate comprising an adiponectin polypeptide, and a first non-
polypeptide moiety
35 covalently attached to the adiponectin polypeptide in another embodiment.
In particular human adiponectin (apMl) and fragments thereof, as well as
analogs thereof, such
as an adiponectin polypeptide comprising a globular domain having at least 80%
identity to the globular
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
74
domain of apMl (shown in sequence id no 6) and optionally comprising a
collagen domain or fragment
thereof, are preferred embodiments of the adiponectin polypeptide.
In one embodiment the adiponectin polypeptide is not full-length acrp30. In
another
embodiment the adiponectin polypeptide is not acrp30 fragment (104-247). In a
further embodiment the
adiponectin polypeptide is not acrp30 fragments. In a further embodiment the
adiponectin polypeptide is
not human full-length adiponectin. The human full-length adiponectin may be
purified from human
plasma or produced recombinantly from E. Coli cells.
The stable adiponectin polypeptide is a trimer wherein the trimer consists of
three monomers.
Thus, in a further embodiment the adiponectin polypeptide is a trimer
(adiponectin polypeptide trimer).
to Typically, the trimer is a homotrimer. However, the trimer may also be a
heterotrimer.
In a further aspect the present invention relates to a liquid composition
comprising an isolated
complex wherein the complex comprises a) an adiponectin polypeptide or a
conjugate comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, and b) calcium ions.
In a further aspect the present invention relates to a liquid composition
comprising an isolated
complex wherein the complex comprises a) an adiponectin polypeptide, and b)
calcium ions.
In a further aspect the present invention relates to a liquid composition
comprising an isolated
complex wherein the complex comprises a) a conjugate comprising an adiponectin
polypeptide, and a
first non-polypeptide moiety covalently attached'to the adiponectin
polypeptide, and b) calcium ions.
In a further aspect the present invention relates to a liquid composition
comprising an isolated
complex wherein the complex comprises a) an adiponectin polypeptide or a
conjugate comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, and b) calcium ions, provided that the adiponectin polypeptide is
expressed and recovered
from mammalian host cells.
In a further aspect the present invention relates to a liquid composition
comprising an isolated
complex wherein the complex comprises a) an adiponectin polypeptide or a
conjugate comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, and b) calcium ions, provided that the adiponectin polypeptide is
expressed and recovered
from bacterial host cells.
The liquid composition may be a solution or suspension, and may comprise a
buffer. However,
liquid solutions are preferred. Thus, in an embodiment the liquid composition
is a liquid solution, such
as an aqueous solution. In a further embodiment the liquid composition, such
as the liquid solution,
further comprises a buffer. The buffer may be any suitable buffer such as any
one of those mentioned
below in the section "Pharmaceutical composition and uses of a conjugate or
adiponectin polypeptide
fragment of the invention", however, care should be taken that if a phosphate
buffer is used then pH
should not be too low and preferably above 4, such as above 5, even more
preferably above 6. However,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
if calcium ions are added to the composition then the trimer structure will be
stable in a broad pH range,
such as from pH 2-10, preferably from 3-9.
In a further aspect the present invention relates to a pharmaceutical
composition comprising a)
an adiponectin polypeptide or a conjugate comprising an adiponectin
polypeptide, and a first non-
5 polypeptide moiety covalently attached to the adiponectin polypeptide, b)
calcium ions, and c) a
pharmaceutically acceptable Garner. In one embodiment such pharmaceutical
composition is a liquid
composition, such as a liquid solution. In a further embodiment the
pharmaceutical composition
comprises a buffer and has a pH from 2-10, provided that the buffer is not a
phosphate buffer. In another
embodiment the pharmaceutical composition comprises a buffer and has a pH from
4-10, such as 5-10,
1 o preferably 6-9. In a further embodiment the pharmaceutical composition
comprises a buffer and has a
pH from 2-10, such as 3-9, and calcium ions. Typically, a molar surplus of
calcium ions relative to the
adiponectin polypeptide is present in the composition.
The use of calcium ions to prepare an isolated complex comprising an
adiponectin polypeptide
or a conjugate comprising an adiponectin polypeptide, and a first non-
polypeptide moiety covalently
15 attached to the adiponectin polypeptide, provides a stable trimer of the
adiponectin polypeptide wherein
the complex has biological activity. Typically, such biological activity can
be measured in any one of
the test assays described in the experimental section, that is, Test Assay:
Determination of adiponectin's
effect on glucose uptake in C2C12 cells; or Test Assay: Measurement of
inhibition of LPS-induced
TNF-alpha production. Moreover, the effect of calcium ions to stabilize the
trimer structure of the
20 adiponectin polypeptide or the conjugate may be tested by reducing pH in a
phosphate containing buffer
in the absence or presence of calcium ions.
Thus, in a further aspect the present invention relates to use of calcium ions
to prepare an
isolated complex comprising an adiponectin polypeptide or a conjugate
comprising an adiponectin
polypeptide, and a first non-polypeptide moiety covalently attached to the
adiponectin polypeptide,
25 wherein the complex is able to inhibit LPS-induced TNF-alpha production, or
is able to enhance the
glucose uptake in muscle cells. Preferably, the complex is able to enhance the
glucose uptake in muscle
cells, in particular as described in the experimental section.
The adiponectin polypeptide may be prepared as described in the section below:
"Methods of
preparing an adiponectin polypeptide for use in the invention". Moreover, the
conjugate comprising an
3o adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, may be prepared as described in the section below: "Methods of
preparing a conjugate of
the invention".
In a further aspect the present invention relates to a method of preparing an
isolated complex
comprising a) an adiponectin polypeptide or a conjugate comprising an
adiponectin polypeptide, and a
35 first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, and b) calcium ions,
provided the adiponectin polypeptide is expressed and recovered from mammalian
host cells, the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
76
method comprising bringing calcium ions in contact with the adiponectin
polypeptide and optionally
reacting the adiponectin polypeptide with the first non-polypeptide moiety.
In a further aspect the present invention relates to a method of preparing an
isolated complex
comprising a) an adiponectin polypeptide or a conjugate comprising an
adiponectin polypeptide, and a
first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, and b) calcium ions,
provided the adiponectin polypeptide is expressed and recovered from bacterial
host cells, the method
comprising bringing calcium ions in contact with the adiponectin polypeptide
and optionally reacting the
adiponectin polypeptide with the first non-polypeptide moiety.
By making sure that calcium ions are present during the preparation of the
adiponectin
polypeptide or conjugate, such as in the culture medium, (such as DMEM/F-
12(1:1) medium Cat no
21041 (Invitrogen)) or suspension used, in the cells, or added to the
preparation (such as in the form of
calcium chloride (CaClz)), a stable trimer is obtained. In fact, during
culturing of the host cells the
presence of calcium ions will provide a stable trimer. Such stability may be
verified on native gels.
Preferably a medium containing calcium is used, such as DMEM/F-12(1:1) medium
Cat no
21041 (Invitrogen). However, media without calcium may also be used, such as
DMEM Cat no 21068
(Invitrogen), in which case calcium is preferably added to the preparation.
Accordingly, in a further aspect the present invention relates to a culture
comprising a) a
mammalian host cell expressing an adiponectin polypeptide, and b) calcium
ions.
In a further aspect the present invention relates to a culture comprising a) a
bacterial host cell
expressing an adiponectin polypeptide, and b) calcium ions.
In a further aspect the present invention relates to a method of preparing an
isolated complex
comprising a) an adiponectin polypeptide or a conjugate comprising an
adiponectin polypeptide, and a
first non-polypeptide moiety covalently attached to the adiponectin
polypeptide, and b) calcium ions,
comprising
a) preparing a nucleotide sequence encoding: a signal peptide and the
adiponectin polypeptide,
b) inserting the nucleotide sequence into a vector,
c) transfecting the vector into a mammalian cell,
d) expressing and optionally secreting the adiponectin polypeptide,
e) recovering the complex, and optionally
3o f) reacting the adiponectin polypeptide with the molecule to which it is to
be conjugated under
conditions conducive for the conjugation to take place, and recovering the
conjugate;
provided that any one of steps d), e), or f) is carried out in a calcium ion
rich environment.
The term "calcium ion rich environment" is intended to mean that calcium ions
(preferably in a
molar surplus relative to the adiponectin polypeptide) are present during the
preparation of the
adiponectin polypeptide, and in particular are present during any one of steps
d), e), or f), such as in the
culture medium or suspension used, or added during steps d), e), or f), (such
as in the form of calcium
chloride (CaClz)).
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
77
It is intended that the particular embodiments of the adiponectin polypeptide
and the conjugate
comprising an adiponectin polypeptide, and a first non-polypeptide moiety
covalently attached to the
adiponectin polypeptide mentioned in the above sections "Adiponectin
polypeptide fragments) of the
invention", "First group of conjugates) of the invention", "Second group of
conjugates) of the
invention", "Third group of conjugates) of the invention", and "Fourth group
of conjugates) of the
invention", also apply to this calcium composition aspect of the invention.
Moreover, when the non-
polypeptide is a polymer, then the embodiments mentioned in the above sections
in connection with a
polymer also applies to the polymer attached to the adiponectin polypeptide.
Thus, the embodiments
described below should not be seen as limiting this particular aspect of the
invention in any way.
t o Thus, in any one of the aspects of the calcium composition aspects
mentioned above the
adiponectin polypeptide or conjugate may be selected from the below
embodiments.
In an embodiment the adiponectin polypeptide is selected from any one of the
seq id no 2-8, 10-
12, or 13, and sequences having at least 80% identity to any one of seq id no
2-8, 10-12, or 13.
Typically, the adiponectin polypeptide is selected from any one of the seq id
no S, 10, 11, 12, or 13, and
15 sequences having at least 80% identity to any one of seq id no 5, 10, 11,
12, or 13.
In a further embodiment the adiponectin polypeptide is selected from any one
of the seq id no 2-
8, 10-12, or 13, and sequences having at least 90% identity to any one of seq
id no 2-8, 10-12, or 13.
Typically, the adiponectin polypeptide is selected from any one of the seq id
no 3, 4, 5, 6, 10, 11, 12, or
13, and sequences having at least 90% identity to any one of seq id no 3, 4,
5, 6, 10, 11, 12, or 13.
20 In a further embodiment the adiponectin polypeptide is selected from any
one of the seq id no 3,
4, 5, 6, 10, 11, 12, or 13, and sequences having at least 92% identity to any
one of seq id no 3, 4, 5, 6,
10, 11, 12, or 13. The percent identity as stated above is determined using
the CLUSTALW program.
In a further embodiment the adiponectin polypeptide is selected from any one
of the seq id no 2-
8, 10-12, or 13. In a further embodiment the adiponectin polypeptide is seq id
no 5. In a further
25 embodiment the adiponectin polypeptide is seq id no 10. In a further
embodiment the adiponectin
polypeptide is seq id no 11. In a further embodiment the adiponectin
polypeptide is seq id no 12. In a
further embodiment the adiponectin polypeptide is selected from any one of the
seq id no 13.
In a further embodiment the adiponectin polypeptide is selected from an
adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
3o wherein the globular domain comprises an amino acid sequence as indicated
in seq id no 1 from
position A108 to N244 as well as sequences that differs from the amino acid
sequence in one or more
substitution(s), and
wherein the collagen domain comprises from 7 amino acids corresponding to
position K101 as
indicated in seq id no 1 to 66 amino acids corresponding to position G42 as
indicated in seq id no 1, and
35 wherein the collagen domain comprises a lysine which is hydroxylated and
glycosylated.
In a further embodiment the adiponectin polypeptide is selected from an
adiponectin
polypeptide having an amino acid sequence selected from the seq id no 10,
wherein the adiponectin
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
78
polypeptide comprises the mutation T121C, such as the adiponectin polypeptide
having the amino acid
sequence of seq id no 17; in another embodiment the adiponectin polypeptide is
selected from an
adiponectin polypeptide having an amino acid sequence selected from the seq id
no 10, wherein the
adiponectin polypeptide comprises the mutation S 146C, such as the adiponectin
polypeptide having the
amino acid sequence of seq id no 18; in a further embodiment the adiponectin
polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 10, wherein
the adiponectin polypeptide comprises the mutation T243C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 19; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 10, wherein
to the adiponectin polypeptide comprises the mutation N127C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 35; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 10, wherein
the adiponectin polypeptide comprises the mutation N141C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 36; in a further embodiment the
adiponectin polypeptide is selected
15 from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 10, wherein
the adiponectin polypeptide comprises the mutation N228C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 37; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
the adiponectin polypeptide comprises the mutation T121 C, such as the
adiponectin polypeptide having
2o the amino acid sequence of seq id no 23; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
the adiponectin polypeptide comprises the mutation S 146C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 24; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
zs the adiponectin polypeptide comprises the mutation T243C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 25; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
the adiponectin polypeptide comprises the mutation N127C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 41; in a further embodiment the
adiponectin polypeptide is selected
3o from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
the adiponectin polypeptide comprises the mutation N141 C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 42; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 5, wherein
the adiponectin polypeptide comprises the mutation N228C, such as the
adiponectin polypeptide having
35 the amino acid sequence of seq id no 43; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
the adiponectin polypeptide comprises the mutation T121C, such as the
adiponectin polypeptide having
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
79
the amino acid sequence of seq id no 32; in a fizrther embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
the adiponectin polypeptide comprises the mutation S146C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 33; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
the adiponectin polypeptide comprises the mutation T243C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 34; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
the adiponectin polypeptide comprises the mutation N127C, such as the
adiponectin polypeptide having
1 o the amino acid sequence of seq id no 50; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
the adiponectin polypeptide comprises the mutation N141C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 51; in a further embodiment the
adiponectin polypeptide is selected
from an adiponectin polypeptide having an amino acid sequence selected from
the seq id no 13, wherein
t5 the adiponectin polypeptide comprises the mutation N228C, such as the
adiponectin polypeptide having
the amino acid sequence of seq id no 52.
In a further embodiment the adiponectin polypeptide is selected from an
adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence as indicated in
seq id no 1 from
20 position A108 to N244, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 66 amino acids corresponding
to position G42 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated,
wherein the adiponectin polypeptide fragment comprises a mutation selected
from any one of
25 A108C, Y109C, V110C, Y111C, R112C, E120C, T121C, Y122C, V123C, T124C,
I125C, P126C,
N127C, M128C, R131C, T133C, K134C, I135C, Q139C, N141C, D144C, G145C, S146C,
T147C,
K149C, H151C, N153C, P155C, Y167C, M168C, K169C, D170C, K178C, D179C, K180C,
A181C,
F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C, N193C, V194C, H204C, E206C,
V207C,
G208C, Q210C, V215C, Y216C, G217C, E218C, G219C, E220C, R221C, N222C, L224C,
Y225C,
3o D227C, N228C, D229C, N230C, H241C, D242C, T243C, or N244C.
In a further embodiment the adiponectin polypeptide is selected from an
adiponectin
polypeptide fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
35 to position K101 as indicated in seq id no 1 to 56 amino acids
corresponding to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation T121 C. Examples
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
of such adiponectin polypeptide fragments are any one of the sequences seq id
no 17, 23, 26, 29, or 32.
In another embodiment the adiponectin polypeptide is selected from an
adiponeetin polypeptide
fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 50 amino acids corresponding
to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponeetin polypeptide comprises the mutation
S146C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 18,
27, or 33. In a further
l0 embodiment the adiponectin polypeptide is selected from an adiponectin
polypeptide fragment
comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
15 indicated in seq id no 1; and wherein the collagen domain comprises a
lysine which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
T243C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 19,
or 28. In a further
embodiment the adiponectin polypeptide is selected from an adiponectin
polypeptide fragment
comprising a globular domain and a collagen domain,
20 wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide fragment comprises the
mutation N127C. Examples
25 of such adiponeetin polypeptide fragments are any one of the sequences seq
id no 35, 41, 44, 47, or 50.
In a further embodiment the adiponectin polypeptide is selected from an
adiponectin polypeptide
fragment comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
3o to position 8100 as indicated in seq id no 1 to 50 amino acids
corresponding to position R58 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N141C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 36,
45, or 51. In a further
embodiment the adiponectin polypeptide is selected from an adiponeetin
polypeptide fragment
35 comprising a globular domain and a collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 8
amino acids corresponding
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
81
to position 8100 as indicated in seq id no 1 to 29 amino acids corresponding
to position D79 as
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated, wherein the adiponectin polypeptide comprises the mutation
N228C. Examples of such
adiponectin polypeptide fragments are any one of the sequences seq id no 37,
or 46.
When the adiponectin polypeptide is part of a conjugate, that is, a conjugate
comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, the first non-polypeptide moiety is selected from a polymer
molecule, a lipophilic
compound, a sugar moiety and an organic derivatizing agent.
The first non-polypeptide moiety may be attached to an amino acid which is one
of the naturally
occurring present in the adiponectin polypeptide, as described in the above
sections "First group of
conjugates) of the invention", "Second group of conjugates) of the invention",
and "Third group of
conjugates) of the invention", or to an introduced amino acid as described in
the above section "Fourth
group of conjugates) of the invention".
To further illustrate the conjugate part of the complex comprising a conjugate
comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, and calcium ions, some non-exhaustive embodiments are disclosed
below.
In one embodiment the conjugate comprises
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence as indicated in
seq id no 1 from position
2o A108 to N244, and wherein the collagen domain comprises from 7 amino acids
corresponding to
position K101 as indicated in seq id no 1 to 66 amino acids corresponding to
position G42 as indicated
in seq id no 1, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated,
wherein the adiponectin polypeptide fragment comprises a mutation selected
from any one of
A108C, Y109C, V110C, Y111C, R112C, E120C, T121C, Y122C, V123C, T124C, I125C,
P126C,
N127C, M128C, R131C, T133C, K134C, I135C, Q139C, N141C, D144C, G145C, S146C,
T147C,
K149C, H151C, N153C, P155C, Y167C, M168C, K169C, D170C, K178C, D179C, K180C,
A181C,
F184C, Y186C, Q188C, Y189C, Q190C, E191C, N192C, N193C, V194C, H204C, E206C,
V207C,
G208C, Q210C, V215C, Y216C, G217C, E218C, G219C, E220C, R221C, N222C, L224C,
Y225C,
3o D227C, N228C, D229C, N230C, H241 C, D242C, T243C, or N244C; and
a first non-polypeptide moiety covalently attached to the introduced cystein
residue. Preferably,
the first non-polypeptide moiety is a polymer, typically a linear or branched
polyethylene glycol.
In another embodiment the conjugate comprises
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1, and wherein the collagen domain comprises from 7
amino acids corresponding
to position K101 as indicated in seq id no 1 to 56 amino acids corresponding
to position A52 as
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
82
indicated in seq id no 1, and wherein the collagen domain comprises a lysine
which is hydroxylated and
glycosylated,
the adiponectin polypeptide fragment comprising a mutation selected from any
one of
A108N+V110T, Y109N+Y111T, V110N+R112T, Y111N, Y111N+S113T, R112N+A114T,
L119N+T121S, L119N, E120N+Y122T, T121N+V123T, Y122N, Y122N+T124S, T124N+p126T,
P126N+M128T, P129T, M128N+I130T, I130N+F132T, R131N, R131N+T133S, T133N+I135T,
K134N+F136T, I135N+Y137T, F136N+N138T, Y137N+Q139T, Q140T, Q139N+N141T,
Q140N+H142T, Y143T, H142N+D144T, D144N, D144N+S146T, G145N, G145N+T147S,
S146N+G148T, T147N+K149T, K149N+H151T, H151N+N153T, P155T, P155N+L157T,
to Y159N+A161T, I164N+V166T, T165N+Y167T, Y167N+K169T, M168N+D170T,
K169N+V171T,
D170N+K172T, V171N+V173T, K172N, K172N+S174T, F176N+K178T, K177N+D179T,
K178N+K180T, D179N+A181T, K180N+M182T, A181N+L183T, M182N+F184T, F184N+Y186T,
Y186N+Q188T, Q188N+Q190T, Y189N+E191T, Q190N+N192T, E191N+N193T, V194T, or
V 194N+Q 196T;
and a sugar moiety covalently attached to the introduced N-glycosylation site.
A further specific
conjugate comprises an adiponectin polypeptide selected from the sequence seq
id no 10 comprising a
mutation selected from Y111N, such as the adiponectin polypeptide having the
seq id no 53, and a sugar
moiety covalently attached to the introduced N-glycosylation site. In a
further embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
2o selected from Y122N, such as the adiponectin polypeptide having the seq id
no 54, and a sugar moiety
covalently attached to the introduced N-glycosylation site. In a further
embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from R131N, such as the adiponectin polypeptide having the seq id no
55, and a sugar moiety
covalently attached to the introduced N-glycosylation site. In a further
embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from D 144N+S 146T, such as the adiponectin polypeptide having the
seq id no 56, and a sugar
moiety covalently attached to the introduced N-glycosylation site. In a
further embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from H151N+N153T, such as the adiponectin polypeptide having the seq
id no 57, and a sugar
moiety covalently attached to the introduced N-glycosylation site. In a
further embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from K178N+K180T, such as the adiponectin polypeptide having the seq
id no 58, and a sugar
moiety covalently attached to the introduced N-glycosylation site. In a
further embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from P129T, such as the adiponectin polypeptide having the seq id no
59, and a sugar moiety
covalently attached to the introduced N-glycosylation site. In a further
embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
83
selected from G145N, such as the adiponectin polypeptide having the seq id no
60, and a sugar moiety
covalently attached to the introduced N-glycosylation site. In a further
embodiment the conjugate
comprises an adiponectin polypeptide selected from the sequence seq id no 10
comprising a mutation
selected from P155T, such as the adiponectin polypeptide having the seq id no
61, and a sugar moiety
covalently attached to the introduced N-glycosylation site.
In a further embodiment the conjugate comprises
an adiponectin polypeptide fragment comprising a globular domain and a
collagen domain,
wherein the globular domain comprises an amino acid sequence from position
A108 to N244 as
indicated in seq id no 1 as well as sequences that differs from the amino acid
sequence in one or more
1 o substitution(s), and wherein the collagen domain comprises from 7 amino
acids corresponding to
position K101 as indicated in seq id no 1 to 56 amino acids corresponding to
position A52 as indicated
in seq id no 1, and wherein the collagen domain comprises a lysine which is
hydroxylated and
glycosylated, and
a first non-polypeptide moiety covalently attached to the adiponectin
polypeptide fragment,
15 wherein the adiponectin polypeptide fragment comprises an amino acid
residue having an
attachment group for said first non-polypeptide moiety, wherein the amino acid
residue is the N-terminal
amino acid residue. Preferably, the first non-polypeptide moiety is a polymer,
typically a linear or
branched polyethylene glycol.
Further preferred embodiments of the adiponectin polypeptide fragment are any
one of the
20 sequences selected from seq id no 2, 3, 4, 5, 6, 10, 11, 12, 13, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, or 61. These sequences are also preferred embodiments when
being the part of the
conjugate.
During pegylation of the adiponectin polypeptide fragment of the present
invention, in the
25 presence of calcium ions, to produce a conjugate, we discovered that it was
possible to introduce one
PEG molecule into a trimer of the polypeptide without destroying the trimer,
and the biological activity
was maintained at least in part. The PEG molecule was attached to the N-
terminal residue of one of the
adiponectin polypeptide monomers, thus producing a trimer containing one PEG
molecule. Moreover,
we also succeded in introducing two and three PEG molecule into a trimer of
the polypeptide, as shown
3o in example 11.
Accordingly, in a further embodiment the conjugate comprises an adiponectin
polypeptide
trimer, and one polymer covalently attached to the adiponectin polypeptide
trimer.
In a further embodiment the conjugate consists of
a) an adiponectin polypeptide trimer wherein the adiponectin polypeptide
trimer contains three
35 adiponectin polypeptide monomers, and
b) one polymer covalently attached to any one of the three monomers of the
adiponectin polypeptide
trimer in such a way that the adiponectin polypeptide trimer only contains one
polymer.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
84
In a still further embodiment the conjugate comprises an adiponectin
polypeptide trimer, and
two polymers covalently attached to the adiponectin polypeptide trimer.
In a further embodiment the conjugate consists of
a) an adiponectin polypeptide trimer wherein the adiponectin polypeptide
trimer contains three
adiponectin polypeptide monomers, and
b) two polymers covalently attached to any one of the three monomers of the
adiponectin polypeptide
trimer in such a way that the adiponectin polypeptide trimer only contains two
polymers.
In a further embodiment the conjugate comprises an adiponectin polypeptide
trimer, and three
polymers covalently attached to the adiponectin polypeptide trimer.
In a further embodiment the conjugate consists of
a) an adiponectin polypeptide trimer wherein the adiponectin polypeptide
trimer contains three
adiponectin polypeptide monomers, and
b) three polymers covalently attached to any one of the three monomers of the
adiponectin polypeptide
trimer in such a way that the adiponectin polypeptide trimer contains three
polymers.
Even though this embodiment relates to trimeric adiponectin polypeptides it is
intended that the
particular embodiments mentioned in the above sections "Adiponectin
polypeptide fragments) of the
invention", "First group of conjugates) of the invention", "Second group of
conjugates) of the
invention", "Third group of conjugates) of the invention", and "Fourth group
of conjugates) of the
invention", also apply to this trimeric embodiment of the invention, for
instance, the embodiments
mentioned in connection with an adiponectin polypeptide also applies to the
adiponectin polypeptide
monomer as part of the trimer. Moreover, when the non-polypeptide is a
polymer, then the embodiments
mentioned in the above sections in connection with a polymer also applies to
the one, two, or three
polymers) attached to the adiponectin polypeptide trimer. Thus, the
embodiments described below
should not be seen as limiting this particular aspect of the invention in any
way.
As described in the above sections "First group of conjugates) of the
invention", "Second group
of conjugates) of the invention", and "Third group of conjugates) of the
invention", then in a further
embodiment the adiponectin polypeptide monomer comprises an amino acid residue
having an
attachment group for the polymer. Such amino acid residue may be any amino
acid residue suitable for
polymer conjugation, preferably the amino acid residue is selected from a
lysine, a cysteine, or an N-
3o terminal residue. In a further embodiment the amino acid residue having an
attachment group for the
polymer is selected from an N-terminal residue.
In a further embodiment the adiponectin polypeptide monomer is selected from
any one of the
seq id no 1-8, 10-12, or 13, such as the seq id no 2, 3, 4, 5, 6, 10, 11, 12,
or 13, in particular seq id no 10,
11, 12, or 13.
As described in the above section "Fourth group of conjugates) of the
invention", then in a
further embodiment the adiponectin polypeptide monomer comprises an amino acid
residue having an
attachment group for a polymer, wherein said amino acid residue has been
introduced in a position that
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
in the parent adiponectin is occupied by a surface exposed amino acid residue,
such introduced amino
acid is typically, selected from C, K, D, or E, preferably C.
Thus, in a further embodiment the adiponectin polypeptide monomer is selected
from any one of
the seq id no 17-52, such as 17, 18, 19, 35, 36, or 37. In this respect, the
polymer is attached to the
5 introduced cysteine.
If one, two, or three polymers are attached to the trimer it is preferred that
such polymer is
selected from a polyethylene glycol. Thus, in a further embodiment the polymer
comprises a
polyethylene glycol, such as a linear or branched polyethylene glycol. In a
further embodiment the
polymer is a polyethylene glycol, such as a linear or branched polyethylene
glycol.
1o In a still further embodiment the polymer, such as a polyethylene glycol,
has a molecular weight
of from lkDa to 200kDa, such as from lkDa to 20kDa, e.g. from SkDa to 20kDa,
such as SkDa, lOkDa,
or 20kDa.
The composition comprising the conjugate may be any suitable composition such
as any one of
those mentioned below in the section "Pharmaceutical composition and uses of a
conjugate or
15 adiponectin polypeptide fragment of the invention". Thus, the composition
may be formulated in a
variety of forms, such as liquid or solid compositions. The term "liquid" is
intended to include aqueous.
In a further embodiment the composition is selected from a liquid composition.
In a further embodiment the liquid composition is a solution or suspension,
such as an aqueous
solution.
Further embodiments of any one of the above conjugates of the invention.
By removing and/or introducing amino acid residues comprising an attachment
group for the
non-polypeptide moiety it is possible to specifically adapt the polypeptide so
as to make the molecule
more susceptible to conjugation to the non-polypeptide moiety of choice, to
optimize the conjugation
pattern (e.g. to ensure an optimal distribution of non-polypeptide moieties on
the surface of the
adiponectin polypeptide and thereby, e.g., effectively shield epitopes and
other surface parts of the
polypeptide without significantly impairing the function thereof). For
instance, by introduction of
attachment groups, the adiponectin polypeptide is boosted or otherwise altered
in the content of the
specific amino acid residues to which the relevant non-polypeptide moiety
binds, whereby a more
3o efficient, specific and/or extensive conjugation is achieved. By removal of
one or more attachment
groups it is possible to avoid conjugation to the non-polypeptide moiety in
parts of the polypeptide in
which such conjugation is disadvantageous, e.g. to an amino acid residue
located at or near a functional
site of the polypeptide (since conjugation at such a site may result in
inactivation or reduced activity of
the resulting conjugate due to impaired receptor recognition). Further, it may
be advantageous to remove
an attachment group located closely to another attachment group in order to
avoid heterogeneous
conjugation to such groups.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
86
It will be understood that the amino acid residue comprising an attachment
group for a non-
polypeptide moiety, either it be removed or introduced, is selected on the
basis of the nature of the non-
polypeptide moiety and, in most instances, on the basis of the conjugation
method to be used. For
instance, when the non-polypeptide moiety is a polymer molecule, such as a
polyethylene glycol or
polyalkylene oxide derived molecule, amino acid residues capable of
functioning as an attachment group
may be selected from the group consisting of lysine, cysteine, aspartic acid,
glutamic acid and arginine.
When the non-polypeptide moiety is a sugar moiety the attachment group is an
in vivo glycosylation
site, preferably an N-glycosylation site.
Alternatively or additionally, the position to be modified is identified on
the basis of an analysis
to of an adiponectin protein sequence family. More specifically, the position
to be modified can be one,
which in one or more members of the family other than the parent adiponectin,
is occupied by an amino
acid residue comprising the relevant attachment group (when such amino acid
residue is to be
introduced) or which in the parent adiponectin, but not in one or more other
members of the family, is
occupied by an amino acid residue comprising the relevant attachment group
(when such amino acid
t 5 residue is to be removed).
In order to determine an optimal distribution of attachment groups, the
distance between amino
acid residues located at the surface of the adiponectin polypeptide is
calculated on the basis of a 3D
structure of the adiponectin polypeptide. More specifically, the distance
between the CB's of the amino
acid residues comprising such attachment groups, or the distance between the
functional group (NZ for
20 lysine, CG for aspartic acid, CD for glutamic acid, SG for cysteine) of one
and the CB of another amino
acid residue comprising an attachment group are determined. In case of
glycine, CA is used instead of
CB. In the adiponectin polypeptide part of a conjugate of the invention, any
of said distances is
preferably more than 8 A, in particular more than l Ot~ in order to avoid or
reduce heterogeneous
conjugation.
25 Furthermore, in the adiponectin polypeptide part of a conjugate of the
invention attachment
groups located at the receptor-binding site of adiponectin has preferably been
removed, preferably by
substitution of the amino acid residue comprising such group.
A still further generally applicable approach for modifying an adiponectin
polypeptide is to
shield, and thereby destroy or otherwise inactivate an epitope present in the
parent adiponectin, by
3o conjugation to a non-polypeptide moiety. Epitopes of human adiponectin may
be identified by use of
methods known in the art, also known as epitope mapping, see, e.g. Romagnoli
et al., J. Biol Chem,
1999, 380(5):553-9, DeLisser HM, Methods Mol Biol, 1999, 96:11-20, Van de
Water et al., Clin
Immunol Immunopathol, 1997, 85(3):229-35, Saint-Remy JM, Toxicology, 1997,
119(1):77-81, and
Lane DP and Stephen CW, Curr Opin Immunol, 1993, S(2):268-71. One method is to
establish a phage
35 display library expressing random oligopeptides of e.g. 9 amino acid
residues. IgGI antibodies from
specific antisera towards human adiponectin are purified by
immunoprecipitation and the reactive
phages are identified by immunoblotting. By sequencing the DNA of the purified
reactive phages, the
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
87
sequence of the oligopeptide can be determined followed by localization of the
sequence on the 3D-
structure of the adiponectin. Alternatively, epitopes can be identified
according to the method described
in US 5,041,376. The thereby identified region on the structure constitutes an
epitope that then can be
selected as a target region for introduction of an attachment group for the
non-polypeptide moiety.
Preferably, at least one epitope, such as two, three or four epitopes of human
recombinant adiponectin
are shielded by a non-polypeptide moiety according to the present invention.
Accordingly, in one
embodiment, the conjugate of the invention has at least one shielded epitope
as compared to wild type
human adiponectin.
In case of removal of an attachment group, the relevant amino acid residue
comprising such
t o group and occupying a position as defined above is preferably substituted
with a different amino acid
residue that does not comprise an attachment group for the non-polypeptide
moiety in question.
In case of introduction of an attachment group, an amino acid residue
comprising such group is
introduced into the position, preferably by substitution of the amino acid
residue occupying such
position. However, such introduction of an attachment group may also be
through addition of an amino
t 5 acid residue to the N- or C-terminal of the polypeptide, such as the
globular, collagen, or non-
homologuous domain of the polypeptide.
The exact number of attachment groups available for conjugation and present in
the adiponectin
polypeptide is dependent on the effect desired to be achieved by conjugation.
The effect to be obtained
is, e.g., dependent on the nature and degree of conjugation (e.g. the identity
of the non-polypeptide
2o moiety, the number of non-polypeptide moieties desirable or possible to
conjugate to the polypeptide,
where they should be conjugated or where conjugation should be avoided, etc.).
For instance, if reduced
immunogenicity is desired, the number (and location ofJ attachment groups
should be sufficient to shield
most or all epitopes. This is normally obtained when a greater proportion of
the adiponectin polypeptide
is shielded. Effective shielding of epitopes is normally achieved when the
total number of attachment
25 groups available for conjugation is in the range of 1-10 attachment groups.
Functional in vivo half life is
i.a. dependent on the molecular weight of the conjugate and the number of
attachment groups needed for
providing increased half life thus depends on the molecular weight of the non-
polypeptide moiety in
question.
In one embodiment, the conjugate of the invention has a molecular weight of at
least 67 kDa, in
30 particular at least 70 kDa as measured by SDS-PAGE according to Laemmli,
U.K., Nature Vol 227
( 1970), p680-85.
In order to avoid too much disruption of the structure and function of the
parent human
adiponectin the total number of amino acid residues to be altered in
accordance with the present
invention (as compared to the amino acid sequence shown in SEQ ID NO 6)
typically does not exceed
35 15. Preferably, when an analog is desired, the adiponectin polypeptide
comprises an amino acid
sequence, which differs in 1-15 amino acid residues from the amino acid
sequence shown in SEQ ID
NO 6, such as in 1-11, 1-8 or in 2-8 amino acid residues, e.g. in 1-5 or in 2-
5 amino acid residues from
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
88
the amino acid sequence shown in SEQ ID NO 6. Thus, normally the adiponectin
polypeptide comprises
an amino acid sequence that differs from the amino acid sequence shown in SEQ
ID NO 6 in 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. Typically, the
above numbers represent either
the total number of introduced or the total number of removed amino acid
residues comprising an
attachment group for the relevant non-polypeptide moiety, or the total number
of introduced and
removed amino acid residues comprising such group.
In the conjugate of the invention it is preferred that at least about 50% of
all conjugatable
attachment groups, such as at least about 80% and preferably all of such
groups are occupied by the
relevant non-polypeptide moiety. Accordingly, in a preferred embodiment the
conjugate of the invention
t0 comprises, e.g., 1-10 non-polypeptide moieties.
Conjugate of the invention, wherein the non polypeptide moiety is a molecule
that has lysine as an
attachment group
In one embodiment the first non-polypeptide moiety has lysine as an attachment
group, and thus
15 the adiponectin polypeptide is one that comprises an amino acid sequence
that differs from that of
wildtype human adiponectin in at least one introduced and/or at least one
removed lysine residue. While
the non-polypeptide moiety may be any of those binding to a lysine residue,
e.g. the s-amino group
thereof, such as a polymer molecule, a lipophilic group, an organic
derivatizing agent or a carbohydrate
moiety, it is preferably any of the polymer molecule mentioned in the section
entitled "Conjugation to a
2o polymer molecule", in particular a branched or linear PEG or polyalkylene
oxide. Most preferably, the
polymer molecule is PEG and the activated molecule to be used for conjugation
is SS-PEG, NPC-PEG,
aldehyd-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC from Shearwater Polymers, Inc, SC-
PEG from
Enzon, Inc., tresylated mPEG as described in US 5,880,255, or oxycarbonyl-oxy-
N-dicarboxyimide-
PEG (US 5,122,614). Normally, for conjugation to a lysine residue the non-
polypeptide moiety has a
25 molecular weight of about 5, 10, 20, or 40 kDa.
The lysine residues) may be replaced with any other amino acid residue, but is
preferably replaced by
an arginine or a glutamine residue in order to give rise to the least
structural difference.
Conjugate of the invention wherein the non polypeptide moiety binds to a
cysteine residue
30 While the first non-polypeptide moiety according to this aspect of the
invention may be any
molecule which, when using the given conjugation method has cysteine as an
attachment group (such as
a carbohydrate moiety, a lipophilic group or an organic derivatizing agent),
it is preferred that the non-
polypeptide moiety is a polymer molecule. The polymer molecule may be any of
the molecules
mentioned in the section entitled "Conjugation to a polymer molecule", but is
preferably selected from
35 the group consisting of linear or branched polyethylene glycol or
polyalkylene oxide. Typically, the
polymer molecule is VS-PEG. The conjugation between the polypeptide and the
polymer may be
achieved in any suitable manner, e.g. as described in the section entitled
"Conjugation to a polymer
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
89
molecule", e.g. in using a one step method or in the stepwise manner referred
to in said section. When
the adiponectin polypeptide comprises only one conjugatable cysteine residue,
this is preferably
conjugated to a first non-polypeptide moiety with a molecular weight of from 1
to 20kDa or more, either
directly conjugated or indirectly through a low molecular weight polymer (as
disclosed in WO
99/55377). However, the conjugation of a cysteine to a first non-polypeptide
moiety having a molecular
weight of at least 5 kDa is also an embodiment of the invention. When the
conjugate comprises two or
more first non-polypeptide moieties, normally each of these has a molecular
weight of 5, 10, or 20 kDa.
Conjugate of the invention wherein the non polypeptide moiety binds to an acid
group
1 o In case of removal of an amino acid residue, the amino acid sequence of
the adiponectin
polypeptide differs from that of human wildtype adiponectin in at least one
removed aspartic acid or
glutamic acid residue, such as 1-5 removed residues, in particular 1-4 or 1-3
removed aspartic acid or
glutamic acid residues. The aspartic acid or glutamic acid residues) may be
replaced with any other
amino acid residue, but is preferably replaced by an arginine or a glutamine
residue.first non-
15 polypeptide moiety can be any non-polypeptide moiety with such property, it
is presently preferred that
the non-polypeptide moiety is a polymer molecule or an organic derivatizing
agent having an acid group
as an attachment group, in particular a polymer molecule such as PEG, and the
conjugate is prepared,
e.g., as described by Sakane and Pardridge, Pharmceutical Research, Vol. 14,
No. 8, 1997, pp 1085-
1091. Normally, for conjugation to an acid group the non-polypeptide moiety
has a molecular weight of
2o about 5, 10, or 20 kDa.
Conjugate of the invention comprising a second non polypeptide moiety
In addition to a first non-polypeptide moiety (as described in the preceding
sections), the
conjugate of the invention may comprise a second non-polypeptide moiety of a
different type as
25 compared to the first non-polypeptide moiety. Preferably, in any of the
above described conjugates
wherein the first non-polypeptide moiety is, e.g., a polymer molecule such as
PEG, a second non-
polypeptide moiety is a sugar moiety, in particular an N-linked sugar moiety.
Such site is e.g. any of
those described in the immediately preceding section entitled "Conjugate of
the invention wherein the
non-polypeptide moiety is a sugar moiety".
3o It will be understood that in order to obtain an optimal distribution of
attached first and second
non-polypeptide moieties, the adiponectin polypeptide may be modified in the
number and distribution
of attachment groups for the first as well as the second non-polypeptide
moiety so as to have e.g. at least
one removed attachment group for the first non-polypeptide moiety and at least
one introduced
attachment group for the second non-polypeptide moiety or vice versa.
Conjugate of the invention wherein the non polypeptide moiety is a sugar
moiety
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
When the conjugate of the invention comprises at least one sugar moiety
attached to an in vivo
glycosylation site, in particular an N-glycosylation site, this is a new in
vivo glycosylation site
introduced into the adiponectin polypeptide. The in vivo glycosylation site
may be an O-glycosylation
site, but is preferably an N-glycosylation site.
For instance, an in vivo glycosylation site is introduced into a position of
the parent adiponectin
occupied by an amino acid residue exposed to the surface of the molecule,
preferably with more than
25% of the side chain exposed to the solvent, in particular more than 50%
exposed to the solvent (these
positions are identified in the Experimentals/Methods section herein). The N-
glycosylation site is
introduced in such a way that the N-residue of said site is located in said
position. Analogously, an O-
1o glycosylation site is introduced so that the S or T residue making up such
site is located in said position.
Still more preferably, the in vivo glycosylation site is introduced into a
position wherein only one
mutation is required to create the site (i.e. where any other amino acid
residues required for creating a
functional glycosylation site is already present in the molecule).
t 5 Non-polypeptide moiety of the conjugate of the invention
As indicated further above the non-polypeptide moiety of the conjugate of the
invention is
preferably selected from the group consisting of a polymer molecule, a
lipophilic compound, a sugar
moiety (by way of in vivo glycosylation) and an organic derivatizing agent.
All of these agents may
confer desirable properties to the polypeptide part of the conjugate, in
particular reduced
2o immunogenicity and/or increased functional in vivo half life and/or
increased serum half life. The
polypeptide part of the conjugate may be conjugated to only one type of non-
polypeptide moiety, but
may also be conjugated to two or more different types of non-polypeptide
moieties, e.g. to a polymer
molecule and a sugar moiety, to a lipophilic group and a sugar moiety, to an
organic derivating agent
and a sugar moiety, to a lipophilic group and a polymer molecule, etc. The
conjugation to two or more
25 different non-polypeptide moieties may be done simultaneous or
sequentially. The choice of non-
polypeptide moiety/ies, e.g. depends on the effect desired to be achieved by
the conjugation. For
instance, sugar moieties have been found particularly useful for reducing
immunogenicity, whereas
polymer molecules such as PEG are of particular use for increasing functional
in vivo half life and/or
serum half life. Using a polymer molecule as a first non-polypeptide moiety
and a sugar moiety as a
3o second non-polypeptide moiey may result in reduced immunogenicity and
increased functional in vivo
or serum half life.
Methods ofpreparing a conjugate of the invention
In the following sections "Conjugation to a lipophilic compound", "Conjugation
to a polymer
35 molecule", "Conjugation to a sugar moiety" and "Conjugation to an organic
derivatizing agent"
conjugation to specific types of non-polypeptide moieties is described.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
91
In a further aspect the invention relates to a method for preparing a
conjugate comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, wherein the adiponectin polypeptide is reacted with the first non-
polypeptide moiety to
which it is to be conjugated under conditions conducive for the conjugation to
take place, and the
conjugate is recovered.
In further aspects the invention relates to a method for preparing a conjugate
comprising an
adiponectin polypeptide, and a first non-polypeptide moiety covalently
attached to the adiponectin
polypeptide, as described above in connection with the first, second, third,
and fourth group of
conjugates) of the invention.
l0
Conjugation to a lipophilic compound
For conjugation to a lipophilic compound the following polypeptide groups may
function as
attachment groups: the N-terminal or C-terminal of the polypeptide, the
hydroxy groups of the amino
acid residues Ser, Thr or Tyr, the E-amino group of Lys, the SH group of Cys
or the carboxyl group of
~ 5 Asp and Glu. The polypeptide and the lipophilic compound may be conjugated
to each other, either
directly or by use of a linker. The lipophilic compound may be a natural
compound such as a saturated
or unsaturated fatty acid, a fatty acid diketone, a terpene, a prostaglandin,
a vitamine, a carotenoide or
steroide, or a synthetic compound such as a carbon acid, an alcohol, an amine
and sulphonic acid with
one or more alkyl-, aryl-, alkenyl- or other multiple unsaturated compounds.
The conjugation between
2o the polypeptide and the lipophilic compound, optionally through a linker
may be done according to
methods known in the art, e.g. as described by Bodanszky in Peptide Synthesis,
John Wiley, New York,
1976 and in WO 96/12505.
Conjugation to a polymer molecule
25 The polymer molecule to be coupled to the polypeptide may be any suitable
polymer molecule,
such as a natural or synthetic homo-polymer or heteropolymer, typically with a
molecular weight in the
range of 300-200,000 Da, such as lkDa to 200kDa.
Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e.
poly-NHZ) and a
polycarboxylic acid (i.e. poly-COOH). A hetero-polymer is a polymer, which
comprises one or more
3o different coupling groups, such as, e.g., a hydroxyl group and an amine
group.
Examples of suitable polymer molecules include polymer molecules selected from
the group
consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG),
such as polyethylene glycol
(PEG) and polypropylene glycol (PPG), branched PEGS, poly-vinyl alcohol (PVA),
poly-carboxylate, poly-
(vinylpyrolidone), polyethylene-co-malefic acid anhydride, polystyrene-co-
malic acid anhydride, dextran
35 including carboxymethyl-dextran, or any other biopolymer suitable for
reducing immunogenicity and/or
increasing functional in vivo half life and/or serum half life. Generally,
polyalkylene glycol-derived
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
92
polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have
various water solubility
properties, and are easily excreted from living organisms.
PEG is the preferred polymer molecule to be used, since it has only few
reactive groups capable of
cross-linking compared, e.g., to polysaccharides such as dextran, and the
like. In particular, monofunctional
PEG, e.g monomethoxypolyethylene glycol (mPEG), is of interest since its
coupling chemistry is relatively
simple (only one reactive group is available for conjugating with attachment
groups on the polypeptide).
Consequently, the risk of cross-linking is eliminated, the resulting
polypeptide conjugates are more
homogeneous and the reaction of the polymer molecules with the polypeptide is
easier to control.
To effect covalent attachment of the polymer molecules) to the polypeptide,
the hydroxyl end
t 0 groups of the polymer molecule must be provided in activated form, i.e.
with reactive functional groups
(examples of which include primary amino groups, hydrazide (HZ), thiol,
succinate (SUC), succinimidyl
succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate
(SPA), succinimidy
carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide
(NHS), aldehyde,
nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitably activated polymer
molecules are
15 commercially available, e.g. from Shearwater Polymers, Inc., Huntsville,
AL, USA. Alternatively, the
polymer molecules can be activated by conventional methods known in the art,
e.g. as disclosed in WO
90/13540. Specific examples of activated linear or branched polymer molecules
for use in the present
invention are described in the Shearwater Polymers, Inc. 1997 and 2000
Catalogs (Functionalized
Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol
and Derivatives,
2o incorporated herein by reference). Specific examples of activated PEG
polymers include the following
linear PEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-
PEG, SG-
PEG, and SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG,
ALD-
PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-
NHS and
those disclosed in US 5,932,462 and US 5,643,575, both of which references are
incorporated herein by
25 reference. Furthermore, the following publications, incorporated herein by
reference, disclose useful
polymer molecules and/or PEGylation chemistries: US 5,824,778, US 5,476,653,
WO 97/32607, EP
229,108, EP 402,378, US 4,902,502, US 5,281;698, US 5,122,614, US 5,219,564,
WO 92/16555, WO
94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO
95/11924,
W095/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837,
WO
30 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058,
EP 439 508,
WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, US 5,736,625, WO 98/05363,
EP 809 996,
US 5,629,384, WO 96/41813, WO 96/07670, US 5,473,034, US 5,516,673, EP 605
963, US 5,382,657,
EP 510 356, EP 400 472, EP 183 503 and EP 154 316.
The conjugation of the polypeptide and the activated polymer molecules is
conducted by use of any
35 conventional method, e.g. as described in the following references (which
also describe suitable methods for
activation of polymer molecules): Harris and Zalipsky, eds., Polyethylene
glycol) Chemistry and Biological
Applications, AZC, Washington; R.F. Taylor, (1991), "Protein immobilisation.
Fundamental and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
93
applications", Marcel Dekker, N.Y.; S.S. along, (1992), "Chemistry of Protein
Conjugation and
Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993),
"Immobilized Affinity Ligand
Techniques", Academic Press, N.Y.). The skilled person will be aware that the
activation method and/or
conjugation chemistry to be used depends on the attachment groups) of the
adiponectin polypeptide as well
as the functional groups of the polymer (e.g. being amino, hydroxyl, carboxyl,
aldehyde or sulfydryl). The
PEGylation may be directed towards conjugation to all available attachment
groups on the polypeptide
(i.e. such attachment groups that are exposed at the surface of the
polypeptide) or may be directed
towards specific attachment groups, e.g. the N-terminal amino group (US
5,985,265). Furthermore, the
conjugation may be achieved in one step or in a stepwise manner (e.g. as
described in WO 99/55377).
1o It will be understood that the PEGylation is designed so as to produce the
optimal molecule with
respect to the number of PEG molecules attached, the size and form (e.g.
whether they are linear or
branched) of such molecules, and where in the polypeptide such molecules are
attached. For instance,
the molecular weight of the polymer to be used may be chosen on the basis of
the desired effect to be
achieved. For instance, if the primary purpose of the conjugation is to
achieve a conjugate having a high
~ 5 molecular weight (e.g. to reduce renal clearance) it is usually desirable
to conjugate as few high Mw
polymer molecules as possible to obtain the desired molecular weight. When a
high degree of epitope
shielding is desirable this may be obtained by use of a sufficiently high
number of low molecular weight
polymer (e.g. with a molecular weight of about 5,000 Da) to effectively shield
all or most epitopes of the
polypeptide. For instance, 2-8, such as 3-6 such polymers may be used.
20 In connection with conjugation to only a single attachment group on the
protein (as described in US
5,985,265), it may be advantageous that the polymer molecule, which may be
linear or branched, has a
high molecular weight, e.g. about 20 kDa.
Normally, the polymer conjugation is performed under conditions aiming at
reacting all available polymer
attachment groups with polymer molecules. Typically, the molar ratio of
activated polymer molecules to
25 polypeptide is 1000-1, in particular 200-1, preferably 100-1, such as 10-1
or 5-1 in order to obtain optimal
reaction. However, also equimolar ratios may be used.
It is also contemplated according to the invention to couple the polymer
molecules to the polypeptide
through a linker. Suitable linkers are well known to the skilled person. A
preferred example is cyanuric
chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; US
4,179,337; Shafer et al., (1986), J.
3o Polym. Sci. Polym. Chem. Ed., 24, 375-378.
Subsequent to the conjugation residual activated polymer molecules are blocked
according to methods
known in the art, e.g. by addition of primary amine to the reaction mixture,
and the resulting inactivated
polymer molecules removed by a suitable method.
The general technology described in WO 99/55377 is also applicable in respect
of producing the
35 conjugates of the present invention. Accordingly, in a further aspect the
invention relates to a method for
stepwise attachment of polyethylene glycol (PEG) moieties in series to an
adiponectin polypeptide,
comprising the steps of:
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
94
reacting an adiponectin polypeptide with a low molecular weight
heterobifunctional or
homobifunctional PEG moiety having the following formula: W-
CHZCH20(CHZCH20)"CHZCHZ-X,
where W and X are groups that independently react with an amine, sulthydryl,
carboxyl or hydroxyl
functional group to attach the low molecular weight PEG moiety to the
adiponectin polypeptide; and
reacting the low molecular weight PEG moiety attached to the adiponectin
polypeptide with a
monofunctional or bifunctional PEG moiety to attach the monofunctional or
bifunctional PEG moiety to
a free terminus of the low molecular weight PEG moiety and form a PEG-
adiponectin polypeptide
conjugate. The n is an integer, which will depend on the weight of the low
molecular weight PEG
moiety. In one embodiment the monofunctional or bifunctional PEG moiety has
the following formula:
Y-CHZCHzO(CHzCHzO)"CHZCHz-Z, wherein Y is reactive to a terminal group on the
free terminus of
the low molecular weight PEG moiety attached to the adiponectin polypeptide
and Z is-OCH3 or a
group reactive with X to form a bifunctional conjugate. In a further
embodiment the monofunctional or
bifunctional PEG moiety is methoxy PEG, branched PEG, hydrolytically or
enzymatically degradable
PEG, pendant PEG, or dendrimer PEG. In a further embodiment W and X are
independently selected
15 from the group consisting of orthopyridyl disulfide, maleimides,
vinylsulfones, iodoacetamides,
hydrazides, aldehydes, succinimidyl esters, epoxides, amines, thiols,
carboxyls, active esters,
benzotriazole carbonates, p-nitrophenol carbonates, isocyanates, and biotin.
In a further embodiment the
low molecular weight PEG moiety has a molecular weight in a range of about 100
to 5,000 daltons, one
example being OPSS-PEG-hydrazide. In a further embodiment the monofunctional
or bifunctional PEG
2o moiety has a molecular weight in a range of about 100 daltons to 200
kilodaltons. In a further
embodiment the low molecular weight PEG moiety and/or the monofunctional or
bifunctional PEG
moiety is a copolymer of polyethylene glycol, such copolymer of polyethylene
glycol is typically,
selected from the group consisting of polyethylene glycol/polypropylene glycol
copolymers and
polyethylene glycol/poly (lactic/glycolic acid) copolymers. In a further
embodiment the method further
25 comprises a step of purifying the PEG-adiponectin polypeptide conjugate
following the stepwise
attachment of two PEG moieties in series to an adiponectin polypeptide. The
term "OPSS-PEG-
hydrazide in combination with mPEG-ALD" as used above and throughout this
description is intended
to means that the stepwise technologi disclosed in WO 99/55377 may be used.
The disclosure of WO
99/55377 is incorporated herein by reference.
30 Covalent in vitro coupling of a carbohydrate moiety to amino acid residues
of adiponectin
polypeptide may be used to modify or increase the number or profile of
carbohydrate substituents.
Depending on the coupling mode used, the carbohydrates) may be attached to a)
arginine and histidine
(Lundblad and Noyes, Chemical Reagents for Protein Modification, CRC Press
Inc. Boca Raton, FI), b)
free carboxyl groups (e.g. of the C-terminal amino acid residue, asparagine or
glutamine), c) free
35 sulfhydryl groups such as that of cysteine, d) free hydroxyl groups such as
those of serine, threonine,
tyrosine or hydroxyproline, e) aromatic residues such as those of
phenylalanine or tryptophan or f) the
amide group of glutamine. These amino acid residues constitute examples of
attachment groups for a
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
carbohydrate moiety, which may be introduced in the adiponectin polypeptide.
Suitable methods of in
vitro coupling are described in WO 87/05330 and in Aplin etl al., CRC Crit
Rev. Biochem., pp. 259-306,
1981. The in vitro coupling of carbohydrate moieties or PEG to protein- and
peptide-bound Gln-residues
can also be carried out by transglutaminases (TGases), e.g. as described by
Sato et al., 1996
Biochemistry 35, 13072-13080 or in EP 725145
Coupling to a sugar moiety
In order to achieve in vivo glycosylation of an adiponectin polypeptide that
has been modified by
introduction of one or more glycosylation sites (see the section "Conjugates
of the invention wherein the
10 non-polypeptide moiety is a sugar moiety"), the nucleotide sequence
encoding the polypeptide part of
the conjugate must be inserted in a glycosylating, eucaryotic expression host.
The expression host cell
may be selected from fungal (filamentous fungal or yeast), insect, mammalian
animal cells, from
transgenic plant cells or from transgenic animals. Furthermore, the
glycosylation may be achieved in the
human body when using a nucleotide sequence encoding the polypeptide part of a
conjugate of the
15 invention or a polypeptide of the invention in gene therapy. In one
embodiment the host cell is a
mammalian cell, such as an CHO cell, BHK or HEK cell, e.g. HEK293, or an
insect cell, such as an SF9
cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any
other suitable glycosylating
host, e.g. as described further below. Optionally, sugar moieties attached to
the adiponectin polypeptide
by in vivo glycosylation are further modified by use of glycosyltransferases,
e.g. using the
2o glycoAdvanceT"'' technology marketed by Neose, Horsham, PA, USA. Thereby,
it is possible to, e.g.,
increase the sialyation of the glycosylated adiponectin polypeptide following
expression and in vivo
glycosylation by CHO cells.
Coupling to an organic derivatizing agent
25 Covalent modification of the adiponectin polypeptide may be performed by
reacting (an)
attachment groups) of the polypeptide with an organic derivatizing agent.
Suitable derivatizing agents
and methods are well known in the art. For example, cysteinyl residues most
commonly are reacted with
a-haloacetates (and corresponding amines), such as chloroacetic acid or
chloroacetamide, to give
carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction
30 with bromotrifluoroacetone, a-bromo-(3-(4-imidozoyl)propionic acid,
chloroacetyl phosphate, N-
alkylmaleimides, 3-vitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-
chloromercuribenzoate, 2-
chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized
by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is
relatively specific for the
histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction
is preferably performed in
35 0.1 M sodium cacodylate at pH 6.O.Lysinyl and amino terminal residues are
reacted with succinic or
other carboxylic acid anhydrides. Derivatization with these agents has the
effect of reversing the charge
of the lysinyl residues. Other suitable reagents for derivatizing a-amino-
containing residues include
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
96
imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and
transaminase-catalyzed reaction
with glyoxylate. Arginyl residues are modified by reaction with one or several
conventional reagents,
among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
ninhydrin. Derivatization of
arginine residues requires that the reaction be performed in alkaline
conditions because of the high pKa
of the guanidine functional group. Furthermore, these reagents may react with
the groups of lysine as
well as the arginine guanidino group. Carboxyl side groups (aspartyl or
glutamyl or C-terminal amino
acid residue) are selectively modified by reaction with carbodiimides (R-N=C=N-
R'), where R and R'
are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)
carbodiimide or 1-ethyl-3-(4-
1o azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl
residues are converted to
asparaginyl and glutaminyl residues by reaction with ammonium ions.
Blocking offunctional site
Excessive polymer conjugation can lead to a loss of activity of the
adiponectin polypeptide to
15 which the polymer is conjugated. This problem can be eliminated, e.g., by
removal of attachment groups
located at the functional site or by blocking the functional site prior to
conjugation. These latter
strategies constitute further embodiments of the invention (the first strategy
being exemplified further
above, e.g. by removal of lysine residues which may be located close to a
functional site). More
specifically, according to the second strategy the conjugation between the
adiponectin polypeptide and
20 the non-polypeptide moiety is conducted under conditions where the
functional site of the polypeptide is
blocked by a helper molecule capable of binding to the functional site of the
polypeptide. Typically, the
helper molecule is one, which specifically recognizes a functional site of the
polypeptide, such as a
receptor. Alternatively, the helper molecule may be an antibody, in particular
a monoclonal antibody
recognizing the adiponectin polypeptide. In particular, the helper molecule
may be a neutralizing
25 monoclonal antibody.
The polypeptide is allowed to interact with the helper molecule before
effecting conjugation.
This ensures that the functional site of the polypeptide is shielded or
protected and consequently
unavailable for derivatization by the non-polypeptide moiety such, as a
polymer. Following its elution
from the helper molecule, the conjugate between the non-polypeptide moiety and
the polypeptide can be
30 recovered with at least a partially preserved functional site.
The subsequent conjugation of the polypeptide having a blocked functional site
to a polymer, a
lipophilic compound, an organic derivatizing agent or any other compound is
conducted in the normal
way, e.g. as described in the sections above entitled "Conjugation to ....".
Irrespectively of the nature of the helper molecule to be used to shield the
functional site of the
35 polypeptide from conjugation, it is desirable that the helper molecule is
free from or comprises only a
few attachment groups for the non-polypeptide moiety of choice in parts) of
the molecule, where the
conjugation to such groups will hamper the desorption of the conjugated
polypeptide from the helper
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
97
molecule. Hereby, selective conjugation to attachment groups present in non-
shielded parts of the
polypeptide can be obtained and it is possible to reuse the helper molecule
for repeated cycles of
conjugation. For instance, if the non-polypeptide moiety is a polymer molecule
such as PEG, which has
the epsilon amino group of a lysine or N-terminal amino acid residue as an
attachment group, it is
desirable that the helper molecule is substantially free from conjugatable
epsilon amino groups,
preferably free from any epsilon amino groups. Accordingly, in a preferred
embodiment the helper
molecule is a protein or peptide capable of binding to the functional site of
the polypeptide, which
protein or peptide is free from any conjugatable attachment groups for the non-
polypeptide moiety of
choice.
1 o In a further embodiment the helper molecule is first covalently linked to
a solid phase such as
column packing materials, for instance Sephadex or agarose beads, or a
surface, e.g. reaction vessel.
Subsequently, the polypeptide is loaded onto the column material carrying the
helper molecule and
conjugation carried out according to methods known in the art, e.g. as
described in the sections above
entitled "Conjugation to ....". This procedure allows the polypeptide
conjugate to be separated from the
15 helper molecule by elution. The polypeptide conjugate is eluated by
conventional techniques under
physico-chemical conditions that do not lead to a substantive degradation of
the polypeptide conjugate.
The fluid phase containing the polypeptide conjugate is separated from the
solid phase to which the
helper molecule remains covalently linked. The separation can be achieved in
other ways: For instance,
the helper molecule may be derivatised with a second molecule (e.g. biotin)
that can be recognized by a
2o specific binder (e.g. streptavidin). The specific binder may be linked to a
solid phase thereby allowing
the separation of the polypeptide conjugate from the helper molecule-second
molecule complex through
passage over a second helper-solid phase column which will retain, upon
subsequent elution, the helper
molecule-second molecule complex, but not the polypeptide conjugate. The
polypeptide conjugate may
be released from the helper molecule in any appropriate fashion. De-protection
may be achieved by
25 providing conditions in which the helper molecule dissociates from the
functional site of the adiponectin
to which it is bound. For instance, a complex between an antibody to which a
polymer is conjugated and
an anti-idiotypic antibody can be dissociated by adjusting the pH to an acid
or alkaline pH.
Conjugation of a tagged adiponectin polypeptide
3o In an alternative embodiment the adiponectin polypeptide is expressed, as a
fusion protein, with
a tag, i.e. an amino acid sequence or peptide stretch made up of typically I-
30, such as 1-20 or 1-15 or
1-10 amino acid residues. Besides allowing for fast and easy purification, the
tag is a convenient tool for
achieving conjugation between the tagged polypeptide and the non-polypeptide
moiety. In particular, the
tag may be used for achieving conjugation in microtiter plates or other
carriers, such as paramagnetic
35 beads, to which the tagged polypeptide can be immobilised via the tag. The
conjugation to the tagged
polypeptide in, e.g., microtiter plates has the advantage that the tagged
polypeptide can be immobilised
in the microtiter plates directly from the culture broth (in principle without
any purification) and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
98
subjected to conjugation. Thereby, the total number of process steps (from
expression to conjugation)
can be reduced. Furthermore, the tag may function as a spacer molecule
ensuring an improved
accessibility to the immobilised polypeptide to be conjugated. The conjugation
using a tagged
polypeptide may be to any of the non-polypeptide moieties disclosed herein,
e.g. to a polymer molecule
such as PEG.
The identity of the specific tag to be used is not critical as long as the tag
is capable of being
expressed with the polypeptide and is capable of being immobilised on a
suitable surface or carrier
material. A number of suitable tags are commercially available, e.g. from
Unizyme Laboratories,
Denmark. For instance, the tag may be any of the following sequences:
1o His-His-His-His-His-His
Met-Lys-His-His-His-His-His-His
Met-Lys-His-His-Ala-His-His-Gln-His-His
Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln
(vectors useful for providing such tags are available from Unizyme
Laboratories, Denmark)
t 5 or any of the following:
EQKLI SEEDL (a C-terminal tag described in Mol. Cell. Biol. 5:3610-16, 1985)
DYKDDDDK (a C- or N-terminal tag)
YPYDVPDYA
Antibodies against the above tags are commercially available, e.g. from ADI,
Aves Lab and
20 Research Diagnostics.
The subsequent cleavage of the tag from the polypeptide may be achieved by use
of
commercially available enzymes.
Also, the polypeptide may be expressed with a tag, e.g. as described in the
section further above
entitled "Conjugation of a tagged adiponectin polypeptide".
25 It will be understood that any of the polypeptides of the invention
disclosed herein may be used
to prepare a conjugate of the invention, i.e. be covalently coupled to any of
the non-polypeptide moieties
disclosed herein. In particular, when a polypeptide of the invention is
expressed in a glycosylating
microorganism the polypeptide may be provided in glycosylated form.
3o Methods of preQarin a~ponectin polypeptide for use in the invention
The polypeptide of the present invention or the polypeptide part of a
conjugate of the invention,
optionally in glycosylated form, may be produced by any suitable method known
in the art. Such
methods include constructing a nucleotide sequence encoding the polypeptide
and expressing the
sequence in a suitable transformed or transfected host. However, polypeptides
of the invention may be
35 produced, albeit less efficiently, by chemical synthesis or a combination
of chemical synthesis or a
combination of chemical synthesis and recombinant DNA technology.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
99
In a further aspect the invention relates to a nucleotide sequence encoding
the adiponectin
polypeptide part of a conjugate of the invention.
In a further aspect the invention relates to a nucleotide sequence encoding
the adiponectin
polypeptide fragment of the invention.
In a fizrther aspect the invention relates to an expression vector comprising
a nucleotide
sequence encoding the adiponectin polypeptide part of a conjugate of the
invention.
In a further aspect the invention relates to an expression vector comprising a
nucleotide
sequence encoding the adiponectin polypeptide fragment of the invention.
In a further aspect the invention relates to a host cell comprising a
nucleotide sequence encoding
the adiponectin polypeptide part of a conjugate of the invention or an
expression vector comprising a
nucleotide sequence encoding the adiponectin polypeptide part of a conjugate
of the invention.
In a further aspect the invention relates to a host cell comprising a
nucleotide sequence encoding
the adiponectin polypeptide fragment of the invention or an expression vector
comprising a nucleotide
sequence encoding the adiponectin polypeptide fragment of the invention.
In one embodiment the nucleotide sequence comprises a sequence selected from
any one of seq
id no 14, 1 S or 16, as well as sequences having at least 70% homology with
any one of seq id no 14, 15
or 16. More preferred are sequences having at least 80% homology, such as 90%,
92%, 95% or 98%
homology with any one of seq id no 14, 15 or 16.
In another embodiment the nucleotide sequence comprises a sequence selected
from any one of
2o seq id no 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71, as well as sequences
having at least 70% homology
with any one of seq id no 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. More
preferred are sequences having
at least 80% homology, such as 90%, 92%, 95% or 98% homology with any one of
seq id no 62, 63, 64,
65, 66, 67, 68, 69, 70, or 71.
In a further embodiment the host cell is selected from a yeast cell, a
bacterial cell, eg E.coli, a
mammalian cell, eg a CHO, BHK, HEK293 cell or an SF9 cell. In a further
embodiment the host cell is
selected from a bacterial cell, such as E.coli. In a further embodiment the
host cell is selected from a
mammalian cell, such as CHO K1. Further embodiments of a suitable host cell of
the invention is
disclosed below.
The nucleotide sequence of the invention encoding an adiponectin polypeptide
may be
constructed by isolating or synthesizing a nucleotide sequence encoding the
parent adiponectin, e.g. with
the amino acid sequence shown in SEQ ID NO 6, and then changing the nucleotide
sequence so as to
effect introduction (i.e. insertion or substitution) or deletion (i.e. removal
or substitution) of the relevant
amino acid residue(s).
The nucleotide sequence may conveniently be modified by site-directed
mutagenesis in
accordance with well-known methods.
Alternatively, the nucleotide sequence is prepared by chemical synthesis, e.g.
by using an
oligonucleotide synthesizer, wherein oligonucleotides are designed based on
the amino acid sequence of
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
100
the desired polypeptide, and preferably selecting those codons that are
favored in the host cell in which
the recombinant polypeptide will be produced. For example, several small
oligonucleotides coding for
portions of the desired polypeptide may be synthesized and assembled by PCR,
ligation or ligation chain
reaction (LCR). The individual oligonucleotides typically contain 5' or 3'
overhangs for complementary
assembly.
Once assembled (by synthesis, site-directed mutagenesis or another method),
the nucleotide
sequence encoding the adiponectin polypeptide is inserted into a recombinant
vector and operably linked
to control sequences necessary for expression of the adiponectin in the
desired transformed host cell.
It should of course be understood that not all vectors and expression control
sequences function
l0 equally well to express the nucleotide sequence encoding a polypeptide
variant described herein. Neither
will all hosts function equally well with the same expression system. However,
one of skill in the art
may make a selection among these vectors, expression control sequences and
hosts without undue
experimentation. For example, in selecting a vector, the host must be
considered because the vector
must replicate in it or be able to integrate into the chromosome. The vector's
copy number, the ability to
15 control that copy number, and the expression of any other proteins encoded
by the vector, such as
antibiotic markers, should also be considered. In selecting an expression
control sequence, a variety of
factors should also be considered. These include, for example, the relative
strength of the sequence, its
controllability, and its compatibility with the nucleotide sequence encoding
the polypeptide, particularly
as regards potential secondary structures. Hosts should be selected by
consideration of their
20 compatibility with the chosen vector, the toxicity of the product coded for
by the nucleotide sequence,
their secretion characteristics, their ability to fold the polypeptide
correctly, their fermentation or culture
requirements, and the ease of purification of the products coded for by the
nucleotide sequence.
The recombinant vector may be an autonomously replicating vector, i.e. a
vector which exists as
an extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a
25 plasmid. Alternatively, the vector is one which, when introduced into a
host cell, is integrated into the
host cell genome and replicated together with the chromosomes) into which it
has been integrated.
The vector is preferably an expression vector, in which the nucleotide
sequence encoding the
polypeptide of the invention is operably linked to additional segments
required for transcription of the
nucleotide sequence. The vector is typically derived from plasmid or viral
DNA. A number of suitable
3o expression vectors for expression in the host cells mentioned herein are
commercially available or
described in the literature. Useful expression vectors for eukaryotic hosts,
include, for example, vectors
comprising expression control sequences from SV40, bovine papilloma virus,
adenovirus and
cytomegalovirus. Specific vectors are, e.g., pCDNA3.1(+)~Iiyg (Invitrogen,
Carlsbad, CA, USA) and
pCI-neo (Promega, La Jola, CA, USA). Useful expression vectors for bacterial
hosts include known
35 bacterial plasmids, such as plasmids from E. coli, including pBR322, pET3a
and pETl2a (both from
Novagen Inc., WI, USA), wider host range plasmids, such as RP4, phage DNAs,
e.g., the numerous
derivatives of phage lambda, e.g. , NM989, and other DNA phages, such as M13
and filamentous single
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
101
stranded DNA phages. Useful expression vectors for yeast cells include the 2~
plasmid and derivatives
thereof, the POT1 vector (IJS 4,931,373), the pJS037 vector described in
(Okkels, Ann. New York
Acad. Sci. 782, 202-207, 1996) and pPICZ A, B or C (Invitrogen). Useful
vectors for insect cells include
pVL941, pBG311 (Cate et al., "Isolation of the Bovine and Human Genes for
Mullerian Inhibiting
Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685-
98 (1986), pBluebac
4.5 and pMelbac (both available from Invitrogen).
Other vectors for use in this invention include those that allow the
nucleotide sequence encoding
the polypeptide to be amplified in copy number. Such amplifiable vectors are
well known in the art.
They include, for example, vectors able to be amplified by DHFR amplification
(see, e.g., Kaufman,
l0 U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular
Dihydrofolate Reductase
cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell.
Biol., 2, pp. 1304-19
(1982)) and glutamine synthetase ("GS") amplification (see, e.g., US 5,122,464
and EP 338,841).
The recombinant vector may further comprise a DNA sequence enabling the vector
to replicate
in the host cell in question. An example of such a sequence (when the host
cell is a mammalian cell) is
15 the SV40 origin of replication. When the host cell is a yeast cell,
suitable sequences enabling the vector
to replicate are the yeast plasmid 2p replication genes REP 1-3 and origin of
replication.
The vector may also comprise a selectable marker, e.g. a gene the product of
which
complements a defect in the host cell, such as the gene coding for
dihydrofolate reductase (DHFR) or
the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40,
1985, pp. 125-130), or
20 one which confers resistance to a drug, e.g. ampicillin, kanamycin,
tetracyclin, chloramphenicol,
neomycin, hygromycin or methotrexate. For filamentous fungi, selectable
markers include amdS, pyre,
arcB, niaD, sC.
The term "control sequences" is defined herein to include all components,
which are necessary
or advantageous for the expression of the polypeptide of the invention. Each
control sequence may be
25 native or foreign to the nucleic acid sequence encoding the polypeptide.
Such control sequences include,
but are not limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter, enhancer or
upstream activating sequence, signal peptide sequence, and transcription
terminator. At a minimum, the
control sequences include a promoter.
A wide variety of expression control sequences may be used in the present
invention. Such
30 useful expression control sequences include the expression control
sequences associated with structural
genes of the foregoing expression vectors as well as any sequence known to
control the expression of
genes of prokaryotic or eukaryotic cells or their viruses, and various
combinations thereof.
Examples of suitable control sequences for directing transcription in
mammalian cells include
the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2
major late promoter, the MT-
35 1 (metallothionein gene) promoter, the human cytomegalovirus immediate-
early gene promoter (CMV),
the human elongation factor 1 a (EF-1 a) promoter, the Drosophila minimal heat
shock protein 70
promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)
promoter, the human
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
102
growth hormone terminator, SV40 or adenovirus Elb region polyadenylation
signals and the Kozak
consensus sequence (Kozak, M. JMoI Biol 1987 Aug 20;196(4):947-50).
In order to improve expression in mammalian cells a synthetic intron may be
inserted in the 5'
untranslated region of the nucleotide sequence encoding the polypeptide of
interest. An example of a
synthetic intron is the synthetic intron from the plasmid pCI-Neo (available
from Promega Corporation,
WI, USA).
Examples of suitable control sequences for directing transcription in insect
cells include the
polyhedrin promoter, the P10 promoter, the Autographa californica polyhedrosis
virus basic protein
promoter, the baculovirus immediate early gene 1 promoter and the baculovirus
39K delayed-early gene
l0 promoter, and the SV40 polyadenylation sequence.
Examples of suitable control sequences for use in yeast host cells include the
promoters of the
yeast a-mating system, the yeast triose phosphate isomerase (TPI) promoter,
promoters from yeast
glycolytic genes or alcohol dehydogenase genes, the ADH2-4c promoter and the
inducible GAL
promoter.
15 Examples of suitable control sequences for use in filamentous fungal host
cells include the
ADH3 promoter and terminator, a promoter derived from the genes encoding
Aspergillus oryzae TAKA
amylase triose phosphate isomerase or alkaline protease, an A. niger a-
amylase, A. niger or A. nidulans
glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase
or lipase, the TPI1
terminator and the ADH3 terminator.
2o Examples of suitable control sequences for use in bacterial host cells
include promoters of the lac
system, the trp system, the TAC or TRC system and the major promoter regions
of phage lambda.
The nucleotide sequence of the invention encoding an adiponectin polypeptide,
whether
prepared by site-directed mutagenesis, synthesis or other methods, may or may
not also include a
nucleotide sequence that encode a signal peptide. The signal peptide is
present when the polypeptide is
25 to be secreted from the cells in which it is expressed. Such signal
peptide, if present, should be one
recognized by the cell chosen for expression of the polypeptide. The signal
peptide may be homologous
(e.g. be that normally associated with human adiponectin) or heterologous
(i.e. originating from another
source than human adiponectin) to the polypeptide or may be homologous or
heterologous to the host
cell, i.e. be a signal peptide normally expressed from the host cell or one
which is not normally
3o expressed from the host cell. Accordingly, the signal peptide may be
prokaryotic, e.g. derived from a
bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or
insect or yeast cell.
The presence or absence of a signal peptide will, e.g., depend on the
expression host cell used
for the production of the polypeptide, the protein to be expressed (whether it
is an intracellular or
extracellular protein) and whether it is desirable to obtain secretion. For
use in filamentous fungi, the
35 signal peptide may conveniently be derived from a gene encoding an
Aspergillus sp. amylase or
glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease or a
Humicola lanuginosa lipase.
The signal peptide is preferably derived from a gene encoding A. oryzae TAKA
amylase, A. niger
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
103
neutral a-amylase, A. niger acid-stable amylase, or A. niger glucoamylase. For
use in insect cells, the
signal peptide may conveniently be derived from an insect gene (cf. WO
90/05783), such as the
lepidopteran Manduca sexta adipokinetic hormone precursor, (c~ US 5,023,328),
the honeybee melittin
(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., Protein
Expression and
Purification 4, 349-357 (1993) or human pancreatic lipase (hpl) (Methods in
Enzymology 284, pp. 262-
272, 1997).
A preferred signal peptide for use in mammalian cells is that of human
adiponectin apparent
from the examples hereinafter or the murine Ig kappa light chain signal
peptide (Coloma, M (1992) J.
Imm. Methods 152:89-104). For use in yeast cells suitable signal peptides have
been found to be the a-
to factor signal peptide from S. cereviciae. (cf. US 4,870,008), the signal
peptide of mouse salivary
amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified
carboxypeptidase
signal peptide (c~ L.A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast
BARI signal peptide (c~ WO
87/02670), and the yeast aspartic protease 3 (YAP3) signal peptide (c~ M. Egel-
Mitani et al., Yeast 6,
1990, pp. 127-137).
15 Any suitable host may be used to produce the adiponectin polypeptide,
including bacteria, fungi
(including yeasts), plant, insect, mammal, or other appropriate animal cells
or cell lines, as well as
transgenic animals or plants. Examples of bacterial host cells include
grampositive bacteria such as
strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or
Streptomyces, or gramnegative bacteria,
such as strains of E. coli. The introduction of a vector into a bacterial host
cell may, for instance, be
2o effected by protoplast transformation (see, e.g., Chang and Cohen, 1979,
Molecular General Genetics
168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961,
Journal ofBacteriology 81:
823-829, or Dubnau and Davidoff Abelson, 1971, Journal ofMolecularBiology 56:
209-221),
electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-
751), or conjugation (see,
e.g., Koehler and Thorne, 1987, Journal ofBacteriology 169: 5771-5278).
25 Examples of suitable filamentous fungal host cells include strains of
Aspergillus, e.g. A. oryzae,
A. niger, or A. nidulans, Fusarium or Trichoderma. Fungal cells may be
transformed by a process
involving protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a
manner known per se. Suitable procedures for transformation of Aspergillus
host cells are described in
EP 238 023 and US 5,679,543. Suitable methods for transforming Fusarium
species are described by
3o Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be
transformed using the
procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.L,
editors, Guide to Yeast
Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187,
Academic Press,
Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen
et al., 1978, Proceedings
of the National Academy of Sciences USA 75: 1920.
35 Examples of suitable yeast host cells include strains of Saccharomyces,
e.g. S. cerevisiae,
Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P.
methanolica, Hansenula, such as
H. Polymorpha or Yarrowia. Methods for transforming yeast cells with
heterologous DNA and
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
104
producing heterologous polypeptides therefrom are disclosed by Clontech
Laboratories, Inc, Palo Alto,
CA, USA (in the product protocol for the Yeastmaker~ Yeast Tranformation
System Kit), and by
Reeves et al., FEMS Microbiology Letters 99 (1992) 193-198, Manivasakam and
Schiestl, Nucleic
Acids Research, 1993, Vol. 21, No. 18, pp. 4414-4415 and Ganeva et al., FEMS
Microbiology Letters
121 (1994) 159-164.
Examples of suitable insect host cells include a Lepidoptora cell line, such
as Spodoptera
frugiperda (Sf~ or Sf21 ) or Trichoplusioa ni cells (High Five) (LJS
5,077,214). Transformation of insect
cells and production of heterologous polypeptides therein may be performed as
described by Invitrogen.
Examples of suitable mammalian host cells include Chinese hamster ovary (CHO)
cell lines,
(e.g. CHO-Kl; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC
CRL-1650), COS
7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell
lines (e.g. ATCC
CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as
well as plant
cells in tissue culture. Additional suitable cell lines are known in the art
and available from public
depositories such as the American Type Culture Collection, Rockville,
Maryland. Also, the mammalian
cell, such as a CHO cell, may be modified to express sialyltransferase, e.g.
2,3-sialyltransferase or 2,6-
sialyltransferase, e.g. as described in US 5,047,335, in order to provide
improved glycosylation of the
adiponectin polypeptide.
Methods for introducing exogeneous DNA into mammalian host cells include
calcium
phosphate-mediated transfection, electroporation, DEAF-dextran mediated
transfection, liposome-
mediated transfection, viral vectors and the transfection methods described by
Life Technologies Ltd,
Paisley, UK using Lipofectamin 2000 and Roche Diagnostics Corporation,
Indianapolis, USA using
FuGENE 6. These methods are well known in the art and e.g. described by Ausbel
et al. (eds.), 1996,
Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The
cultivation of
mammalian cells are conducted according to established methods, e.g. as
disclosed in (Animal Cell
Biotechnology, Methods and Protocols, Edited by Nigel Jerkins, 1999, Human
Press Inc, Totowa, New
Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture,
Cambridge University
Press 1997).
In the production methods of the present invention, the cells are cultivated
in a nutrient medium
suitable for production of the polypeptide using methods known in the art. For
example, the cell may be
3o cultivated by shake flask cultivation, small-scale or large-scale
fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or industrial
fermenters performed in a
suitable medium and under conditions allowing the polypeptide to be expressed
and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising carbon and
nitrogen sources and
inorganic salts, using procedures known in the art. Suitable media are
available from commercial
suppliers or may be prepared according to published compositions (e.g., in
catalogues of the American
Type Culture Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can
be recovered directly from the medium. If the polypeptide is not secreted, it
can be recovered from cell
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
105
lysates. Preferably a medium containing calcium is used, such as DMEM/F-
12(1:1) medium Cat no
21041 (Invitrogen). However, media without calcium may also be used, such as
DMEM Cat no 21068
(Invitrogen).
The resulting polypeptide may be recovered by methods known in the art. For
example, the
polypeptide may be recovered from the nutrient medium by conventional
procedures including, but not
limited to, centrifizgation, filtration, extraction, spray drying,
evaporation, or precipitation.
The polypeptides may be purified by a variety of procedures known in the art
including, but not
limited to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative isoelectric
focusing), differential solubility (e.g.,
l0 ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,
Protein Purification, J.-C. Janson
and Lars Ryden, editors, VCH Publishers, New York, 1989).
In connection with the preparation of the conjugate(s), or adiponectin
polypeptide fragment of
the invention a novel method of preparing a desired adiponectin polypeptide
(such as any one of seq id
no 2-8, 10-13, and 17-61), in a mammalian cell has been established.
Basically, a cDNA encoding the
signal peptide apMl(1-17) wherein the last two C-terminal amino acids are HD
and an adiponectin
polypeptide having a Gly residue as the N-terminal amino acid was prepared as
described in the
examples. Using the SignalP V1.1 World Wide Web Server to predict a suitable
signal peptide, we
discovered that the creation of an HDG amino acid sequence at the C-terminal
part of the signal peptide
and the desired adiponectin polypeptide resulted in a cleavage site after the
G of HDG. By constructing
2o a cDNA as outlined in the examples using the naturally occurnng Glycin
residues in the collagenous
domain of apMl, cleavage sites can be established after e.g. G42, G45, G48,
G51, G54, G57, G60, or
G63 of the collagenous domain of apMl (thus leaving the N-terminal amino acid
residues: I43, H46,
H49, A52, R55, R58, T61, or E64, respectively), in this respect the signal
peptide should include His
and Asp as the last two C-terminal amino acids, or alternatively, if a
cleavage site should be established
after G57, or G60, the signal peptide may include His as the last C-terminal
amino acid, and make use
of the Asp56, or Asp 59, respectively. However, any desired fragment of the
adiponectin polypeptide
may be prepared, in which case the last three C-terminal amino acids of the
signal peptide should be
HDG; a non-limiting example is preparation of apMl(101-244) by preparing a
cDNA sequence
encoding a signal peptide wherein the last three C-terminal amino acids are
HDG, and apMl(101-244)
thus making it possible for the mammalian cells, such as any one of those
mentioned above, preferably
CHO cells, to cleave between the C-terminal G of the signal peptide and K101
of apMl(101-244). Thus,
a signal peptide wherein the last three C-terminal amino acids of the signal
peptide are HDG, also covers
the above mentioned situations where a G or DG is the N-terminal amino acids)
of the adiponectin
polypeptide.
Accordingly, in a further aspect the present invention relates to a method of
preparing an
adiponectin polypeptide, comprising
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
106
a) preparing a nucleotide sequence encoding: a signal peptide and the
adiponectin polypeptide, wherein
the last three C-terminal amino acids of the signal peptide are HDG,
b) inserting the nucleotide sequence into a vector,
c) transfecting the vector into a mammalian cell, and
d) expressing and optionally secreting the adiponectin polypeptide.
In a further aspect the present invention relates to a method of preparing an
adiponectin
polypeptide, comprising
a) preparing a nucleotide sequence encoding: a signal peptide and the
adiponectin polypeptide, wherein
the last three C-terminal amino acids of the signal peptide are HDG,
1 o b) inserting the nucleotide sequence into a vector,
c) transfecting the vector into a mammalian cell,
d) expressing and optionally secreting the adiponectin polypeptide, and
e) obtaining the adiponectin polypeptide.
In one embodiment step d involves expressing and secreting the adiponectin
polypeptide.
15 Obtaining the adiponectin polypeptide in step e, typically comprises
recovering and purifying
the expressed and optionally secreted adiponectin polypeptide. Such methods of
recovering or purifying
are available to the skilled person, and suitable examples are outlined above.
In a further embodiment the nucleotide sequence is selected from RNA, DNA, or
cDNA,
preferably cDNA. In a further embodiment the RNA, DNA, or cDNA comprises a
sequence selected
2o from seq id no 9, 14, 15, or 16, as well as sequences having at least 70%
homology with any one of seq
id no 9, 14, 15 or 16. More preferred are sequences having at least 80%
homology, such as 90%, 92%,
95% or 98% homology with any one of seq id no 9, 14, 15 or 16.
In a further embodiment the RNA, DNA, or cDNA comprises a sequence selected
from seq id
no 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71, as well as sequences having at
least 70% homology with any
25 one of seq id no 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. More preferred
are sequences having at least
80% homology, such as 90%, 92%, 95% or 98% homology with any one of seq id no
62, 63, 64, 65, 66,
67, 68, 69, 70, or 71.
In a further embodiment the signal peptide is selected from the sequence
MLLLGAVLLLLALPGHDG, or MLLLQALLFLLILPSI~G, preferably
so MLLLGAVLLLLALPGHDG.
In a further embodiment the vector is an expressions vector, such as a plasmid
or viral DNA.
Any of the above mentioned mammalian cells are suitable as the host cell
expressing the
adiponectin polypeptide. In a further embodiment the mammalian cell is
selected from a CHO cell.
35 Other methods of the invention
In a still further aspect the invention relates to a method of reducing
immunogenicity and/or of
increasing functional in vivo half life and/or serum half life of an
adiponectin polypeptide, which
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
107
method comprises introducing an amino acid residue constituting an attachment
group for a non-
polypeptide moiety into a position exposed at the surface of the protein that
does not contain such group
and removing an amino acid residue constituting an attachment group for a non-
polypeptide moiety and
subjecting the resulting modified polypeptide to conjugation with the non-
polypeptide moiety.
In one embodiment the non-polypeptide moiety is selected from the group
consisting of a
polymer molecule, a sugar moiety, a lipophilic group and an organic
derivatizing agent.
Preferably, the amino acid residue to be introduced and/or removed is as
defined in the present
application.
In a further aspect the invention relates to a composition comprising any one
of the above
l0 conjugates, or adiponectin polypeptide fragments. Such composition is
typically, selected from a
pharmaceutical composition as described below, but may be a bulk composition,
such as a freeze dried
bulk composition, or liquid composition.
Pharmaceutical composition and uses of a conjugate or adiponectin polypeptide
fragment of the
15 invention
In the following sections reference is only made to a conjugate of the
invention, however, in
connection with the description of a pharmaceutical composition the phrase,
conjugate, also includes
adiponectin polypeptides as well as fragments. The conjugate of the invention
is administered at a dose
typically in the range of 0.001 mg/kg to 0.5 mg/kg body weight. The exact dose
to be administered
2o depends on the circumstances. Normally, the dose should be capable of
preventing or lessening the
severity or spread of the condition or indication being treated. It will be
apparent to those of skill in the
art that an effective amount of a conjugate or composition of the invention
depends, inter alia, upon the
disease, the dose, the administration schedule, whether the conjugate or
composition is administered
alone or in conjunction with other therapeutic agents, the serum half life of
the compositions, and the
25 general health of the patient.
The conjugate of the invention can be used "as is" and/or in a salt form
thereof. Suitable salts
include, but are not limited to, salts with alkali metals or alkaline earth
metals, such as sodium,
potassium, lithium, calcium and magnesium, as well as e.g. zinc salts. These
salts or complexes may by
present as a crystalline and/or amorphous structure.
3o The conjugate of the invention is preferably administered in a composition
including a
pharmaceutically acceptable carrier or excipient. "Pharmaceutically
acceptable" means a Garner or
excipient that does not cause any untoward effects in patients to whom it is
administered. Such
pharmaceutically acceptable carriers and excipients are well known in the art.
The conjugate of the invention can be formulated into pharmaceutical
compositions by well-
35 known methods. Suitable formulations are described in US 5,183,746,
Remington's Pharmaceutical
Sciences by E.W.Martin, 18''' edition, A. R. Gennaro, Ed., Mack Publishing
Company [1990];
Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer
and L. Hovgaard, Eds.,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
108
Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd
edition, A. Kibbe, Ed.,
Pharmaceutical Press [2000]).
The pharmaceutical composition of the conjugate of the invention may be
formulated in a
variety of forms, including liquid, gel, lyophilized, pulmonary dispersion, or
any other suitable form,
e.g. as a compressed solid. The preferred form will depend upon the particular
indication being treated
and will be apparent to one of skill in the art.
The pharmaceutical composition containing the conjugate of the invention may
be administered
orally, intravenously, intracerebrally, intramuscularly, intraperitoneally,
intradermally, subcutaneously,
intranasally, intrapulmonary, by inhalation, or in any other acceptable
manner, e.g. using PowderJect or
t o Protease technology. The preferred mode of administration will depend upon
the particular indication
being treated and will be apparent to one of skill in the art.
Parenterals
An example of a pharmaceutical composition is a solution designed for
parenteral
administration. Although in many cases pharmaceutical solution formulations
are provided in liquid
form, appropriate for immediate use, such parenteral formulations may also be
provided in frozen or in
lyophilized form. In the former case, the composition must be thawed prior to
use. The latter form is
often used to enhance the stability of the active compound contained in the
composition under a wider
variety of storage conditions, as it is recognized by those skilled in the art
that lyophilized preparations
2o are generally more stable than their liquid counterparts. Such lyophilized
preparations are reconstituted
prior to use by the addition of one or more suitable pharmaceutically
acceptable diluents such as sterile
water for injection or sterile physiological saline solution.
In case of parenterals, they are prepared for storage as lyophilized
formulations or aqueous
solutions by mixing, as appropriate, the conjugate having the desired degree
of purity with one or more
pharmaceutically acceptable carriers, excipients or stabilizers typically
employed in the art (all of which
are termed "excipients"), for example buffering agents, stabilizing agents,
preservatives, isotonifiers,
non-ionic detergents, antioxidants and/or other miscellaneous additives.
Buffering agents help to maintain the pH in the range which approximates
physiological
conditions. They are typically present at a concentration ranging from about 2
mM to about 50 mM.
3o Suitable buffering agents for use with the present invention include both
organic and inorganic acids and
salts thereof such as citrate buffers (e.g., monosodium citrate-disodium
citrate mixture, citric acid-
trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.),
succinate buffers (e.g., succinic
acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture,
succinic acid-disodium
succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium
tartrate mixture, tartaric acid-
potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
fumarate buffers (e.g., fumaric
acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture,
monosodium fumarate-
disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-
sodium glyconate mixture,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
109
gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate
mixture, etc.), oxalate
buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide
mixture, oxalic acid-
potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium
lactate mixture, lactic acid-
sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and
acetate buffers (e.g., acetic
acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.).
Additional possibilities are
phosphate buffers, histidine buffers and trimethylamine salts such as Tris.
Preservatives are added to
retard microbial growth, and are typically added in amounts of about 0.2%-1 %
(w/v). Suitable
preservatives for use with the present invention include phenol, benzyl
alcohol, meta-cresol, methyl
paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride,
benzalkonium halides (e.g.
to benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl
parabens such as methyl or
propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
Isotonicifiers are added to ensure isotonicity of liquid compositions and
include polyhydric
sugar alcohols, preferably trihydric or higher sugar alcohols, such as
glycerin, erythritol, arabitol,
xylitol, sorbitol and mannitol. Polyhydric alcohols can be present in an
amount between 0.1 % and 25%
15 by weight, typically 1 % to 5%, taking into account the relative amounts of
the other ingredients.
Stabilizers refer to a broad category of excipients which can range in
function from a bulking
agent to an additive which solubilizes the therapeutic agent or helps to
prevent denaturation or
adherence to the container wall. Typical stabilizers can be polyhydric sugar
alcohols (enumerated
above); amino acids such as arginine, lysine, glycine, glutamine, asparagine,
histidine, alanine, omithine,
20 L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars
or sugar alcohols, such as
lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,
myoinisitol, galactitol, glycerol and the
like, including cyclitols such as inositol; polyethylene glycol; amino acid
polymers; sulfur-containing
reducing agents, such as urea, glutathione, thioctic acid, sodium
thioglycolate, thioglycerol, a-
monothioglycerol and sodium thiosulfate; low molecular weight polypeptides
(i.e. <10 residues);
25 proteins such as human serum albumin, bovine serum albumin, gelatin or
immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; monosaccharides such as xylose,
mannose, fructose and
glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides
such as raffinose, and
polysaccharides such as dextran. Stabilizers are typically present in the
range of from 0.1 to 10,000 parts
by weight based on the active protein weight.
30 Non-ionic surfactants or detergents (also known as "wetting agents") may be
present to help
solubilize the therapeutic agent as well as to protect the therapeutic
polypeptide against agitation-
induced aggregation, which also permits the formulation to be exposed to shear
surface stress without
causing denaturation of the polypeptide. Suitable non-ionic surfactants
include polysorbates (20, 80,
etc.), polyoxamers (184, 188 etc:), Pluronic~ polyols, polyoxyethylene
sorbitan monoethers (Tween~-
35 20, Tween~-80, etc.).
Additional miscellaneous excipients include bulking agents or fillers (e.g.
starch), chelating agents (e.g.
EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and
cosolvents. The active ingredient
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
110
may also be entrapped in microcapsules prepared, for example, by coascervation
techniques or by
interfacial polymerization, for example hydroxymethylcellulose, gelatin or
poly-(methylmethacylate)
microcapsules, in colloidal drug delivery systems (for example liposomes,
albumin microspheres,
microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
Parenteral formulations to be used for in vivo administration must be sterile.
This is readily
accomplished, for example, by filtration through sterile filtration membranes.
Sustained release preparations
1 o Suitable examples of sustained-release preparations include semi-permeable
matrices of solid
hydrophobic polymers containing the conjugate, the matrices having a suitable
form such as a film or
microcapsules. Examples of sustained-release matrices include polyesters,
hydrogels (for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides,
copolymers of L-glutamic acid
and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic acid-glycolic acid
15 copolymers such as the ProLease~ technology or Lupron Depot~ (injectable
microspheres composed of
lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-
hydroxybutyric acid. While
polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for
long periods such as up to or over 100 days, certain hydrogels release
proteins for shorter time periods.
When encapsulated polypeptides remain in the body for a long time, they may
denature or aggregate as a
2o result of exposure to moisture at 37°C, resulting in a loss of
biological activity and possible changes in
immunogenicity. Rational strategies can be devised for stabilization depending
on the mechanism
involved. For example, if the aggregation mechanism is discovered to be
intermolecular S-S bond
formation through thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture content,
using appropriate additives,
25 and developing specific polymer matrix compositions.
Pulmonary delivery
Conjugate formulations suitable for use with a nebulizer, either jet or
ultrasonic, will typically
comprise the conjugate dissolved in water at a concentration of, e.g., about
0.01 to 25 mg of conjugate
3o per mL of solution, preferably about 0.1 to 10 mg/mL. The formulation may
also include a buffer and a
simple sugar (e.g., for protein stabilization and regulation of osmotic
pressure), and/or human serum
albumin ranging in concentration from 0.1 to 10 mg/ml. Examples of buffers
that may be used are
sodium acetate, citrate and glycine. Preferably, the buffer will have a
composition and molarity suitable
to adjust the solution to a pH in the range of 3 to 9. Generally, buffer
molarities of from 1 mM to 50 mM
35 are suitable for this purpose. Examples of sugars which can be utilized are
lactose, maltose, mannitol,
sorbitol, trehalose, and xylose, usually in amounts ranging from 1 % to 10% by
weight of the
formulation.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
111
The nebulizer formulation may also contain a surfactant to reduce or prevent
surface induced
aggregation of the protein caused by atomization of the solution in forming
the aerosol. Various
conventional surfactants can be employed, such as polyoxyethylene fatty acid
esters and alcohols, and
polyoxyethylene sorbitan fatty acid esters. Amounts will generally range
between 0.001 % and 4% by
weight of the formulation. An especially preferred surfactant for purposes of
this invention is
polyoxyethylene sorbitan monooleate.
Specific formulations and methods of generating suitable dispersions of liquid
particles of the invention
are described in WO 9420069, US 5915378, US 5960792, US 5957124, US 5934272,
US 5915378, US
5855564, US 5826570 and US 5522385 which are hereby incorporated by reference.
1 o Three specific examples of commercially available nebulizers suitable for
the practice of this
invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St.
Louis, Mo., the Acorn II
nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado, and
the AERx
pulmonary drug delivery system manufactured by Aradigm Corporation, Hayward,
California.
Conjugate formulations for use with a metered dose inhaler device will
generally comprise a
finely divided powder. This powder may be produced by lyophilizing and then
milling a liquid
conjugate formulation and may also contain a stabilizer such as human serum
albumin (HSA).
Typically, more than 0.5% (w/w) HSA is added. Additionally, one or more sugars
or sugar alcohols may
be added to the preparation if necessary. Examples include lactose maltose,
mannitol, sorbitol, sorbitose,
trehalose, xylitol, and xylose. The amount added to the formulation can range
from about 0.01 to 200%
(w/w), preferably from approximately 1 to 50%, of the conjugate present. Such
formulations are then
lyophilized and milled to the desired particle size.
The properly sized particles are then suspended in a propellant with the aid
of a surfactant. The
propellant may be any conventional material employed for this purpose, such as
a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-
tetrafluoroethane, or combinations
thereof. Suitable surfactants include sorbitan trioleate and soya lecithin.
Oleic acid may also be useful as
a surfactant. This mixture is then loaded into the delivery device. An example
of a commercially
available metered dose inhaler suitable for use in the present invention is
the Ventolin metered dose
inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.
Such conjugate formulations for powder inhalers will comprise a finely divided
dry powder
containing conjugate and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or
mannitol in amounts which facilitate dispersal of the powder from the device,
e.g., 50% to 90% by
weight of the formulation. The particles of the powder shall have aerodynamic
properties in the lung
corresponding to particles with a density of about 1 g/cm2 having a median
diameter less than 10
micrometers, preferably between 0.5 and 5 micrometers, most preferably of
between 1.5 and 3.5
micrometers.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
112
An example of a powder inhaler suitable for use in accordance with the
teachings herein is the
Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
The powders for these devices may be generated and/or delivered by methods
disclosed in US 5997848,
US 5993783, US 5985248, US 5976574, US 5922354, US 5785049 and US 55654007.
The pharmaceutical composition containing the conjugate of the invention may
be administered
by a wide range of mechanical devices designed for pulmonary delivery of
therapeutic products,
including but limited to nebulizers, metered dose inhalers, and powder
inhalers, all of which are familiar
to those of skill in the art.
Some specific examples of commercially available devices suitable for the
practice of this
invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St.
Louis, Missouri; the
Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood,
Colorado; the Ventolin
metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park,
North Carolina; the
Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford,
Massachusetts; the "standing cloud"
device of Inhale Therapeutic Systems, Inc., San Carlos, California; the AIR
inhaler manufactured by
t 5 Alkermes, Cambridge, Massachusetts; and the AERx pulmonary drug delivery
system manufactured by
Aradigm Corporation, Hayward, California.
The pharmaceutical composition of the invention may be administered in
conjunction with other
therapeutic agents. These agents may be incorporated as part of the same
pharmaceutical composition or
may be administered separately from the conjugate of the invention, either
concurrently or in accordance
2o with any other acceptable treatment schedule.
In a further aspect the conjugate or adiponectin polypeptide fragment of the
invention is
administered together with insulin, eg human recombinant insulin. In addition,
the conjugate, or
adiponectin polypeptide fragment, or pharmaceutical composition of the
invention may be used as an
adjunct to other therapies.
25 In a further aspect the invention relates to a pharmaceutical composition
comprising a conjugate
of the invention and a pharmaceutically acceptable diluent, carrier or
adjuvant.
In a further aspect the invention relates to a pharmaceutical composition
comprising an
adiponectin polypeptide fragment of the invention and a pharmaceutically
acceptable diluent, Garner or
adjuvant.
3o The adiponectin polypeptide fragment as part of the pharmaceutical
composition may be
selected from any one of the aspects or embodiments.disclosed in the above
sections "Adiponectin
polypeptide fragments) of the invention" and "Calcium composition aspects".
Moreover, the conjugate
as part of the pharmaceutical composition may be selected from any one of the
aspects or embodiments
disclosed in the above sections "First group of conjugates) of the invention",
"Second group of
35 conjugates) of the invention", "Third group of conjugates) of the
invention", and "Fourth group of
conjugates) of the invention", and "Calcium composition aspects".
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
113
Accordingly, this invention provides compositions and methods for treating
type 1 diabetes;
impaired glucose tolerance; type 2 diabetes; syndrome X; obesity;
cardiovascular disease, such as
atherosclerosis; dyslipidemia; or for lowering body weight without reducing
food intake; rheumatoid
arthritis; Crohn's disease; systemic lupus erythematosus; Sjogren's disease;
cachexia; septic shock;
myasthenia gravis; post-traumatic brain damage; myocardial infarction; post-
surgical brain-damage; and
other destructive processes related to stress or activation of the
inflammatory system; in particular IGT,
type 2 diabetes, syndrome X, dyslipidemia, septic shock, or cardiovascular
disease, such as
atherosclerosis.
In a further aspect the invention relates to a method of treating a mammal
with type 1 diabetes,
1 o IGT, type 2 diabetes, syndrome X, obesity, or dyslipidemia, or for
lowering body weight of a mammal
without reducing food intake, which method comprises administering an
effective amount of a conjugate
or adiponectin polypeptide fragment of the invention.
In a further aspect the invention relates to a method of treating a mammal
with rheumatoid
arthritis, Crohn's disease, systemic lupus erythematosus, Sjogren's disease,
cachexia, septic shock,
15 diabetes, myasthenia gravis, post-traumatic brain damage, myocardial
infarction, post-surgical brain-
damage, and other destructive processes related to stress or activation of the
inflammatory system,
which method comprises administering an effective amount of a conjugate or
adiponectin polypeptide
fragment of the invention.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
2o fragment of the invention for the manufacture of a medicament for treatment
of type 1 diabetes.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for treatment of
IGT.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for treatment of
type 2 diabetes.
25 In a further aspect the invention relates to use of a conjugate or
adiponectin polypeptide
fragment of the invention for the manufacture of a medicament for treatment of
syndrome X.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for treatment of
obesity.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
3o fragment of the invention for the manufacture of a medicament for treatment
of dyslipidemia.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for treatment of
septic shock.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for lowering
body weight of a mammal
35 without reducing food intake.
In a further aspect the invention relates to use of a conjugate or adiponectin
polypeptide
fragment of the invention for the manufacture of a medicament for treament of
rheumatoid arthritis,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
114
Crohn's disease, systemic lupus erythematosus, Sjogren's disease, cachexia,
myasthenia gravis, post-
traumatic brain damage, myocardial infarction, post-surgical brain-damage, and
other destructive
processes related to stress or activation of the inflammatory system. Any one
of the specified conditions,
diseases or disorders is considered separate embodiments of the invention, and
as such can be the
subject of individual claims.
In fact, we have discovered that the adiponectin polypeptide of the invention,
including the
composition comprising the adiponectin polypeptide trimer stabilized with
calcium ions, and the
adiponectin polypeptide fragments of the invention have excellent effect as a
TNF-alpha inhibitor and
were able to inhibit LPS-induced TNF-alpha production in a monocytic cell
line. It is quite unexpected
1 o that the present adiponectin polypeptides have this effect, and it means
that the present adiponectin
polypeptides will be effective as medicaments in the treatment of diseases,
disorders, or conditions
caused by expression or release of TNF-alpha in a human cell, such as septic
shock..
Accordingly, in a further aspect the invention relates to use of an
adiponectin polypeptide or
conjugate such as any one of those mentioned in the above sections
"Adiponectin polypeptide
15 fragments) of the invention", "First group of conjugates) of the
invention", "Second group of
conjugates) of the invention", "Third group of conjugates) of the invention",
and "Fourth group of
conjugates) of the invention", and "Calcium composition aspects" for preparing
a medicament for
treatment of a disease, disorder, or condition caused by expression or release
of TNF-alpha in a human
cell, wherein said medicament inhibits expression or release of TNF-alpha.
20 In a further aspect the invention relates to use of an isolated complex
comprising
a) a conjugate comprising an adiponectin polypeptide trimer wherein the
adiponectin polypeptide trimer
contains three adiponectin polypeptide monomers, and one first polymer
covalently attached to any one
of the three monomers of the adiponectin polypeptide trimer in such a way that
the resulting trimer only
contains one polymer, and
25 b) calcium ions,
for preparing a medicament for treatment of a disease, disorder, or condition
caused by
expression or release of TNF-alpha in a human cell, wherein said medicament
inhibits expression or
release of TNF-alpha.
Diseases, disorders, or conditions which are caused by expression or release
of TNF-alpha in a
3o human cell are for instance, type 1 diabetes, IGT, type 2 diabetes,
syndrome X, obesity, or dyslipidemia
in any suitable animal, preferably mammal, and in particular human, or for
lowering body weight of a
mammal, in particular a human, without reducing food intake, rheumatoid
arthritis, Crohn's disease,
systemic lupus erythematosus, Sjogren's disease, cachexia, septic shock,
myasthenia gravis, post-
traumatic brain damage, myocardial infarction, post-surgical brain-damage, and
other destructive
35 processes related to stress or activation of the inflammatory system. As a
test assay for measuring
inhibitory effect of a composition or a conjugate comprising an adiponectin
polypeptide, the assay
described in example 23 may be used.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
115
The invention is further described in the following examples. The examples
should not, in any
manner, be understood as limiting the generality of the present specification
and claims.
Experimental
Structures:
There exist no experimental structures of any part of human adiponectin. An
experimental
structure determined by X-ray crystallography of the globular part of the
homologous protein from
mouse have been reported by Shapiro & Scherer (1998) Current Biology, 8, 335-
338. They report the
l0 structure of an asymmetrical homotrimer of the region equivalent to V 110
to N244 in sequence id no 1
determined to a resolution of 2.1~. In this region the mouse sequence has 12
differences as compared to
the human sequence, i.e. 91% sequence identity (see also scheme 1). The
structure showed an
assymmetrical trimer of beta-sandwich structured monomers, each monomer having
a ten-strand jelly-
roll folding topology identical to the known TNF (tumor necrosis factor)
structures (e.g. Banner et.al.
~5 (1993) Cell, 73, 431-445). In the structure several regions were not
detected, most probably due to
dynamical behaviour of these regions that are located in loop regions and at
the C-terminal. These
regions were (using the residue numbering of the homologous human protein as
shown in sequence id
no 1 and in brackets are shown the original numbering from the structure file
deposited in PDB (Berman
et.al. (2000) Nucleic Acids Research, 28, 235-242) having accession code
1C28): In molecule A:
2o K192{A195}, G217{A220}-L224{A227}, N244{A247}. In molecule B: E120{B123}-
V125{B128},
A181 {B184}, N193 {B196}, G217{B220}-N230{B233}, T243 {B246}-N244{B247}. In
molecule C:
V125{C128}-N127{C130}, Y167{C170}-K169{C172}, Y186{C189}-D195{C198},
V215{C218}-
V229{C232}, T243{C246}-N244{C247} (see also scheme 1).
Besides the known structure of mouse adiponectin there have been reported a
structure of
25 another molecule homologous to human adiponectin. Bogin et.al. (2002)
Structure, 10, 165-173, reports
the structure of the globular part of the homologous Collagen X. In this
region this molecule has 59
residues identical to human adiponectin i.e. 44% sequence identity (see scheme
3). They report a well
resolved symmetrical trimer molecule where the most noticeable differences to
the reported marine
adiponectin are the presence of four calcium ions and one sodium ion. These
ions are located in the
3o region where the marine adiponectin are disordered in the structure. Three
of the calcium ions are
related by symmetry and are coordinated by the sidechains of two aspartic acid
residues (D626 and
D634), by the backbone carbonyl oxygen of E627 and by one water molecule. The
fourth calcium ion is
positioned on the three fold symmetry axis and is also coordinated by the side
chain of the three copies
of D634 as well as the same three water molecules which are also coordinated
to the other three calcium
35 ions. The sodium ion is also placed on the three fold symmetry axis 5.98
from the central calcium ion,
coordinated by the backbone oxygen atom of the three copies of Q635 and to
four water molecules.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
116
From our experiments (cf. examples 24 and 25) we have concluded that human
adiponectin,
such as fragment apMl(82-244) is stabilized on a trimeric form in the presence
of calcium ions, and that
destabilizing, such as by lowering the pH in the presence of phosphate ions,
results in a destabilized
trimer. As the residues coordinating the calcium ions are conserved between
collagen X and human
adiponectin, we conclude that D187 and D195 in the globular domain of human
adiponectin are
involved in the binding of calcium ions, and that mutation in one or both of
these positions results in
reduced affinity to calcium ions. Without being bound by theory, we believe
that adiponectin in additon
to binding calcium ions, probably also binds a sodium ion in the same manner
as Collagen X. A buried
conserved histidine residue (H163) located close to the symmetry axis at a
distance of app. 10~$ from the
t o metal ions can be an important player in the metal ion binding and general
stability of the trimer. Under
normal conditions this residue is neutral, but at low pH and low calcium ion
concentrations this residue
could be protonated and thereby destabilize the core of the trimer resulting
in a structure similar to the
experimentally determined murine adiponectin structure where the structural
parts surrounding the metal
ion binding sites becomes flexible and unstructured. On the other hand the
binding of the metal ions (i.e.
15 high metal ion concentrations) can lower the pKa of the histidine to a
level where it will not get
protonated even at low pH.
There exist a few experimental structures of collagen like molecules, all of
which are based on
synthetically produced collagen like fragments. A summary of the known
structures can be found in the
SCOP data base Murzin et.al. (1995). "SCOP: a structural classification of
proteins database for the
2o investigation of sequences and structures". J. Mol. Biol. 247, 536-540. In
this work the structure
reported by Berisio et.al. (2002) Protein Sci. 11, 262-270 are used for
modelling of the collagen part of
human adiponectin.
Methods
Accessible Surface Area (ASA)
The computer program Access (B. Lee and F.M.Richards, J. Mol.Biol. 55: 379-400
(1971))
version 2 (Copyright (c) 1983 Yale University) are used to compute the
accessible surface area (ASA) of
the individual atoms in the structure. This method typically uses a probe-size
of 1.41 and defines the
3o Accessible Surface Area (ASA) as the area formed by the centre of the
probe. Prior to this calculation all
water molecules and all hydrogen atoms should be removed from the coordinate
set, as should other
atoms not directly related to the protein.
Fractional ASA of side chain
The fractional ASA of the side chain atoms is computed by division of the sum
of the ASA of
the atoms in the side chain with a value representing the ASA of the side
chain atoms of that residue
type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton
(1991) J.Mol.Biol.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
117
220, 507-530. For this example the CA atom is regarded as a part of the side
chain of Glycine residues
but not for the remaining residues. The following table are used as standard
100% ASA for the side
chain:
Ala 69.23
~rz
Arg 200.35
t~z
Asn 106.25
~z
Asp 102.06
AZ
Cys 96.69
Az
Gln 140.58
~Z
Glu 134.61
~2
Gly 32.28
Az
His 147.00
~Z
Ile 137.91
AZ
Leu 140.76
~Z
Lys 162.50
~Z
Met 156.08
~r2
Phe 163.90
~2
Pro 119.65
t~2
Ser 78.16
AZ
Thr 101.67
AZ
Trp 210.89
t~2
Tyr 176.61
AZ
Val 114.14
~Z
Residues not detected in the structure are typically defined as having 100%
exposure as they are thought
to reside in flexible regions.
Determining distances between atoms:
1 o The distance between atoms is most easily determined using molecular
graphics software e.g.
InsightII v. 98.0, MSI INC, or InsightII v 2000.1, Accelrys INC.
HomoloQV modelling
Homology modelling based on sequence alignment to the sequence or sequences of
one or more
known structures are performed using the software Modeller 98, MSI INC, or
Modeler 2000.1, Accelrys
INC.
Modelling of the~lobular part of human adiponectin and determination of
surface accessibility:
Based on the known structure of the globular part of the murine adiponectin
molecule a
zo structure alignment to the human sequence was constructed as shown in
Scheme 1. From this alignment
a series of 20 model structures was build using Modeller 98 using the input
files shown in Scheme 2.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
118
Only the part from V 110 to N244 was modelled. To simplify further analysis
the individual monomers
was restrained to be identical using the subroutine "defsym" thereby resulting
in a symmetrical trimer as
is also seen for most of the homologous TNF like structures. The structure
with the lowest
"MODELLER OBJECTIVE FUNCTION" having a value of 9004 was structure number O5.
This
structure was selected as the best representative after an analysis with
PROCHECK ver. 3.4 (Laskowski
et.al. (1993). J. Appl. Cryst., 26, 283-291) showed an acceptable geometry for
all residues, with no
residues in the disallowed region of the Ramachandran plot.
In this example, we determined the relative surface accessibility of the side
chains in the first monomer
molecule of the model number OS in the context of the intact trimer. The
surface accessibility for the
other monomers was also calculated and the values correlated generally well
with the values for the first
molecule except in some loop regions, typically those where the residue
position had no structure
determined in at least one of the monomers in the template. As the quality of
the modelling in these
regions are expected to be of a lower quality, and as these residues in the
template structure at least in
one monomer have showed extreme flexibility, any position which is
undetermined in at least one of the
template monomers are defined as having 100% surface accessibility. These
residues are E120, T121,
Y122, V123, T124, I125, P126, N127, Y167, M168, K169, A181, Y186, D187, Q188,
Y189, Q190,
E191, N192, N193, V194, D195, V215, Y216, 6217, E218, 6219, E220, 8221, N222,
6223, L224,
Y225, A226, D227, N228, D229, N230, T243, N244 (here as in the rest of the
example the residue
numbering of sequence id no 1 is used). The residues A108 and Y109 were not
included in the model,
2o and are also defined as having 100% surface accessibility.
Surface exposure:
The following list describe the result of the surface area calculation,
revised with respect to the
above residues defined as having 100% surface accessibility.
Performing fractional ASA calculations on the first monomer of the 05 model
resulted in the
following residues having 0% of their side chain exposed to the surface: S
113, A114, F 11 S, S 116, V 117,
6118, F132, Y143, 6148, F150, C152, 6156, L157, Y158, F160, Y162, V166, V173,
S174, L175,
L183, A197, V201, L202, L203, L205, V211, L213, 5232, T235, 6236.
The following residues had more than 25% of their side chain exposed to the
surface: A108,
3o Y109, V110, Y1l l, 8112, E120, T121, Y122, V123, T124, I125, P126, N127,
M128, 8131, T133,
K134, I135, Q139, N141, D144, 6145, 5146, T147, K149, H151, N153, P155, Y167,
M168, K169,
D170, K178, D179, K180, A181, F184, Y186, D187, Q188, Y189, Q190, E191, N192,
N193, V194,
D195, H204, E206, V207, 6208, Q210, V215, Y216, 6217, E218, 6219, E220, 8221,
N222, 6223,
L224, Y225, A226, D227, N228, D229, N230, H241, D242, T243, N244.
The following residues had more than 50% of their side chain exposed to the
surface: A108,
Y109, V110, Y111, E120, T121, Y122, V123, T124, I125, P126, N127, M128, 8131,
Q139, N141,
D144, 6145, S146, N153, Y167, M168, K169, K178, D179, K180, A181, Y186, D187,
Q188, Y189,
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
119
Q190, E191, N192, N193, V194, D195, E206, V207, 6208, V215, Y216, 6217, E218,
6219, E220,
8221, N222, 6223, L224, Y225, A226, D227, N228, D229, N230, H241, T243, N244.
The following residues had 100% of their side chain exposed to the surface:
A108, Y109, E120,
T121, Y122, V123, T124, I125, P126, N127, Y167, M168, K169, A181, Y186, D187,
Q188, Y189,
Q190, E191, N192, N193, V194, D195, V215, Y216, 6217, E218, 6219, E220, 8221,
N222, 6223,
L224, Y225, A226, D227, N228, D229, N230, T243, N244.
Modelling of a calcium bound truncated form of human adiponectin (E82-N244)
and determination of
surface accessibility:
In order to model the globular domain of human adiponectin in the context of
the collagen part
and in the context of bound metal ions as in the known structure of collagen
X, a modelling of the
fragment E82-N244 has been performed. The modelling was based on the known
crystal structure of the
globular part of the marine adiponectin molecule Shapiro & Scherer (1998)
Current Biology, 8, 335-
338, the crystal structure of the globular part of collagen X (Bogin et.al.
,2002, Structure, 10, 165-173),
15 and on the crystal structure of the collagen triple helix [(Pro-Pro-
Gly)~o]3 as reported by Berisio et.al.
(2002) Protein Sci. 11, 262-270.
The structure of the globular part of collagen X reports only one of the
monomers in the
symmetrical trimer. The other two can be constructed by application of the
appropriate symmetry
operations using e.g. the software Swiss-PdbViewer v.3.7 (Guex et al., 1997,
Electrophoresis 18,2714-
20 2723).
From the collagen structure, the monomers labelled A, B and C was used in the
modelling.
An alignment of the amino acid sequences of the above structures to the human
adiponectin
sequence was constructed as shown in Scheme 3 and was the basis of the
modelling. Prior to the
modelling the marine adiponectin structure and the collagen X structure was
structurally aligned using
25 Modeler 2000.1 and the collagen structure was placed in an orientation
relative to the two other
molecules that was app. 100 away to minimize any bias on the resulting
structures from the original
configuration of the template structures.
A modelling strategy where extra constraints was added to keep the globular
part in a
symmetrical trimer structure was enforced by constraining residues V29-N244 in
each of the trimers to
3o be in the same conformation by use of the DEFINE-SYMMETRY command in the
Modeler software.
The three individual calcium ions was also constrained to each other.
From this alignment a series of 20 model structures was build using Modeller
2000.1 with the
input files shown in Scheme 4. The structure with the lowest "MODELLER
OBJECTIVE FUNCTION"
having a value of 17751 was structure number 03. This structure was selected
as the best representative
35 after an analysis with PROCHECK ver. 3.4 (Laskowski et.al. (1993). J. Appl.
Cryst., 26, 283-291)
showed an acceptable geometry for all residues, with only few residues in the
disallowed region of the
Ramachandran plot, and most of these belonging to the collagen part.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
120
In this example, we determined the absolute and relative surface accessibility
of the side chains
in all three monomer molecules of the model number 03 in the context of the
intact trimer and including
the five metal ions. Besides the few residues having contact to the collagen
stalk the surface accessibility
for the three monomers was generally quite identical in the globular region
(V110-N244) having an
average difference in side chain accessible surface area of 1.0 .~2.
Surface exposure:
Performing fractional ASA calculations on the three monomers of the model 03
resulted in the
following residues having 0% of their side chain exposed to the surface in all
three monomers: G87,
1o G90, G93, S113, A114, F115, 5116, V117, 6118, F132, Y143, 6148, C152, 6156,
L157, Y158, F160,
V166, 5174, L175, D187, D195, 5198, V201, L202, L205, V211, L213, 6223, A226,
5232, F234,
6236, F237.
The following residues had more than 25% of their side chain exposed to the
surface in at least
one of the monomers: E82, T83, G84, V85, P86, A88, E89, P91, R92, F94, P95,
I97, Q98, 8100, K101,
E103, P104, 6105, E106, 6107, A108, Y109, Y111, E120, T121, Y122, V123, I125,
N127, M128,
8131, T133, K134, I135, Q139, N141, D144, 6145, 5146, T147, K149, H151, N153,
P155, Y167,
K169, K178, D179, K180, M182, Q188, E191, N192, H204, E206, V207, 6208, 6217,
E218, 6219,
E220, 8221, Y225, D227, N228, D229, H241, D242, T243, N244.
The following residues had more than 50% of their side chain exposed to the
surface in at least one of
2o the monomers: E82, T83, G84, V85, P86, A88, E89, P91, R92, F94, P95, I97,
Q98, 8100, K101, E103,
P104, 6105, E106, 6107, A108, Y109, Y111, E120, T121, Y122, I125, N127, M128,
8131, N141,
6145, 5146, N153, K169, K178, D179, K180, E191, N192, E206, V207, 6208, 6217,
E218, 6219,
8221, N228, D229, H241, T243, N244.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
121
Scheme 1
Sequence alignment the globular domain of human adiponectin (residues V110-
N244) to marine
adiponectin. The residues present in each of the three independent monomers in
the structure of the
marine protein are shown on the lines labelled 1 C28 A, 1 C28 B and 1 C28 C.
Human . VYRSAFSVGL ETYVTIPNMP IRFTKIFYNQ QNHYDGSTGK
Mouse . MYRSAFSVGL ETRVTVPNVP IRFTKIFYNQ QNHYDGSTGK
1C28 A . MYRSAFSVGL ETRVTVPNVP IRFTKIFYNQ QNHYDGSTGK
1C28 B . MYRSAFSVGL ------PNVP IRFTKIFYNQ QNHYDGSTGK
1C28 C . MYRSAFSVGL ETRVT---VP IRFTKIFYNQ QNHYDGSTGK
Human FHCNIPGLYY FAYHITVYMK DVKVSLFKKD KAMLFTYDQY
.
Mouse . FYCNIPGLYY FSYHITVYMK DVKVSLFKKD KAVLFTYDQY
1C28 A . FYCNIPGLYY FSYHITVYMK DVKVSLFKKD KAVLFTYDQY
1C28 B . FYCNIPGLYY FSYHITVYMK DVKVSLFKKD K-VLFTYDQY
1C28 C . FYCNIPGLYY FSYHITV--- DVKVSLFKKD KAVLFT----
Human . QENNVDQASGSVLLHLEVGD QVT~)LQVYGEG ERNGLYADND
Mouse . QEKNVDQASGSVLLHLEVGD QVV~ILQVYGDG DHNGLYADNV
1C28 A . QE-NVDQASGSVLLHLEVGD QVV~ILQVY--- -----YADNV
1C28 . QEK-VDQASGSVLLHLEVGD QVWLQVY--- ----------
B
1C28 C . ------QASGSVLLHLEVGD QVWLQ----- ----------
Human . NDSTFTGFLL YHDTN
3o Mouse . NDSTFTGFLLYHDTN
1C28 A . NDSTFTGFLLYHDT-
1C28 B . -DSTFTGFLLYHD--
1C28 C . NDSTFTGFLLYHD--
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
122
Scheme 2
Input files for Modeler 98
# model. top
S #
# Modelling of Human ACRP based on X-ray structure of Mouce ACRP:
# 1C28.PDB Mouse trimer enforcing trifold symmetry.
INCLUDE
SET OUTPUT_CONTROL= 1 1 1 1
SET ALNFILE = 'align.pir'
SET PDB_EXT = '.pdb'
SET KNOWNS = '1C28'
SET SEQUENCE = 'ACRP HUN1AN'
SET MODEL = 'ACRP HUMAN.pdb'
SET ATOM_FILES_DIRECTORY = './'
SET CSRFILE = 'model.rsr'
SET STARTING_MODEL = 1
SET ENDING MODEL = 20
SET FINAL MALIGN3D = 1
# Don't use spline functions to convert restraints
SET SPLINE_ON_SITE = off
#
# Set through variable target function
SET LIBRARY SCHEDULE = 1
# use all restraints to define Hot-spots
SET RSTRS_REFINED = 2
# Write report on individual optimizations
#
SET OUTPUT = 'NO REPORT SHORT'
SET MD LEVEL = 'refinel'
CALL ROUTINE = 'model'
STOP
SUBROUTINE ROUTINE = 'special restraints'
CALL ROUTINE = 'defsym'
RETURN
END SUBROUTINE
SUBROUTINE ROUTINE = 'defsym'
SET RES_TYPES = 'ALL'
SET ATOM_TYPES = 'ALL'
SET SELECTION_STATUS = 'INITIALIZE'
SET SELECTION SEARCH = 'SEGMENT'
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
123
SET SYMMETRY 0.5
WEIGHT =
_ ATOMSSET 2,SELECTION_SEGMENT'l:' 135:'
PICK = = '
ATOMS PICK
_ ATOMS_ 3,SELECTION_SEGMENT'136:''270:'
_ _SET =
PICK =
ATOMS PICK_
_ ADD = on off
DEFINE SYMMETRY
SYMMETRY
_ _ _SET 2,SELECTION_SEGMENT'136:''270:'
PICK ATOMS= =
ATOMS PICK_
_ ATOMS_SET 3,SELECTION_SEGMENT'271:''405:'
PICK = =
ATOMS PICK_
_ ADD = on off ,
DEFINE SYMMETRY
SYMMETRY
_ _ SET 2,SELECTION_SEGMENT'271:''405:'
PICK ATOMS_= =
ATOMS PICK_
_ ATOMS_SET 3,SELECTION_SEGMENT'l:' 135:'
ATOMS PICK_ = = '
PICK
_ ADD_SYMMETRY = on off
DEFINE_SYMMETRY
RETURN
END SUBROUTINE
Alignment file 'align.pir':
>P1;1C28
structureX:1C28_ABC.pdb:113 :A:245 :C:undefined:undefined:-1.00:-1.00
MYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHIT
VYMKDVKVSLFKKDKAVLFTYDQYQE-NVDQASGSVLLHLEVGDQVWLQVY--------Y
ADNVNDSTFTGFLLYHDT-/
MYRSAFSVGL------PNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHIT
VYMKDVKVSLFKKDK-VLFTYDQYQEK-VDQASGSVLLHLEVGDQVWLQVY---------
-----DSTFTGFLLYHD--/
MYRSAFSVGLETRVT---VPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHIT
V---DVKVSLFKKDKAVLFT----------QASGSVLLHLEVGDQVWLQ-----------
----NDSTFTGFLLYHD--
>P1~ACRP_HUMAN
sequence ACRP_HUMAN: . . . . . .-1.00:-1.00
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHIT
VYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLY
ADNDNDSTFTGFLLYHDTN/
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHIT
VYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLY
ADNDNDSTFTGFLLYHDTN/
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHIT
VYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLY
ADNDNDSTFTGFLLYHDTN*
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
124
Scheme 3
Sequence alignment of residues E82-N244 of human adiponectin to the globular
parts of
murine adiponectin and collagen X. The residues present in each of the three
independent
monomers in the structure of the murine protein are shown on the lines
labelled 1 C28 A,
s 1 C28 B and 1 C28 C. The sequence from the collagen X structure is labelled
1 GR3. The
sequence of the collagen template structure is labelled 1 K6F.
Human . ---ETGVPGA EGPRGFPGIQ GRKGEPGEGA Y
Mouse : _________- __________ __________ _
1C28 A . __________ __________ __________ _
1C28 B : __________ __________ __________ _
1C28 C : __________ ___--_____ __________ _
1GR3 ; __________ __________ __________ _
1K6F . PPGPPGPPGP PGPPGPPGPP GPPGPPGPP- -
Human . VYRSAFSVGL ETYVTIPNMP IRFTKIFYNQ QNHYDGSTGK
Mouse . MYRSAFSVGL ETRVTVPNVP IRFTKIFYNQ QNHYDGSTGK
1C28 A . MYRSAFSVGL ETRVTVPNVP IRFTKIFYNQ QNHYDGSTGK
1C28 MYRSAFSVGL ------PNVP IRFTKIFYNQ QNHYDGSTGK
B .
1C28 C . MYRSAFSVGL ETRVT---VP IRFTKIFYNQ QNHYDGSTGK
1GR3 . MPVSAFTVIL SKAYPAIGTP IPFDKILYNR QQHYDPRTGI
Human . FHCNIPGLYY FAYHITVYMK DVKVSLFKKD KAMLFTYDQY
Mouse FYCNIPGLYY FSYHITVYMKDVKVSLFKKD KAVLFTYDQY
.
1C28 A . FYCNIPGLYY FSYHITVYMKDVKVSLFKKD KAVLFTYDQY
1C28 B . FYCNIPGLYY FSYHITVYMKDVKVSLFKKD K-VLFTYDQY
1C28 C . FYCNIPGLYY FSYHITV---DVKVSLFKKD KAVLFT----
1GR3 . FTCQIPGIYY FSYHVHVKGTHVWVGLYKNG TPVMYTYDEY
Human . QENNVDQASG SVLLHLEVGDQVWLQVYGEG ERNGLYADND
Mouse . QEKNVDQASG SVLLHLEVGDQVWLQVYGDG DHNGLYADNV
1C28 A . QE-NVDQASG SVLLHLEVGDQVWLQVY--- -----YADNV
1C28 B . QEK-VDQASG SVLLHLEVGDQVWLQVY--- ----------
1C28 . ------QASGSVLLHLEVGDQVWLQ----- ----------
C
1GR3 . TKGYLDQASGSAIIDLTENDQVWLQLPNAE S-NGLYSSEY
Human . NDSTFTGFLL YHDTN
Mouse . NDSTFTGFLLYHDTN
1C28 . NDSTFTGFLLYHDT-
A
1C28 B . -DSTFTGFLLYHD--
1C28 C . NDSTFTGFLLYHD--
1GR3 . VHSSFSGFLVAPM--
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
125
Scheme 4
Input files for Modeler 2000.1
# Modelling of Human ACRP
#
INCLUDE
SET OUTPUT CONTROL= 1 1 1 1
SET ALNFILE = 'align.pir'
SET PDB EXT = '.pdb'
SET KNOWNS = '1C28' '1GR3 ABC ions' '1K6F ABC'
SET SEQUENCE = 'ACRP HUMAN'
SET MODEL = 'ACRP HUMAN.pdb'
SET ATOM FILES DIRECTORY = './'
SET CSRFILE = 'model.rsr'
SET HETATM IO = ON
READ ALIGNMENT FILE = ALNFILE, ALIGN CODES = KNOWNS SEQUENCE
CHECK ALIGNMENT
WRITE ALIGNMENT FILE = 'Malign.ali'
SET STARTING MODEL = 1
SET ENDING MODEL = 20
SET INITIAL MALIGN3D = 0
SET FINAL MALIGN3D = 0
SET GENERATE METHOD = 'transfer xyz'
SET OUTPUT CONTROL = 1 1 1 1 1
SET LIBRARY SCHEDULE = 1
SET MAX VAR ITERATIONS = 1000
SET MD LEVEL = 'refine 3'
CALL ROUTINE = 'model'
STOP
SUBROUTINE ROUTINE = 'special restraints'
CALL ROUTINE = 'defsym'
RETURN
END SUBROUTINE
SUBROUTINE ROUTINE = 'defsym'
SET RES TYPES = 'ALL'
SET ATOM TYPES = 'ALL'
SET SELECTION STATUS = 'INITIALIZE'
SET SELECTION SEARCH = 'SEGMENT'
SET SYMMETRY WEIGHT = 0.5
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
126
PICK ATOMS PICK ATOMS SET 2,SELECTIONSEGMENT'29:' '163:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'193:''327:'
= =
DEFINE SYMMETRY ADD SYMMETRY= on off
PICK ATOMS PICK ATOMS SET 2,SELECTIONSEGMENT'193:''327:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'357:''491:'
= =
DEFINE SYMMETRY ADD SYMMETRY= on off
PICK ATOMS PICK ATOMS SET 2,SELECTIONSEGMENT'357:''491:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'29:' '163:'
= =
DEFINE SYMMETRY ADD SYMMETRY= on off
PICK ATOMS PICKATOMS SET 2,SELECTIONSEGMENT'164:''164:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'328:''328:'
= =
DEFINE SYMMETRY ADD SYMMETRY= on off
PICK ATOMS PICK ATOMS SET 2,SELECTIONSEGMENT'328:''328:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'492:''492:'
= =
IS DEFINE SYMMETRYADD SYMMETRY= on off
PICK ATOMS PICK ATOMS SET 2,SELECTIONSEGMENT'492:''492:'
= =
PICK ATOMS PICK ATOMS SET 3,SELECTIONSEGMENT'164:''164:'
= =
DEFINE SYMMETRY ADD SYMMETRY= on off
RETURN
END SUBROUTINE
Alignment file 'align.pir':
>P1;1C28
structureX:1C28 ABC edited2.pdb:113 :A:245 :C: . .
_______________________________
MYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMK
DVKVSLFKKDKAVLFTYDQYQE-NVDQASGSVLLHLEVGDQVWLQVY--------YADNV
NDSTFTGFLLYHDT-/-/
MYRSAFSVGL------PNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMK
DVKVSLFKKDK-VLFTYDQYQEK-VDQASGSVLLHLEVGDQVWLQVY-------------
-DSTFTGFLLYHD--/-/
MYRSAFSVGLETRVT---VPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITV--
DVKVSLFKKDKAVLFT----------QASGSVLLHLEVGDQVWLQ--------------
NDSTFTGFLLYHD--/-/
_/_*
>P1;1GR3 ABC ions
structureX:lGR3 ABC ions edited.pdb:549 :A:902 :D: . . .
_-__________-__________________
MPVSAFTVILSKAYPAIGTPIPFDKILYNRQQHYDPRTGIFTCQIPGIYYFSYHVHVKGT
HVWVGLYKNGTPVMYTYDEYTKGYLDQASGSAIIDLTENDQVWLQLPNAES-NGLYSSEY
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
127
VHSSFSGFLVAPM--/3/
MPVSAFTVILSKAYPAIGTPIPFDKILYNRQQHYDPRTGIFTCQIPGIYYFSYHVHVKGT
HVWVGLYKNGTPVMYTYDEYTKGYLDQASGSAIIDLTENDQVWLQLPNAES-NGLYSSEY
VHSSFSGFLVAPM--/3/
MPVSAFTVILSKAYPAIGTPIPFDKILYNRQQHYDPRTGIFTCQIPGIYYFSYHVHVKGT
HVWVGLYKNGTPVMYTYDEYTKGYLDQASGSAIIDLTENDQVWLQLPNAES-NGLYSSEY
VHSSFSGFLVAPM--/3/
3/9*
>P1;1K6F ABC
structureX:lK6F ABC edited2.pdb:l :A:29 :C: . . .
PPGPPGPPGPPGPPGPPGPPGPPGPPGPP--
_______________________________________-____________-____-__
_____-__-______/_/
PPGPPGPPGPPGPPGPPGPPGPPGPPGPP--
__________-____/_/
PPGPPGPPGPPGPPGPPGPPGPPGPPGPP--
_____-________-/_/
-/-
>P1;ACRP HUMAN
sequence:ACRP HUMAN: . . . . . .-1.00:-1.00
---ETGVPGAEGPRGFPGIQGRKGEPGEGAY
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMK
DVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADND
NDSTFTGFLLYHDTN/3/
---ETGVPGAEGPRGFPGIQGRKGEPGEGAY
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMK
DVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADND
NDSTFTGFLLYHDTN/3/
---ETGVPGAEGPRGFPGIQGRKGEPGEGAY
VYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMK
DVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADND
NDSTFTGFLLYHDTN/3/
3/9*
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
128
Test Assay A: Determination of adiponectin's effect on glucose uptake in C2C12
cells.
In order to investigate if an adiponectin polypeptide or a conjugate is able
to enhance the
glucose uptake in muscle cells, both the basal level and the insulin
stimulated level, we used the C2C12
cell line (ATCC, Rockville, MD). Briefly, quadruple samples of C2C12 cells
(105/well) were
differentiated in 12-well plates in 1 ml DMEM medium, supplemented with 5%
horse serum, at 37°C for
4 days. Differentiated C2C 12 cells were then incubated in different
concentrations of adiponectin
polypeptide or conjugate for 24 hours, preferably for 4 hours. After washing,
the wells were stimulated
in the presence/absence of 100 nM insulin for 30 minutes. The wells were
washed and incubated for 15
1o minutes in the presence of 0.5 p.Ci/ml 3H-D-Glucose. Glucose uptake was
terminated by aspiration of
the solution. Cells were then washed three times, and radioactivity associated
with the cells was
determined by cell lysis in 0.1 M NaOH, followed by scintillation counting.
Aliquots of cell lysates were
used for protein determination.
15 Test Assay B: Measurement of inhibition of LPS-induced TNF-alpha
production.
In order to investigate if an adiponectin polypeptide or a conjugate is able
to inhibit LPS-
induced TNF-alpha production we used the monocytic cell line THP-1 (ATCC,
Rockville, MD). Briefly,
triplicate samples of THP-1 cells (105/well) were incubated in 96 well-plates
at 37° C with titrated
amounts of adiponectin (highest concentration SOOnM (25,5 ~g/ml) in serum free
cell culture medium
20 (RPMI-1640, containing 10 mM HEPES).
Following 18 h pre-incubation with adiponectin the cultures were incubated for
additional 4 h
with a final concentration of 0,5 pg/ml lipopolysaccharide (LPS) (List
Biologieals) and then 50 ~1
supernatant where withdrawn and frozen at -20° C for subsequent
analysis of TNF-alpha.
The diluted cell culture supernatants where analyzed for TNF-alpha content
using a standard
25 ELISA (R&D), and the IC50 of adiponectin where calculated using a 4-
parameter non linear regression
data analysis.
Test Assay C: Measurement of glucose production in primary hepatocytes.
Single-cell suspensions of hepatocytes are obtained from perfusions of Sprague-
Dawley rats
30 using the procedure of Berry and Friend (J. Cell. Biol. 43, 506-520, 1969)
and the perfusion mixture of
Leffert et al. (Methods Enzymol. 58, 536-544, 1979), alternatively pig
hepatocytes may also be used.
The cells are plated on tissue culture plastic for 6 h at a density of 2 x 105
cells per well in a 24-well
culture plate that is pre-coated with rat-tail collagen I. During plating
cells are cultured in RPMI 1640
medium supplemented with 10% FBS, penicillin/streptomycin, 10 microg/ml
insulin and 10 microM
35 dexamethasone. After allowing for adherence, the media is changed to RPMI
with 5 mM glucose, 0.4%
FCS, and no insulin or dexamethasone. The cells are allowed to equilibrate
overnight in this low-glucose
media. The following morning this media is refreshed, insulin and/or a
conjugate of the invention is
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
129
added and treatment lasted another 24 h. After stimulation, glucose production
is measured by
incubating the cells for 6 h in glucose-free RPMI containing 5 mM each of
alanine, valine, glycine,
pyruvate and lactate. Glucose is subsequently measured with a Trinder assay
(Sigma). Reduction of
glucose production is a clear indication that the tested conjugates increases
insulin sensitivity.
EXAMPLE 1
Expression/secretion of apMl(100-244) in CHOKl cells
In order to get the globular domain of human adiponectin (apMl), preceded by
the last 8 amino
acids of the collagenous region, secreted from CHOKI cells the following cDNA
is constructed: In
brief, by using a 5' primer (PBR 196; 5'-CGCGGATCCACCATGCTGTTGCTGGGAGCTGTTCTAC
TGCTATTAGCTCTGCCCGGTCATGACGGCAGGAAAGGAGAACCTGGAGAA-3'), encoding the
signal peptide of apMl (M1-D17) and 8 amino acids of the collagenous region
(G99-E106), together
with a 3' primer (PBR 189; 5'-ATATATCCCAAGCTTTCAGTTGGTGTCATGGTAGA-3') in a PCR
reaction containing QUICK-Clone cDNA (Human fat cell derived; # 7128-1,
Clontech, USA) as
template, a cDNA fragment encoding the signal peptide of apMl (M1-D17), the
nine last amino acids of
the collagenous region (G99-6107) followed by the entire globular domain (A108-
N244) is isolated.
The SignalP World Wide Web server (http://www.cbs.dtu.dk/services/Si ng alP~
predicts the presence
and location of signal peptide cleavage between G99 and 8100. After treatment
of the PCR fragment
with BamHI and HindIII the fragment is inserted into a vector designated
pcDNA3.1 (-)Hygro/Intron (a
2o derivative of pcDNA3.1 (-)Hygro (Invitrogen, USA) in which a chimeric
intron obtained from pCI-neo
(Promega, USA) has been inserted between the BamHI and NheI sites in the MCS
of the vector). The
correct DNA sequence of the inserted PCR fragment is confirmed by usage of an
ABI PRISM 3100
Genetic Analyzer.
This plasmid is then transfected into CHO Kl cells by usage of Fugene 6
(Roche, USA) as
transfection agent. In order to select for stable CHO Kl producers the medium
(from now on containing
360 pg/ml Hygromycin (Gibco, USA)) is exchanged every day until a confluent
monolayer of primary
stable transfectants is obtained. 24 hours later the culture medium is
harvested and assayed by Western
blotting for the presence of the apMl(100-244) protein. As detecting antibody
can be used a polyclonal
(rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; # PAl-054). PA1-054
immunizing peptides
correspond to amino acid residues 18-32 and 187-200 from mouse Acrp30 protein.
E(18) D D V T T T
E E L A P A L V(32) and F(187) T Y D Q Y Q E K N V D Q A(200). The immunizing
peptide located
in the globular domain of Acrp30 only differs in one position from the
corresponding sequence in the
apMl protein (K195N). Preparation of anti-apMl rabbit antiserum can also
easily be done by
immunizing rabbits with a synthetic peptide having the sequence:
CY(225)ADNDNDSTFTGFLLYHDTN(244). Hereafter the stable pool is cloned by the
limited dilution
method in order to isolate the highest producing CHO Kl clones.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
130
Using the commercial polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA) as
detecting antibody in a Western blot (Fig 1) it was possible to show the
expression of apMl(100-244)
from the stable pool (lane 7) and from 4 selected stable clones (lanes 8-11).
Clone B49 (lane 11) was
used for serum-free production of apMl(100-244) in Roller Bottles (see Example
5)
EXAMPLE 2
Expression/secretion of ap~82-244) in CHOK1 cells
In order to get the globular domain of human adiponectin (apM 1 ), preceded by
the last 26 amino
acids of the collagenous region, secreted from CHOK1 cells the following cDNA
is constructed: In
brief, by using a 5' primer (PBR 195; 5'-CGCGGATCCACCATGCTGTTGCTGGGAGCTGTTCTAC
TGCTATTAGCTCTGCCCGGTCATGACGGTGAAACCGGAGTACCCGGGGCT-3'), encoding the
signal peptide of apMl (M1-D17) and 8 amino acids of the collagenous region
(G81-A88), together with
a 3' primer (PBR 189; 5'-ATATATCCCAAGCTTTCAGTTGGTGTCATGGTAGA-3') in a PCR
reaction containing QUICK-Clone cDNA (Human fat cell derived; # 7128-1,
Clontech, USA) as
template, a cDNA fragment encoding the signal peptide of apMl (M1-D17), the 27
last amino acids of
the collagenous region (G81-6107) followed by the entire globular domain (A108-
N244) is isolated.
The SignalP World Wide Web server (http://www.cbs.dtu.dk/services/SienalPn
predicts the presence
and location of signal peptide cleavage between G81 and E82. After treatment
of the PCR fragment with
BamHI and HindIII the fragment is inserted into a vector designated pcDNA3.1(-
)Hygro/Intron (a
2o derivative of pcDNA3.1 (-)Hygro (Invitrogen, USA) in which a chimeric
intron obtained from pCI-neo
(Promega, USA) has been inserted between the BamHI and NheI sites in the MCS
of the vector. The
correct DNA sequence of the inserted PCR fragment is confirmed by usage of an
ABI PRISM 3100
Genetic Analyzer.
This plasmid is then transfected into CHO Kl cells by usage of Fugene 6
(Roche, USA) as
transfection agent. In order to select for stable CHO Kl producers the medium
(from now on containing
360 ~,g/ml Hygromycin (Gibco, USA)) is exchanged every day until a confluent
monolayer of primary
stable transfectants is obtained. 24 hours later the culture medium is
harvested and assayed by Western
blotting for the presence of the apM 1 (82-244) protein. As detecting antibody
can be used a polyclonal
(rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; # PA1-054). PA1-054
immunizing peptides
3o correspond to amino acid residues 18-32 and 187-200 from mouse Acrp30
protein. E(18) D D V T T T
E E L A P A L V(32) and F(187) T Y D Q Y Q E K N V D Q A(200). The immunizing
peptide located
in the globular domain of Acrp30 only differs in one position from the
corresponding sequence in the
apMl protein (K195N). Preparation of anti-apMl rabbit antiserum can also
easily be done by
immunizing rabbits with a synthetic peptide having the sequence:
CY(225)ADNDNDSTFTGFLLYHDTN(244). Hereafter the stable pool is cloned by the
limited dilution
method in order to isolate the highest producing CHO Kl clones.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
131
Using the commercial polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA) as
detecting antibody in a Western blot (Fig 1) it was possible to show the
expression of apMl(82-244)
from the stable pool (lane 2) and from 4 selected stable clones (lanes 3-6).
Clone A6 (lane 3) was used
for serum-free production of apM 1 (82-244) in Roller Bottles (see Example 5)
EXAMPLE 3
Expression/secretion of apMl 58-244) in CHOKl cells
In order to get the globular domain of human adiponectin (apM 1 ), preceded by
the last 50 amino
acids of the collagenous region, secreted from CHOK1 cells the following cDNA
is constructed: In
t o brief, by using a 5' primer (PBR 203; 5'-
CGCGGATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTGC
CCGGTCATGACGGCAGAGATGGCACCCCTGGTGAG-3'), encoding the signal peptide of apMl
(M1-D17) and 8 amino acids of the collagenous region (G57-E64), together with
a 3' primer (PBR 189;
S'-ATATATCCCAAGCTTTCAGTTGGTGTCATGGTAGA-3') in a PCR reaction containing QUICK-
15 Clone cDNA (Human fat cell derived; # 7128-1, Clontech, USA) as template, a
cDNA fragment
encoding the signal peptide of apMl(M1-D17), the 51 last amino acids of the
collagenous region (G57-
G107) followed by the entire globular domain (A108-N244) is isolated. The
SignalP World Wide Web
server (http://www.cbs.dtu.dk/services/SignalP~ predicts the presence and
location of signal peptide
cleavage between G57 and R58. After treatment of the PCR fragment with BamHI
and HindIII the
20 fragment is inserted into a vector designated pcDNA3.1 (-)Hygro/Intron (a
derivative of pcDNA3.1 (-
)Hygro (Invitrogen, USA) in which a chimeric intron obtained from pCI-neo
(Promega, USA) has been
inserted between the BamHI and NheI sites in the MCS of the vector). The
correct DNA sequence of the
inserted PCR fragment is confirmed by usage of an ABI PRISM 3100 Genetic
Analyzer.
This plasmid is then transfected into CHO Kl cells by usage of Fugene 6
(Roche, USA) as transfection
25 agent. In order to select for stable CHO Kl producers the medium (from now
on containing 360 ~g/ml
Hygromycin (Gibco, USA)) is exchanged every day until a confluent monolayer of
primary stable
transfectants is obtained. 24 hours later the culture medium is harvested and
assayed by Western blotting
for the presence of the apM 1 (58-244) protein. As detecting antibody can be
used a polyclonal (rabbit)
anti-Acrp30 antibody (Affinity BioReagents, USA; # PAl-054). PA1-054
immunizing peptides
30 correspond to amino acid residues 18-32 and 187-200 from mouse Acrp30
protein. E(18) D D V T T T
E E L A P A L V(32) and F(187) T Y D Q Y Q E K N V D Q A(200). The immunizing
peptide located
in the globular domain of Acrp30 only differs in one position from the
corresponding sequence in the
apMl protein (K195N). Preparation of anti-apMl rabbit antiserum can also
easily be done by
immunizing rabbits with a synthetic peptide having the sequence:
35 CY(225)ADNDNDSTFTGFLLYHDTN(244). Hereafter the stable pool is cloned by the
limited dilution
method in order to isolate the highest producing CHO K1 clones.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
132
Expression/secretion of apMl(58-244) in CHOK1 cells by usa a of an UCOE
expression vector
In order to increase the expression level of apMl(58-244) the construct
generated above is digested with
NheI and PmeI in order to excise a fragment containing the chimeric intron and
the cDNA encoding
apM 1 (S 8-244). This fragment is then inserted between the NheI and PmeI
sites of the expression vector
CET 720 (obtained from Cobra Therapeutics Limited, UK), which contains a
ubiquitous chromatin
opening element (UCOE, cf also WO 00/05393) in front of the CMV promoter.
This plasmid is then transfected into CHO K1 cells by usage of Fugene 6
(Roche, USA) as transfection
agent. The following day the medium is exchanged to medium containing 12.5
~g/ml Puromycin
(Sigma) in order to select for stable clones. In the next period the selection
medium is exchanged every
l0 day until a confluent primary selection pool is obtained. At this time a 24-
hours medium sample is
taken out and assayed by Western blotting for the presence of the apMl(58-244)
protein. As detecting
antibody is used the polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA; # PAl-054).
A relatively strong band, representing the apM 1 (58-244) protein, is now seen
on the Western blot.
15 EXAMPLE 4
Ex~ression/secretion of a~Ml 52-244) in CHOKl cells
In order to get the globular domain of human adiponectin (apMl), preceded by
the last 56 amino acids
of the collagenous region, secreted from CHOK1 cells the following cDNA is
constructed: In brief, by
using a 5' primer (PBR 202; 5'-
20 CGCGGATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG
CCCGGTCATGACGGGGCCCCAGGCCGTGATGGCAGA-3'), encoding the signal peptide of apMl
(M1-D17) and 8 amino acids of the collagenous region (G51-R58), together with
a 3' primer (PBR 189;
5'-ATATATCCCAAGCTTTCAGTTGGTGTCATGGTAGA-3') in a PCR reaction containing QUICK-
Clone cDNA (Human fat cell derived; # 7128-1, Clontech, USA) as template, a
cDNA fragment
25 encoding the signal peptide of apMl (M1-D17), the 57 last amino acids of
the collagenous region (G51-
G107) followed by the entire globular domain (A108-N244) is isolated. The
SignalP World Wide Web
server (http://www.cbs.dtu.dk/services/Si~nalP~ predicts the presence and
location of signal peptide
cleavage between G51 and A52. After treatment of the PCR fragment with BamHI
and HindIII the
fragment is inserted into a vector designated pcDNA3.1 (-)Hygro/Intron (a
derivative of pcDNA3.1 (-
30 )Hygro (Invitrogen, USA) in which a chimeric intron obtained from pCI-neo
(Promega, USA) has been
inserted between the BamHI and NheI sites in the MCS of the vector). The
correct DNA sequence of the
inserted PCR fragment is confirmed by usage of an ABI PRISM 3100 Genetic
Analyzer.
This plasmid is then transfected into CHO K1 cells by usage of Fugene 6
(Roche, USA) as transfection
agent. In order to select for stable CHO K1 producers the medium (from now on
containing 360 pg/ml
35 Hygromycin (Gibco, USA)) is exchanged every day until a confluent monolayer
of primary stable
transfectants is obtained. 24 hours later the culture medium is harvested and
assayed by Western blotting
for the presence of the apMl(52-244) protein. As detecting antibody can be
used a polyclonal (rabbit)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
133
anti-Acrp30 antibody (Affinity BioReagents, USA; # PA1-054). PA1-054
immunizing peptides
correspond to amino acid residues 18-32 and 187-200 from mouse Acrp30 protein.
E(18) D D V T T T
E E L A P A L V(32) and F(187) T Y D Q Y Q E K N V D Q A(200). The immunizing
peptide located
in the globular domain of Acrp30 only differs in one position from the
corresponding sequence in the
apMl protein (K195N). Preparation of anti-apMl rabbit antiserum can also
easily be done by
immunizing rabbits with a synthetic peptide having the sequence:
CY(225)ADNDNDSTFTGFLLYHDTN(244). Hereafter the stable pool is cloned by the
limited dilution
method in order to isolate the highest producing CHO Kl clones.
l0 Expression/secretion of apMl(52-244) in CHOKl cells by usage of an UCOE
expression vector
In order to increase the expression level of apMl(52-244) the construct
generated above is digested with
NheI and PmeI in order to excise a fragment containing the chimeric intron and
the cDNA encoding
apM 1 (52-244). This fragment is then inserted between the NheI and PmeI sites
of the expression vector
CET 720 (obtained from Cobra Therapeutics Limited, UK), which contains a
ubiquitous chromatin
15 opening element (UCOE, cf also WO 00/05393) in front of the CMV promoter.
This plasmid is then transfected into CHO Kl cells by usage of Fugene 6
(Roche, USA) as transfection
agent. The following day the medium is exchanged to medium containing 12.5
~g/ml Puromycin
(Sigma) in order to select for stable clones. In the next period the selection
medium is exchanged every
day until a confluent primary selection pool is obtained. At this time a 24-
hours medium sample is
2o taken out and assayed by Western blotting for the presence of the apMl(52-
244) protein. As detecting
antibody is used the polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA; # PAl-054).
A relatively strong band, representing the apM 1 (52-244) protein, is now seen
on the Western blot.
EXAMPLE 5
25 Seeding of roller bottles and serum-free~roduction of apM 1 polypeptide
fragments
At confluency in a T-175 flask, apMl polypeptide fragment producing CHO Kl
cells are transferred to a
roller bottle (1700 cm2) in 300 ml DMEM/F-12 medium (Life Tecnologies # 31330)
supplemented with
10% FBS and penicillin/streptomycin (P/S). Medium is exchanged every second
day until the bottle is
nearly confluent (typically after 4 days). The medium is then changed to 300
ml serum-free UltraCHO
30 medium (BioWhittaker # 12-724) supplemented with 1/1000 EX-CYTE
(Serologicals Proteins #
81129N) and P/S. Due to the relatively high protein content in the UltraCHO
medium (300 ~g/ml) this
medium can not be used as a production medium. However, it has appeared that
the usage of this
medium leads to a very thick cell-layer giving a higher yield in the final
production medium. After 4
days (where the medium is exchanged every second day) the roller bottle is
ready for production and the
35 medium is shifted to the production medium: DMEM/F-12 medium without phenol
red (Life
Technologies # 21041; contains 116 mg/1 CaCl2) supplemented with 1/100 ITSA
(Life Technologies #
51300-044) [ITSA: Insulin (1.0 g/L) - Transfernn (0.55 g/L) - Selenium (0.67
mg/L) supplement for
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
134
Adherent cultures], 1/1000 EC-CYTE and P/S. During the production period the
medium is exchanged
every day.
EXAMPLE 6
Expression/secretion of apMl(101-244) in CHOKl cells
In order to get the globular domain of human adiponectin (apMl), preceded by
the last 7 amino acids of
the collagenous region, secreted from CHOKI cells the following cDNA is
constructed: In brief, by
using a 5' primer (PBR 206; 5'-CGCGGATCCACCATGCTG
TTGCTGGGAGCTGTTCTACTGCTATTAGCTCTGCCCGGTCATGACGGCAAAGGAGAACCTG
GAGAA-3') encoding the signal peptide of apMl (M1-D17) followed by a glycine
and 6 amino acids of
the collagenous region (K101-E106), together with a 3' primer (PBR 189; 5'-
ATATATCCCAAGCTTTCAGTTGGTGTCATGGTAGA-3') in a PCR reaction containing a plasmid
designated PF446, harbouring the full-length apMl cDNA, as template, a cDNA
fragment encoding the
signal peptide of apMl (M1-D17), a glycine and the seven last amino acids of
the collagenous region
(K101-6107) followed by the entire globular domain (A108-N244) is isolated.
The SignalP World Wide
Web server (http://www.cbs.dtu.dk/services/Si~nalPn predicts the presence and
location of signal
peptide cleavage between the glycine and K101. After treatment of the PCR
fragment with BamHI and
HindIII the fragment is inserted into a vector designated pcDNA3.1 (-
)Hygro/Intron (a derivative of
pcDNA3.1 (-)Hygro (Invitrogen, USA) in which a chimeric intron obtained from
pCI-neo (Promega,
2o USA) has been inserted between the BamHI and NheI sites in the MCS of the
vector. The correct DNA
sequence of the inserted PCR fragment is confirmed by usage of an ABI PRISM
3100 Genetic Analyzer.
In order to increase the expression level of apMl(101-244) the construct
generated above is digested
with NheI and PmeI in order to excise a fragment containing the chimeric
intron and the apMl(101-244)
cDNA. This fragment is then inserted between the NheI and PmeI sites of the
expression vector CET
720 (obtained from Cobra Therapeutics Limited, UK), which contains a
ubiquitous chromatin opening
element (LJCOE) in front of the CMV promoter.
This plasmid is then transfected into CHO Kl cells by usage of Fugene 6
(Roche, USA) as transfection
agent. The following day the medium is exchanged to medium containing 12.5
pg/ml Puromycin
(Sigma) in order to select for stable clones. In the next period the selection
medium is exchanged every
3o day until a confluent primary selection pool is obtained. At this time a 24-
hours medium sample is
taken out and by Western blotting the presence of the apMl (101-244) protein
is verified. As detecting
antibody is used the polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA; # PAl-054).
The stable pool is directly expanded into three Roller Bottles for serum free
production. The protein is
then purified for charaterization.
EXAMPLE 7
Purification of CHO-expressed apMl 100-244)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
135
1 L of serum free produced CHO cell culture medium is ultrafiltrated on a
Millipore Labscale system
using a Biomax 5 membrane. Buffer shift to 20 mM Tris, 50 mM NaCl, pH 8.0
(Buffer A). Final volume
100 ml. This solution is applied to a 20 ml Q Sepharose FF (Pharmacia) column
previously equilibrated
with 5 column volumes buffer A. Following application the column is washed
with 3 column volumes
Buffer A and eluted with a linear gradient over 20 column volumes from Buffer
A to Buffer A including
containing 500 mM NaCI. 2 ml fractions are collected and pooled from A280 and
SDS-PAGE analysis.
The pool containing apMl(100-244) are concentrated to 2 ml and buffer changed
to SO mM Tris, 100
mM NaCl pH 7.5 using a Viva spin column (5 kDa cut off). Typical yields range
from 0.5 - 2 mg
apMl(100-244) from 1 1 culture medium. Further purification is obtained by gel
permeation
to chromatography applying the 2 ml concentrated eluate from the anion
exchange column to a Sephacryl
S-200 HR (16/60 Hi prep material, Pharmacia) previously equilibrated in 20 mM
Tris, 100 mM NaCI.
Fractions are analyzed by SDS-PAGE and pooled according to purity. The
material is > 90 % pure as
judged by SDS-PAGE. The pooled fractions are concentrated on a Viva spin
column (5 kDa cut off) and
frozen at -80 °C.
EXAMPLE 8
Purification of CHO-expressed apMl(82-244)
Serum free culture medium is clarified on a 0.22~,m filter. The medium is
thereafter concentrated to 10
times by ultrafiltration on a Millipore Labscale system using a Biomax 10
membrane, and diafiltered
2o against 3 volumes of 20 mM Tris pH 7.4 (buffer A). Initial purification is
performed by anion exchange
chromatography. 200 mL of the resulting solution is applied to a 25 mL Q
Sepharose FF (Amersham
Biosciences) column previously equilibrated with 4 column volumes of buffer A.
Following application,
the column is eluted in a linear gradient over 20 column volumes from buffer A
to 20 % buffer A
containing 1 M NaCI. Fractions of 10 mL are collected. The chromatographic
system is a Vision
BioCAD from PerSeptive Biosystems detecting at 280 nxn. A chromatographic peak
eluting at ca. 11 mS
is identified by SDS-PAGE analysis (non-reducing, treated 5 minutes at 95
°C in SDS sample buffer) to
contain a protein at molecular weight slightly less than 20 kDa. Fractions
containing this protein are
pooled. Further purification is obtained by hydrophobic interaction
chromatography. The pool after
anion exchange is added (NH4)ZS04 from a 3.5 M stock solution, to a
concentration of 0.9 M, and
applied to a 8 mL Butyl 650S (TosoHaas) column previously equilibrated with 5
column volumes of 0.9
M (NH~)zS04 and 20 mM NaH2P04 adjusted to pH 7.2 with NaOH (buffer A). The
column is eluted in a
linear gradient over 15 column volumes from buffer A to buffer B (20 mM
NaHZP04 adjusted to pH 7.2
with NaOH). Fractions of 8 mL column volume are collected. A chromatographic
peak eluting at ca.
120 mM (NH4)ZS04 is identified by SDS-PAGE analysis (non-reducing, treated 5
minutes at 95 °C in
SDS sample buffer) to contain an almost pure protein at slightly less than 20
kDa. Relevant fractions are
pooled, and ODzBO measured to 0.33, corresponding to a concentration of 0.26
mg/mL using a theoretical
molar absorbance of 1.28. The purified protein is frozen at -80 °C.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
136
EXAMPLE 9
Alternative generic method for purification of CHO-expressed apM 1 fragments
and analogs, such as
apM 1 (82-244), apM 1 100-244), apM 1 ( 1 O 1-244) or S 146C-apM 1 (82-244)
Serum free culture medium is clarified on a 0.22pm filter. The medium is
thereafter concentrated 10
times by ultrafiltration, and diafiltered against 3 volumes of 20 mM Tris
adjusted to pH 7.4 with HCI,
using for example a Millipore Labscale system with a Biomax 10 membrane.
Initial purification is
performed by anion exchange chromatography. Up to 10 column volumes of the
diafiltrate is applied to
a Q Sepharose FF (Amersham Biosciences) column previously equilibrated with 5
column volumes of 1
to mM CaCl2, 20 mM Tris adjusted to pH 7.4 with HCl (buffer Al). Following
application, the column is
eluted in a linear gradient over 20 column volumes from buffer A, to buffer B1
(buffer A1 containing 0.2
M NaCI). Fractions of about 0.5 column volumes are collected. The
chromatographic system may be an
Akta Purifier (Amersham Biosciences) detecting at 280 nm. SDS-PAGE analysis
(non-reducing, treated
S minutes at 95 °C in SDS sample buffer) is used to select fractions
containing the desired compound.
Such fractions contain a protein band corresponding to the molecular weight of
the apMl fragment or
analog monomer. The selected fractions are pooled. Further purification is
obtained by hydrophobic
interaction chromatography. The pool after anion exchange is added 1 volume 5
M NaCI, to a final
concentration of 2.5 M NaCI, and applied to a similarly sized Butyl 6505
(TosoHaas) column previously
equilibrated with 5 column volumes of 2.5 M NaCI, 1 mM CaCl2, 20 mM Tris
adjusted to pH 7.6 with
2o HCl (buffer AZ). The column is eluted in a linear gradient over 15 column
volumes from buffer AZ to
buffer BZ (1 mM CaCl2, 20 mM Tris adjusted to pH 7.4 with HCl). Fractions of
about 0.5 column
volume are collected. SDS-PAGE analysis as described above is used for the
selection of fractions
containing the target compound. The selected fractions are pooled. The pool
after hydrophobic
interaction chromatography is then concentrated to the desired concentration
(e.g. 0.5-1 mg/mL) and
diafiltered against 4 volumes of 2 mM CaCl2, 10 mM sodium citrate, 150 mM
sodium chloride adjusted
to pH 6.8 with hydrochloric acid. If not used immediately, the purified
protein is frozen at -80 °C.
Characterization of anMl(82-244)
The purified apMl(82-244) was subjected to automated N-terminal amino acid
sequence determination
3o following immobilisation onto a PVDF membrane in a ProSorb device.
The following N-terminal amino acid sequence was found.
Glu-Thr-Gly-Val-(hydroxy-Pro/Pro)-Gly-Ala-Glu-Gly-Pro-Arg-Gly-Phe-(hydroxy-
Pro/Pro)-Gly-Ile-
Gln-Gly-Arg-(glyco-hydroxy-Lys?/Lys)- Gly-Glu-(hydroxy-Pro/Pro)-
Initially, it should be noted that hydroxy-Pro is positively identified during
amino acid sequencing
which is not the case for glycosylated hydroxy-Lys (glyco-hydroxy-Lys).
This means that at the positions where (hydroxy-Pro/Pro) are indicated both
hydroxy-Pro and Pro are
found and positively identified. It also means that at the position where
(glyco-hydroxy-Lys?/Lys) is
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
137
indicated Lys is found and positively identified while the glyco-hydroxy-Lys
is suggested based on the
presence of additional specific but unidentifiable signals (see below).
The amino acid sequence above is identical to the N-terminal amino acid
sequence of apMl(82-244) but
the following comments are necessary.
The Pro-residue in position 86 is to a large extent found in the hydroxylated
form as a hydroxy-Pro-
residue. However, the hydroxylation is partial as Pro is also easily detected
although in lesser amount
than hydroxy-Pro.
The Pro-residue in position 91 is found not to be hydroxylated as hydroxy-Pro
is not detected.
The Pro-residue in position 95 is almost exclusively found in the hydroxylated
form as a hydroxy-Pro-
1o residue. However, Pro is also detected although in very small amounts.
The Pro-residues in position 104 is almost exclusively found in the non-
hydroxylated form as a Pro-
residue. However, hydroxylation is present as hydroxy-Pro is also detected
although in very small
amounts.
The status of the Lys-residue in position 101 is difficult to assess but the
amount of Lys is less than
15 expected. In addition to the lower signal for Lys, several un-identifiable
signals are found which
potentially represents glyco-hydroxy-Lys. Our interpretation of the data is
that Lys 101 is partially
hydroxylated and the hydroxy-Lys subsequently glycosylated . Hydroxy-Lys is
normally only
encountered in the glycosylated form as glyco-hydroxy-Lys.
Purified apMl(82-244) was also subjected to MALDI-TOF mass spectrometry and
found to contain two
2o components with the masses 18457 Da and 18800 Da, respectively. Reduction
of the sample prior to
analysis did not alter this.
The theoretical mass of apM 1 (82-244) is 18424 Da and the mass difference of
33 Da to the form with
mass 18457 Da could be explained by hydroxylation of Pro-residues while the
additional mass
difference of 343 Da to the form with mass 18800 Da could be explained by
hydroxylation and
25 subsequent glycosylation of a Lys-residue. Hydroxy-Lys residues are
normally only found in the
glycosylated form with a glucose-galactose disaccharide attached.
The data obtained by MALDI-TOF mass spectrometry is supported by the result of
the N-terminal
amino acid sequence determination.
Two other pieces of information can be deducted from the MALDI-TOF mass
spectrometry.
3o The first observation is that the potential N-glycosylation site at amino
acid residue Asn230 in the
globular domain of apMl(82-244) is not utilised as that would have been
detected as a significant
increase of mass compared to the theoretical mass.
The second information is that the single Cys-residue at position 152 in
apMl(82-244) is not modified
by attachment of thiol-reactive compounds as the mass of apM 1 (82-244) does
not change upon
35 reduction.
In summary, apMl(82-244) is partially hydroxylated on the three Pro-residues
and partially glyco-
hydroxylated on the Lys101-residue in the collagen-like part of the molecule.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
138
Interestingly, the hydroxylated and glycosylated component, seen in a spectrum
of the apM 1 (82-244)
fragment, is estimated to constitute about 60% relative to the non-hydroxy-
glycosylated component.
Characterization of apMl(100-244)
Purified apMl(100-244) was subjected to MALDI-TOF mass spectrometry and found
to contain one
major component with a mass of 16718 Da. The apMl(100-244) has a theoretical
mass of 16715 Da. A
small component of 17067 Da is also seen and this component originates from
hydroxylated and
glycosylated K101. Relative to the major component (16718 Da) this component
is estimated to
constitute below 5%.
Characterization of apMl(101-244)
By MALDI-TOF mass spectrometry a component with mass 16558.7 Da can be
identified as apM(101-
244) which has a therotical mass of 16558.4 Da. No component is seen that
could represent
hydroxylated and glycosylated lysine in position 101 proving that having K101
as the N-terminal amino
15 acid leads to a fragment without any hydroxy-glycosylation at K101.
EXAMPLE 10
N-terminal PEG~ation of apMl(100-2441 with 20 kDa PEG
apMl(100-244) used in this example is at a protein concentration of 1.5 mg/ml
in 100 mM phosphate
20 buffer at pH 5Ø PEG-aldehyde mW 20kDa obtained from Shearwater Polymers ,
Inc. is added as solid.
A 1 M NaCNBH3 stock solution in 100 mM phosphate buffer at pH 5.0 is used. The
experiment is
carried out as follows: 20 pl 1 M NaCNBH3 stock solution is added to an
Eppendorf tube containing 1
ml of apMl(100-244) solution at 4°C. After mixing, 8 mg of PEG-aldehyde
mW 20 kDa is added and
the solution mixed. The reaction is allowed to continue for 10 h at
4°C. At this time the reaction is
25 stopped by addition of 200 Ul 100 mM HCL. As judged by SDS-PAGE
approximately 90 % of the
apMl(100-244) is mono PEGylated. Further purification is carried out using a
Superose 6 column
(Pharmacia) equilibrated in 100 mM phosphate buffer at pH 5Ø The fractions
containing mono
PEGylated material are pooled based on A280 and SDS-PAGE.
3o EXAMPLE 11
N-terminal PEGylation of apMl(82-244) with 5 and 12 kDa PEG
apMl (82-244) used in this example is at a protein concentration of 1.5 mg/ml
in a 10 mM sodium
acetate buffer, 1 mM CaCIZ, 200 mM NaCI at pH 5Ø Two different mPEG-aldehyde
reagents (5 kDa or
12 kDa) from Shearwater Corporation have been employed. The activated PEG is
added as a solid to the
35 protein solution to obtain a 5 molar surplus (5 mol PEG per mol protein).
The NaCNBH3 reagent is
added to the protein solution from a 1 M stock solution in 10 mM sodium
acetate, 1 mM CaCl2, 200 mM
NaCI at pH 5Ø
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
139
The experiments is carried out as follows: 10 pl 1 M NaCNBH3 stock solution is
added to the Eppendorf
tube containing 0.5 ml (0.75 mg) of apMl(82-244) at 4°C and the
resulting solution is mixed. Then
either 1.1 mg of 5 kDa mPEG-aldehyde or 2.6 mg of 12 kDa mPEG-aldehyde at
4°C is added and the
solution is mixed. The reaction mixture is placed on a rocking platform at
4°C and allowed to continue
for 6 - 7 hours using the 5 kDa PEG or 4 - 5 hours using the 12 kDa PEG.
The degree of PEGylation of the apMl (82-244) trimer can be evaluated using an
analytical Superdex
200 (pre-packed 1.0 cm ID x 30 cm column from Amersham Biosciences) SEC method
run under native
conditions using the following buffer as mobile phase: 10 mM sodium acetate, 1
mM CaCl2, 200 mM
NaCI at pH 5Ø A flow rate of 1 ml/min and UV-detection at 214 nm is
employed.
1 o Using the above PEGylation conditions the protein mixture consists mainly
of un-PEGylated apM 1 (82-
244) trimer (approx. 40 - 50%), apMl(82-244) trimer having one PEG (approx. 40
- SO%) and
apM 1 (82-244) trimer having two PEGs (approx. 10 - 20%).
The degree of PEGylation is highly dependent of the reaction time at
4°C for both SkDa and l2kDa
PEG. The yield of apMl (82-244) trimer having three PEGS increases
significantly with time. For both
15 PEG sizes the yield of apM 1 (82-244) trimer having three PEGS is >_ 50%
after 20 hours reaction time at
4°C.
The apMl(82-244) trimer having one, two, or three PEGS can be further purified
on a semi-preparative
SEC column (2.6 cm ID x 60 cm) using the resin Superdex 200 prep grade
(Amersham Biosciences). A
sample volume <_ 2 ml is loaded on a pre-equilibrated SEC column and a flow
rate of 4 ml/min is used.
20 The mobile phase consists of 10 mM sodium acetate, 1 mM CaClz, 200 mM NaCI
at pH 5Ø The
fractions containing the apMl(82-244) trimer having one, two, and three PEGs
are each individually
pooled based on the results from the analytical SEC method.
Residual free PEG this can be removed on an anion exchanger. First, the sample
is ultrafiltrated and then
diafiltrated using for example Vivaspin 20 ml modules (from Vivascience), with
a 10 kDa cut-off
25 membrane, against a 1 mM CaClz, 20 mM Tris buffer adjusted to pH 7.4 with
HCl prior to the anion
exchange chromatography step. The diafiltrate is applied to a Q Sepharose FF
(Amersham Biosciences)
column previously equilibrated with 5 column volumes of 1 mM CaCl2, 20 mM Tris
adjusted to pH 7.4
with HCl (buffer A). Following application, the column is eluted in a linear
gradient over 20 column
volumes from buffer A to buffer B (buffer A containing 0.2 M NaCI). Fractions
containing the
3o PEGylated apMl(82-244) are pooled based on results from the analytical SEC
method and/or SDS-
PAGE analysis. Since the SDS-PAGE analysis is runned under denatured
conditions (non-reducing,
treated 10 minutes at 70 °C in SDS sample buffer) this method is only
used for PEGylated apM 1 (82-
244) samples without remaining free PEG.
35 EXAMPLE 12
Construction and expression of T121C-apMl(82-2441.
Using apM 1 (82-244) /pcDNA3.1 (-)Hygro/Intron as template, two PCR reactions
are performed with
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
140
two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR211 (5'-
GATAGTAACGTAGCACTCCAATCCCACACT-3') and PBR210 (5'-AGT
GTGGGATTGGAGTGCTACG TTACTA TC-3')/PBR193 (5'-ATATATCCCA
AGCTTTCAGTTGGTGTCATGGTAGAG-3') resulting in two fragments of 375 and 390 base
pairs,
respectively. These two fragments are assembled in a third PCR reaction with
the flanking primers
PBR195 and PBR193. The resulting gene is inserted into the mammalian
expression vector pcDNA3.1 (-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to T121C-
t o apMl (82-244). This construct is transfected into CHOKI cells and stable
pool is selected with
Hygromycin. The title analog, T121 C-apM 1 (82-244), is detected on western
blot by usage of the
polyclonal (rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054).
EXAMPLE 13
Construction and expression of S146C-apMl(82-244).
Using apMl(82-244) /pcDNA3.1(-)Hygro/Intron) as template, two PCR reactions
are performed with
two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR213 (5'-GTG
GAATTTACCAGTGCAGCCATCATAGTG-3') and PBR212 (5'-CACTATGAT
GGCTGCACTGGTAAATTCCAC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 453 and 312 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBRl 93. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1 (-
z5 )Hygro/Intron and confirmed by DNA sequencing to have the correct base
changes leading to S 146C-
apMl(82-244). This construct is transfected into CHOKl cells and stable pool
is selected with
Hygromycin. The title analog, S 146C-apM 1 (82-244), is detected on western
blot by usage of the
polyclonal (rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054).
3o EXAMPLE 14
Construction and expression of T243C-apMl(82-244). -
Using apM 1 (82-244) /pcDNA3.1 (-)Hygro/Intron as template, a PCR reaction is
performed with two
primers PBR195 (5'- CGCGGATCCACCATGCT
GTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTGCCCGGTCATGACGGT
35 GAAACCGGAGTACCCGGGGCT-3' and PBR214 (5'-ATATATCCCAAGCT
TTCAGTTGCAGTCATGGTAGA -3') resulting in a fragment of 735 bp. This fragment is
inserted into
the mammalian expression vector pcDNA3.1 (-)Hygro/ Intron and co~rmed by DNA
sequencing to
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
141
have the correct base changes leading to T243C-apMl(82-244). This cDNA,
together with the upstream
intron, is moved to the UCOE vector CET720. This construct is transfected into
CHOK1 cells and stable
pool is selected with Puromycin. The title analog, T243C-apMl(82-244), is
detected on western blot by
usage of the polyclonal (rabbit) anti-Acrp30 antibody (Affinity BioReagents,
USA; PA1-054).
EXAMPLE 15
Construction and expression of N127C-apMl (82-244).
Using apM 1 (82-244) /pcDNA3. l (-)Hygro/Intron as template, two PCR reactions
are performed with
two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR225 (5-GCG
AATGGGCATGCAGGGGATAGTAACGTA-3') and PBR224 (5-TACGTTACT
ATCCCCTGCATGCCCATTCGC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 393 and 372 base pairs,
respectively.
15 These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBR193. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1(-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to N127C-
apM 1 (82-244). This cDNA, together with the upstream intron, is moved to the
UCOE vector CET720.
This construct is transfected into CHOKI cells and stable pool is selected
with Puromycin. The title
20 analog, N127C-apMl(82-244), is being evaluated on western blot by usage of
the polyclonal (rabbit)
anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054).
EXAMPLE 16
Construction and expression of N141 C-apMl (82-244).
25 Using apM 1 (82-244) /pcDNA3.1 (-)Hygro/Intron as template, two PCR
reactions are performed with
two overlapping primer-sets [PBR195 (S'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR227 (5-GCC
ATCATAGTGGCATTGCTGATTGTAGAA-3') and PBR226 (5-TTCTACAAT
3o CAGCAATGCCACTATGATGGC -3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 435 and 330 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBR193. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1 (-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to N141C-
35 apMl(82-244). This cDNA, together with the upstream intron, is moved to the
UCOE vector CET720.
This construct is transfected into CHOKl cells and stable pool is selected
with Puromycin. The title
analog, N141C-apMl(82-244), is being evaluated on western blot by usage of the
polyclonal (rabbit)
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
142
anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054).
EXAMPLE 17
Construction and expression of N228C-apM 1 (82-244).
Using apM 1 (82-244) /pcDNA3. l (-)Hygro/Intron as template, a PCR reaction is
performed with two
primers PBR195 (5'- CGCGGATCCACCATGCT
GTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTGCCCGGTCATGACGGT
GAAACCGGAGTACCCGGGGCT-3' and PBR231 (5'-ATATATCCCAAGCTT
TCAGTTGGTGTCATGGTAGAGAAGAAAGCCTGTGAAGGTGGAGTCATT
to GTCGCAATCAGCATAGAG-3') resulting in a fragment of 735 bp. This fragment is
inserted into the
mammalian expression vector pcDNA3.1 (-)Hygro/ Intron and confirmed by DNA
sequencing to have
the correct base changes leading to N228C-apMl(82-244). This cDNA, together
with the upstream
intron, is moved to the UCOE vector CET720. This construct is transfected into
CHOKI cells and stable
pool is selected with Puromycin. The title analog, N228C-apMl(82-244), is
being evaluated on western
blot by usage of the polyclonal (rabbit) anti-Acrp30 antibody (Affinity
BioReagents, USA; PA1-054).
EXAMPLE 18
Construction and expression of Y111N-apMl 82-244).
Using apMl(82-244) /pcDNA3.1(-)Hygro/Intron as template, two PCR reactions are
performed with
2o two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR217 (5-GAA
TGCTGAGCGGTTTACATAGGCACCTTC-3') and PBR216 (5- GAAGGTGCC
TATGTAAACCGCTCAGCATTC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 345 and 420 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBR193. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1(-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to Y111N-
apM 1 (82-244). This cDNA, together with the upstream intron, is moved to the
UCOE vector CET720.
3o This construct is transfected into CHOK1 cells and stable pool is selected
with Puromycin. The N-linked
glycosylated title analog, Y111N-apMl(82-244), is detected on western blot by
usage of the polyclonal
(rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; PAl-054). A 100%
glycosylation is seen.
EXAMPLE 19
Construction and expression of Y122N-apMl 82-244).
Using apM 1 (82-244) /pcDNA3.1 (-)Hygro/Intron as template, two PCR reactions
are performed with
two overlapping primer-sets [PBR195 (5'-CGCG
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
143
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')lPBR219 (5-GTT
GGGGATAGTAACGTTAGTCTCCAATCC-3') and PBR218 (5-GGATTGGAG
ACTAACGTTACTATCCCCAAC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 381 and 384 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBR193. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1 (-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to Y122N-
apMl(82-244). This cDNA, together with the upstream intron, is moved to the
UCOE vector CET720.
to This construct is transfected into CHOKI cells and stable pool is selected
with Puromycin. The title
analog, Y122N-apMl(82-244), is being evaluated on western blot by usage of the
polyclonal (rabbit)
anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054).
EXAMPLE 20
Construction and expression ofD144N+S146T-apMl(82-244).
Using apM 1 (82-244) /pcDNA3.1 (-)Hygro/Intron as template, two PCR reactions
are performed with
two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAAACCGGAGTACCCGGGGCT-3')/PBR221 (5-GAA
TTTACCAGTAGTGCCGTTATAGTGGTT-3') and PBR220 (5-AACCACTAT
AACGGCACTACTGGTAAATTC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 450 and 315 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBRl 93. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1 (-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to
D 144N+S 146T-apM 1 (82-244). This cDNA, together with the upstream intron, is
moved to the UCOE
vector CET720. This construct is transfected into CHOK1 cells and~stable pool
is selected with
Puromycin. The title analog, D144N+S146T-apMl(82-244), is being evaluated on
western blot by usage
of the polyclonal (rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA;
PAl-054).
EXAMPLE 21
Construction and expression of R131N-apMl 82-244).
Using apMl(82-244) /pcDNA3.1(-)Hygro/Intron as template, two PCR reactions are
performed with
two overlapping primer-sets [PBR195 (5'-CGCG
GATCCACCATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTG CCCGGTCATGACG
GTGAA.ACCGGAGTACCCGGGGCT-3')/PBR223 (5-GAA
GATCTTGGTAAAGTTAATGGGCATGTT-3') and PBR222 (5-AACATGCCC
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
144
ATTAACTTTACCAAGATCTTC-3')/PBR193 (5'-ATATATCCCAAGCTTTCA
GTTGGTGTCATGGTAGAG-3') resulting in two fragments of 408 and 357 base pairs,
respectively.
These two fragments are assembled in a third PCR reaction with the flanking
primers PBR195 and
PBRl 93. The resulting gene is inserted into the mammalian expression vector
pcDNA3.1 (-
)Hygro/Intron and confirmed by DNA sequencing to have the correct base changes
leading to R131N-
apMl (82-244). This cDNA, together with the upstream intron, is moved to the
UCOE vector CET720.
This construct is transfected into CHOKl cells and stable pool is selected
with Puromycin. The N-linked
glycosylated title analog, R131N-apMl(82-244), is detected on western blot by
usage of the polyclonal
(rabbit) anti-Acrp30 antibody (Affinity BioReagents, USA; PA1-054). A 100%
glycosylation is seen.
to
EXAMPLE 22
Generic method for C ~~s-peQVlation of analogs of apM 1 fragments with an
introduced Cys (Cys-apM 1 ),
such as T121C-apMl(82-244), S146C-apMl(82-244), or T243C-apMl(82-244)
The Cys-apM 1 (as a trimer) used in this example is at a protein concentration
of 0.5 mg/ml in a 20 mM
15 Tris buffer, 1 mM CaClz, 100 mM NaCI, pH 7.4. Prior to PEGylation the Cys-
apMl sample is reduced
with DTT to ensure that the introduced cysteine residues can react and then
the DTT is subsequent
removed on a desalting column as described below. Two different Cys-specific
PEG reagents from
Shearwater Corporation have been employed. There are mPEG-OPSS and mPEG-
vinylsulfone in
different sizes (5, 10 or 20 kDa activated PEG). The activated PEG is added as
a solid to the protein
2o solution to obtain a 25 molar surplus (25 mol PEG per mol protein).
The experiments is carried out at room temperature (20 - 25 °C) as
follows: 20 pl 0.5 M DTT stock
solution is added to the Eppendorf tube containing 0.5 ml (0.25 mg) of Cys-apM
1 and the resulting
solution is mixed. After 30 min reaction time at room temperature the DTT is
removed on an
equilibrated NAP-5 (Amersham Biosciences) desalting column using 20 mM Tris, 1
mM CaCl2, 100
25 mM NaCI, pH 7.4 as buffer. The sample is diluted 2 times over the desalting
column giving a total
eluate volume of 1.0 ml. Before PEGylation, the sample is concentrated using a
Vivaspin 2 ml module
(from Vivascience), with a 10 kDa cut-off membrane, to obtain a protein
concentration of 0.5 mg/ml. To
the Cys-apMl solution is then added either 1.8 mg of 5 kDa, 3.6 mg of 10 kDa
or 7.2 mg of 20 kDa
Cys-specific PEG reagent and the solution is mixed. The reaction mixture is
placed on a rocking
30 platform and allowed to continue for 1 hour at room temperature.
The degree of PEGylation of the Cys-apMl can be evaluated using an analytical
Superdex 200 (pre-
packed 1.0 cm ID x 30 cm column from Amersham Biosciences) SEC method runned
under native
conditions using the following buffer as mobile phase: 10 mM sodium acetate, 1
mM CaCl2, 200 mM
NaCI at pH 5Ø A flow rate of 1 ml/min and UV-detection at 214 nm is
employed.
35 The Cys-apMl having one, two, or three PEGS can be further purified on a
semi-preparative SEC
column (2.6 cm ID x 60 cm) using the resin Superdex 200 prep grade (Amersham
Biosciences). A
sample volume <- 2 ml is loaded on a pre-equilibrated SEC column and a flow
rate of 4 ml/min is used.
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
145
The mobile phase consists of 10 mM sodium acetate, 1 mM CaClz, 200 mM NaCl at
pH 5Ø The
fractions containing the Cys-apMl material having one, two, or three PEGs are
pooled based on the
results from the analytical SEC method.
When the PEGylated sample after the SEC column contains trace amount of
residual free PEG this can
be removed on an anion exchanger. First, the sample is ultrafiltrated and then
diafiltrated using for
example Vivaspin 20 ml modules (from Vivascience), with a 10 kDa cut-off
membrane, against a 1 mM
CaCl2, 20 mM Tris buffer adjusted to pH 7.4 with HCl prior to the anion
exchange chromatography step.
The diafiltrate is applied to a Q Sepharose FF (Amersham Biosciences) column
previously equilibrated
with 5 column volumes of 1 mM CaCl2, 20 mM Tris adjusted to pH 7.4 with HCl
(buffer A). Following
1 o application, the column is eluted in a linear gradient over 20 column
volumes from buffer A to buffer B
(buffer A containing 0.2 M NaCI). Fractions containing the PEGylated Cys-apMl
are pooled based on
results from the analytical SEC method and/or SDS-PAGE analysis. Since the SDS-
PAGE analysis is
runned under denatured conditions (non-reducing, treated 10 minutes at 70
°C in SDS sample buffer)
this method is only used for PEGylated Cys-apMl samples without remaining free
PEG.
EXAMPLE 23
apMl 82-244) inhibits TNF-alpha release from LPS-stimulated monocytic cells
In order to investigate if adiponectin(82-244) also is able to inhibit LPS-
induced TNF-alpha production
we used the monocytic cell line THP-1 (ATCC, Rockville, MD).
Briefly, triplicate samples of THP-1 cells (105/well) were incubated in 96
well-plates at 37° C with
titrated amounts of adiponectin (highest concentration SOOnM (25,5 pg/ml) in
serum free cell culture
medium (RPMI-1640, containing 10 mM HEPES)
Following 18 h pre-incubation with adiponectin the cultures were incubated for
additional 4 h with a
final concentration of 0,5 pg/ml lipopolysaccharide (LPS) (List Biologicals)
and then 50 ~1 supernatant
where withdrawn and frozen at -20° C for subsequent analysis of T'NF-
alpha.
The diluted cell culture supernatants where analyzed for TNF-alpha content
using a standard
ELISA (R&D), and the IC50 of adiponectin where calculated using a 4-parameter
non linear regression
data analysis.
The results are shown in Fig. 2
EXAMPLE 24
Adiponectin trimer complex is de-stabilized by lowerin~the pH, but not in
the~resence of Ca2+ ions.
Purified apMl(82-244), produced in CHO-Kl (see example 2 and 8), was present
in a buffer
containing 120 mM (NHQ)ZS04 + 20 mM NaH2P04 (pH 6.8) . In order to examine the
effect of lowering
the pH without Ca2+ ions present or in the presence of Ca2+ ions the sample
was diluted in six different
buffers, in the ratio 1 volume sample to 4 volumes buffer. The buffers were
prepared by mixing from
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
146
stock solutions of acetic acid, sodium hydroxide and calcium chloride to the
following proportions (pH
was measured before addition of sample):
Buffer no.: 1 2 3 4 5 6
Acetic acid (mM) 50 50 50 50 50 50
Sodium hydroxide (mM) 5 25 45 5 25 45
Calcium chloride (mM) 0 0 0 10 10 10
Resulting pH 3.6 4.85.6 3.64.6 5.7
The resulting six different samples were examined on a coomassie stained Novex
8-16% Tris-Glycine
gel (Invitrogen; Cat. No. EC60452) run under native conditions (Fig. 3, lane 1-
6, respectively).
As seen in Fig. 3 the adiponectin trimer complex is de-stabilized at pH 3.6
and pH 4.8 in the absence of
Ca2+ ions, whereas in the presence of 10 mM CaClz no severe effect on the
stability of the adiponectin
l0 trimer complex is observed at these low pH values. (In Fig. 3, M is SeeBlue
Plus2).
EXAMPLE 25
De-stabilized adinonectin trimer complex can be recovered by addition of Caz+
ions.
Purified apMl(82-244), produced in CHO-K1 (see example 2 and 8), had been gel
filtrated on a
Superdex 75 column and buffer-shifted to near isotonic buffer: 100 mM NaCI, 20
mM NaH2P04, 10
mM NaOH (pH 6.8). After storage at -20 °C for several weeks the
material was verified to have a
heterogeneous appearance on a coomassie stained Novex 8-16% Tris-Glycine gel
(Invitrogen; Cat. No.
EC60452) run under native conditions (Fig. 4, lane 1 ). Supplying the material
(with heterogeneous
appearance) with 20 mM CaCl2, 20 mM MgCl2 and 20 mM ZnCl2, respectively,
showed that only CaClz
was able to re-stabilize the adiponectin trimer (Fig. 4, lane 2, 3 & 4,
respectively). (In Fig. 4, M is
SeeBlue Plus2).
These results indicate that the phosphate present in the isotonic buffer has
withdrawn the Ca2+ ions,
present in the adiponectin trimer, during storage at -20C. During storage at
freezing temperature, the pH
of the solution apparently also could have decreased a bit, leading to an
easier withdrawal of CaZ+ ions.
Then by supplying the solution with Caz+ ions, in the form of CaClz, the
adiponectin trimer complex is
recovered again. Addition of Mgz+ or Zn2+ ions showed no effect.
EXAMPLE 26
3o Acute treatment of db/db mice with apMl(82-244) fra~tnent transiently
normalizes blood glucose level
db/db mice are from 56 to 66 days old at initiation of experiment (approx. 36
g). All mice have been
maintained on a 12:12 light:dark cycle, fed standard rodent diet ad libitum,
and have unlimited access to
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
147
water. At t = 0 the blood glucose levels are measured from tail nick samples
in two groups, Group #1 &
Group #2 (n = 3 for each group), using a Glucometer Elite Monitor (Bayer
Corporation). At t = 30 min,
intraperitoneal (IP) injections are the following: Group #1: Vehicle (200 pl
buffer: 2 mM CaClz, 10 mM
NaCitrate, 150 mM NaCI, pH 7,4) and Group #2: 25 ~g apMl(82-244)
(adiponectin(82-244)) fragment
in 200 pl buffer. At 90, 150, 210, 270 minutes the blood glucose levels are
determined. The resulting
blood glucose levels are shown in graph below.
db/db mice (n=3) injected with single dose
of adiponectin(82-244) fragment
1
~e
1
J
w
O
As seen in the graph a single dose of 25 pg adiponectin(82-244) fragment
transiently normalizes the
1o blood glucose level (6.7 mmol/L) at t = 210 minutes.
SUBSTITUTE SHEET (RULE 26)
0 50 100 150 200 250 300
min
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence list
Sequence id no 1:
Consisting of si n~al (1-17Z non-homoloeous domain (18-41) collagen domain (42-
107), and logy bular
domain (108-244):
MetLeuLeuLeuGlyAlaValLeuLeuLeuLeuAlaLeuProGlyHisAspGln
GluThrThrT'hrGlnGlyProGlyValLeuLeuProLeuProLysGlyAlaCysThrGlyTrpMetAla
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
1o
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 2:
Consisting of non-homologous domain (19-41), collagen domain (42-107), and
globular domain (108-
244
2o GluThrThrThrGlnGlyProGlyValLeuLeuProLeuProLysGlyAlaCysThrGlyTrpMetAla
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGln
AlaSerGlySerValLeuLeuHisLeuGluValGlyAspGlnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 3:
Consisting of collagen domain (42-107), and globular domain (108-244):
GlyIleProGlyHisProGlyHisAsnGIyAlaProGlyArgAspGlyArgAspGIyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGIyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGIyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGInGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
to TrpLeuGlnValTyrGlyGluGlyGIuArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 4:
Consisting of collagen domain (88-107), and globular domain (108-244):
15 AlaGluGIyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGIyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGIuThrTyrVaIThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGIySerThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspVaILysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
20 TrpLeuGlnVaITyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer Thr
PheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 5:
Consisting of collagen domain (101-107), and globular domain (108-244):
25 LysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
3o TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 6:
Consisting of globular domain (108-244):
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerTluGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
t 0 Sequence id no 7:
Consisting~~~lobular domain~115-244):
PheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrLysIlePheTyr
AsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyrTyrPheAlaTyr
HisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeuPheThrTyrAsp
15
GlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspGlnValTrpLeuG
lnVal
TyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSerThrPheThrGly
PheLeuLeuTyrHisAspThrAsn
Sequence id no 8:
20 Consisting of globular domain (132-244):
PheThrLysIlePheTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGly
LeuTyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAla
MetLeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValG
lyAs
pGlnValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsn
25 AspSerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 9:
cDNA encodin ag-pMl(1-244) shown as seq id no 1
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgaccaggaaaccacgactcaagggcccggag
tcctgcttcccctgcccaag
30
ggggcctgcacaggttggatggcgggcatcccagggcatccgggccataatggggccccaggccgtgatggcagagatg
gcacccctggtgaga
agggtgagaaaggagatccaggtcttattggtcctaagggagacatcggtgaaaccggagtacccggggctgaaggtcc
ccgaggctttccgggaa
tccaaggcaggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgt
tactatccccaacatgccca
ttcgctttaccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgg
gctgtactactttgcctaccacatc
acagtctatatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccagg
aaaataatgtggaccaggcct
35
ccggctctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggact
ctatgctgataatgacaat
gactccaccttcacaggctttcttctctaccatgacaccaactga
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 10:
Consisting of collagen domain (82-107), and globular domain (108-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
l0 SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 11:
Consisting of collagen domain (100-107), and globular domain (108-244): .
ArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVal
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 12:
Consisting of collagen domain (52-107), and globular domain (108-244):
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluT'hrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
3o
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 13:
Consisti.~ of collagen domain 58-107), and globular domain (108-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
1o
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 14:
15 cDNA encoding apMl(52-244)
gccccaggccgtgatggcagagatggcacccctggtgagaagggtgagaaaggagatccaggtcttattggtcctaagg
gagacatcggtgaaacc
ggagtacccggggctgaaggtccccgaggctttccgggaatccaaggcaggaaaggagaacctggagaaggtgcctatg
tataccgctcagcattc
agtgtgggattggagacttacgttactatccccaacatgcccattcgctttaccaagatcttctacaatcagcaaaacc
actatgatggctccactggtaaa
ttccactgcaacattcctgggctgtactactttgcctaccacatcacagtctatatgaaggatgtgaaggtcagcctct
tcaagaaggacaaggctatgctc
20
ttcacctatgatcagtaccaggaaaataatgtggaccaggcctccggctctgtgctcctgcatctggaggtgggcgacc
aagtctggctccaggtgtat
ggggaaggagagcgtaatggactctatgctgataatgacaatgactccaccttcacaggctttcttctctaccatgaca
ccaactga
Sequence id no 15:
25 cDNA encoding_apM1~58-244)
agagatggcacccctggtgagaagggtgagaaaggagatccaggtcttattggtcctaagggagacatcggtgaaaccg
gagtacccggggctgaa
ggtccccgaggctttccgggaatccaaggcaggaaaggagaacctggagaaggtgcctatgtataccgctcagcattca
gtgtgggattggagactta
cgttactatccccaacatgcccattcgctttaccaagatcttctacaatcagcaaaaccactatgatggctccactggt
aaattccactgcaacattcctgg
gctgtactactttgcctaccacatcacagtctatatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatg
ctcttcacctatgatcagtacca
3o
ggaaaataatgtggaccaggcctccggctctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtat
ggggaaggagagcgtaat
ggactctatgctgataatgacaatgactccaccttcacaggctttcttctctaccatgacaccaactga
Sequence id no 16:
35 cDNA encodin apM 1 82-244
gaaaccggagtacccggggctgaaggtccccgaggctttccgggaatccaaggcaggaaaggagaacctggagaaggtg
cctatgtataccgctc
agcattcagtgtgggattggagacttacgttactatccccaacatgcccattcgctttaccaagatcttctacaatcag
caaaaccactatgatggctccac
tggtaaattccactgcaacattcctgggctgtactactttgcctaccacatcacagtctatatgaaggatgtgaaggtc
agcctcttcaagaaggacaagg
ctatgctcttcacctatgatcagtaccaggaaaataatgtggaccaggcctccggctctgtgctcctgcatctggaggt
gggcgaccaagtctggctcca
40
ggtgtatggggaaggagagcgtaatggactctatgctgataatgacaatgactccaccttcacaggctttcttctctac
catgacaccaactga
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 17:
T121 C-apM 1 82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheT
hrLysIle
PheTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
to SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 18:
S 146C-apM 1 82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
15
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlyCysThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluVaIGIyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
20 SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 19:
T243C-anMl (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
25
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
30 SerThrPheThrGlyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 20:
T 121 C-a~M 142-244):
GlyIleProGlyHisProGlyHisAsnGlyAlaProGIyArgAspGlyArgAspGlyThrProGlyGlu
LysGIyGluLysGlyAspProGIyLeuIleGlyProLysGlyAspIleGIyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGIyArgLysGlyGluProGlyGluGly
AIaTyrVaITyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePhe
TyrAsnGlnGInAsnHisTyrAspGlySerThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
1o
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheT'hrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 21:
15 S146C-apMl (42-244):
GlyIleProGlyHisProGIyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGIyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGIyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
20 yrAsnGlnGlnAsnHisTyrAspGlyCysT'hrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysVaISerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGInAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
InVaI
TrpLeuGInVaITyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 22:
T243 C-apM 1 42-244):
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGIyGluThrGlyValProGly
3o AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGIuAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGIyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
8
Sequence id no 23:
T 121 C-apM 1 x,101-2441:
LysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheT'hr
LysIlePhe
TyrAsnGlnGlnAsnHisTyrAspGlySerThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleT'hrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGInVaITyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 24:
S 146C-anM 1 ( 101-244
LysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGIuThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlyCysThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysVaISerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGIuAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnVaITyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 25:
T243C-apMl (101-244):
LysGlyGIuProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerVaIGIyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGIySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGIyGIuGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
3o ThrPheThrGlyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
9
Sequence id no 26:
T 121 C-apM 1 100-244):
ArgLysGlyGluProGIyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePhe
TyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGIyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysVaISerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGIySerValLeuLeuHisLeuGIuValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
t o ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 27:
S 146C-apM 1 (,100-244
ArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlyCysThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysVaISerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGInTyrGlnGluAsnAsnValAspGlnAlaSerGIySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 28:
T243C-apMl (100-244):
ArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGInTyrGInGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGInValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
3o ThrPheThrGlyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 29:
T121C-apMl (52-244):
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
5 LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrVaITyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePhe
TyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspVaILysValSerLeuPheLysLysAspLysAlaMetLeu
1o
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGIySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGIyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 30:
S 146C-apM 1 52-244
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGIuThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGInGlyArgLysGlyGIuProGlyGIuGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGIuThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGIyCysThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 31:
T243C-apMl (52-244):
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGIyProArgGIyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerVaIGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGInGIuAsnAsnValAspGlnAlaSerGlySerVaILeuLeuHisLeuGluValGlyAspG
lnVal
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
11
Sequence id no 32:
T121C-apMl (58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluCysTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePhe
TyrAsnGlnGlnAsnHisTyrAspGlySerT'hrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
1o
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 33:
S146C-apM11~58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
2o yrAsnGlnGlnAsnHisTyrAspGlyCysT'hrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 34:
T243C-apMl 58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
3o AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspCysAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
12
Sequence id no 35:
N127C-anMl (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGInGlyArgLysGlyGluProGlyGlu
GIyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGInGIuAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluVaIGIyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 36:
N 141 C-apM 1 82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGIuProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGIuThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnCysHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGInAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
2o ValTrpLeuGlnValTyrGlyGluGlyGIuArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
SerThrPheThrGIyPheLeuLeuTyrHisAspThrAsn
Sequence id no 37:
N228C-apMl (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGIyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluVaIGIyA
spGln
3o ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGIyLeuTyrAlaAspCysAspAsnAsp
SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
13
Sequence id no 38:
N127C-appMl (42-244):
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGIyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlyS erThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
to
PheThrTyrAspGlnTyrGlnGIuAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGIyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 39:
N 141 C-apM 1 (42-244):
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGIyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGIuGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnCysHisTyrAspGlySerThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGIySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 40:
N228C-a~Ml (42-244):
GlyIleProGlyHisProGlyHisAsnGlyAlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGIyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
3o AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGInTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGIuValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspCysAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
14
Sequence id no 41:
N127C-apMl (101-244):
LysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
1o ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 42:
N 141 C-apM 1 ( 101-244)
LysGlyGluProGlyGluGly
15
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnCysHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
2o ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 43:
N228C-apMl (101-244):
LysGlyGluProGlyGluGly
25
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspCysAspAsnAspSer
30 ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 44:
N127C-apM 1 100-244):
ArgLysGlyGluProGlyGluGly
s
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
to ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 45:
N 141 C-apM 1 100-244):
ArgLysGlyGluProGlyGluGly
~s
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnCysHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
2o ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 46:
N228C-apMl (100-244):
ArgLysGlyGluProGlyGluGly
2s
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspCysAspAsnAspSer
30 ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
3s
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
16
Sequence id no 47:
N127C-apM l 52-244):
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGIyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AIaTyrVaITyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
1o
PheThrTyrAspGlnTyrGInGIuAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluVaIGIyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 48:
N14IC-apMl (52-244):
AlaProGlyArgAspGlyArgAspGlyThrProGlyGlu
LysGlyGluLysGIyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGIyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
2o yrAsnGlnGlnCysHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluVaIGIyAspG
lnVa1
TrpLeuGlnVaITyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 49:
N228C-apMl (52-244):
AlaProGlyArgAspGlyArgAspGlyT'hrProGlyGlu
LysGlyGluLysGIyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGIyVaIProGly
3o AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerVaIGlyLeuGluThrTyrVaIThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGIyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnVaITyrGlyGluGIyGluArgAsnGlyLeuTyrAlaAspCysAspAsnAspSer
ThrPheThrGIyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
17
Sequence id no 50:
N127C-apMl (58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProCysMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrIIisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
to
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVal
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 51:
N141C-apMl (58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnCysHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIle'ThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 52:
N228C-apMl (58-244):
ArgAspGlyThrProGlyGlu
LysGlyGluLysGlyAspProGlyLeuIleGlyProLysGlyAspIleGlyGluThrGlyValProGly
3o AlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGluGly
AlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheThrL
ysIlePheT
yrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeuTyr
TyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMetLeu
PheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyAspG
lnVa1
TrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspCysAspAsnAspSer
ThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
18
Sequence id no 53:
Y111N-ap~82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValAsnArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIle
PheTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
t o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 54:
Y 122N-apM 1 (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
15
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrAsnValThrIleProAsnMetProIleArgPheT
hrLysIle
PheTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheT'hrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGly
AspGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
20 SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 55:
R131N-apMl (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
25
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleAsnPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySer'TluGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
3o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
19
Sequence id no 56:
D 144N+S 146T-apM 1 (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrVa1'ThrIleProAsnMetProIleArgPhe
ThrLysIleP
heTyrAsnGlnGlnAsnHisTyrAsnGlyThrThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
1o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 57:
H151N+N153T-apMl 82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheAsnCysThrIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPhe'ThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGly
AspGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
2o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 58:
K178N+K180T-~~82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysAsnAspThrAlaMet
LeuPheThrTyrAspGlnTyrGInGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
3o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
Sequence id no 59:
P 129T-apM 1 (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
5
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetThrIleArgPheT
hrLysIleP
heTyrAsnGInGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
SerThrPheThrGlyPheLeuLeuTyrHisAspT'hrAsn
Sequence id no 60:
G 145N-apM 1 (82-244):
GluThrGlyValProGlyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGIyGluProGlyGlu
15
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAsnGlySerThrGlyLysPheHisCysAsnIleProGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGInGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
2o SerThrPheT'hrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 61:
P 155T-apM l (82-244):
GluThrGlyValProGIyAlaGluGlyProArgGlyPheProGlyIleGlnGlyArgLysGlyGluProGlyGlu
GlyAlaTyrValTyrArgSerAlaPheSerValGlyLeuGluThrTyrValThrIleProAsnMetProIleArgPheT
hrLysIleP
heTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCysAsnIleThrGlyLeu
TyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLysValSerLeuPheLysLysAspLysAlaMet
LeuPheThrTyrAspGlnTyrGlnGluAsnAsnValAspGlnAlaSerGlySerValLeuLeuHisLeuGluValGlyA
spGln
ValTrpLeuGlnValTyrGlyGluGlyGluArgAsnGlyLeuTyrAlaAspAsnAspAsnAsp
3o SerThrPheThrGlyPheLeuLeuTyrHisAspThrAsn
Sequence id no 62:
cDNA encoding T121C-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagtgctacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
ccttcacaggctttcttctctaccatgacaccaactga
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
21
Sequence id no 63:
cDNA encoding S 146C-apM 1 (82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctgcactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
l0 ccttcacaggctttcttctctaccatgacaccaactga
Sequence id no 64:
cDNA encodin~T243C-ap~82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
ccttcacaggctttcttctctaccatgactgcaactga
Sequence id no 65:
cDNA encoding Y111N-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtaaaccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
ccttcacaggctttcttctctaccatgacaccaactga
3o Sequence id no 66:
cDNA encoding Y122N-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagactaacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
ccttcacaggctttcttctctaccatgacaccaactga
Sequence id no 67:
4o cDNA encoding S146T-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactataacggcactactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
ccttcacaggctttcttctctaccatgacaccaactga
SUBSTITUTE SHEET (RULE 26)
CA 02468619 2004-05-27
WO 03/055916 PCT/DK02/00897
22
Sequence id no 68:
cDNA encoding R131N-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattaacttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactcca
t0
ccttcacaggctttcttctctaccatgacaccaactga
Sequence id no 69:
cDNA encoding N127C-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
cctgcatgcccattcgctttac
caagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtactac
tttgcctaccacatcacagtctat
atgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataatg
tggaccaggcctccggctct
gtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgctg
ataatgacaatgactccac
cttcacaggctttcttctctaccatgacaccaactga
Sequence id no 70:
cDNA encoding N141 C-apMl (82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaatgccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtcta
tatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataat
gtggaccaggcctccggctct
gtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgctg
ataatgacaatgactccac
cttcacaggctttcttctctaccatgacaccaactga
Sequence id no 71:
cDNA encoding N228C-apMl(82-244)
atgctgttgctgggagctgttctactgctattagctctgcccggtcatgacggagtacccggggctgaaggtccccgag
gctttccgggaatccaaggc
aggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcc
ccaacatgcccattcgcttta
ccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtacta
ctttgcctaccacatcacagtct
atatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataa
tgtggaccaggcctccggct
ctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgattgcgacaatgactcca
ccttcacaggctttcttctctaccatgacaccaactga
SUBSTITUTE SHEET (RULE 26)
