Note: Descriptions are shown in the official language in which they were submitted.
- W O 92/18154 2 ~ ~ 7 ~ 7 5 P ~ /US92~03122
~ .
HUMAN BONE DERIVED INSULIN LIKE
5GROWTH FACTOR BINDING PROTEIN
Field of the Invention
The present invention relates to bone metabolism, and
more particularly to bone metabolic processes which are
mediated by a novel insulin-like growth factor binding protein
(IGFBP) isolated from human bone. More specifically, the
invention relates to a IGFBP termed human bone derived IGFBP
(hBD-IGFBP) which potentiates the effect of insulin-like growth
factor-II (IGF-II~ on bone cell proliferation.
Backaround of the Invention
IGF-I and IGF-II, the two most abundant growth
factors present in human plasma, constitute a family of
polypeptides that resemble proinsulin in structure and have
both anabolic and acute insulin-like activities in numerous
tissues (Daughaday, et al., Endocrine Rev., 10:68-91 (1989)).
Although previous studies using rats and mice emphasized IGF-I
as the primary IGF, with IGF-II being a fetal hormone, recent
findings have pointed to an important role for IGF-II in adult
human bone metabolism. IGF-II has been found to be the most
abundant growth factor present in human bone, and is the most
abundant growth factor produced by human bone cells. Further,
IGF-II is one of the few growth factors which is mitGgenic to
human bone cells. Also, a recently purified inhibitory IGFBP,
termed IGFBP-4, was found to inhibit basal bone cell
proliferation by about 40% in serum free conditions, suggesting
that endogenous production of IGFs contributed substantially to
cell proliferation in the absence of added growth factors. And
finally, IGF-II receptor blocking antibodies have been shown to
inhibit basal bone cell proliferation, suggesting that IGF-II
is a key bone cell growth factor (Mohan, et al., Growth
Genetics and Hormones, 6:1-9 (1990) and Mohan, et al., Clin.
Orthopedics & Rel. Res., 263:30-48 ~1990)).
: .
W092/l81s4 2 1a7 ~7 ~ 2 PCT/US92/0312
Recently it has also become clear that a family of
structurally related proteins that specifically bind the IGFs
are involved in the modulation of IGF action in different
tissues. Four classes of human IGFBPs (designated hIGFBP-1,
hIGFBP-2, hIGFBP-3 and hIGFBP-4) have been isolated, and the
complete amino acid sequences predicted from the nucleotide
sequences of the isolated cDNA clone~ (See, Mohan et al., Clin.
Orthopedics & Rel. Res. 263:30-48 (1990); Baxter et al., Prog.
Growth Factor Res. 1: 49-68 (ls89); Binkert et al., EM~30 J.
8:2497-2502 (1988); Brewer et al., Biochem. Biophys. Res. Comm.
152:1289-1297 (1988); Brinkman et al., EMBO J. 7:2417-2423
(1988); Lee et al., Mol. Endocri~ol. 2:404-411 (1988); Wood et
al., Mol. ~ndocrinol. 2:1176-1185 (1988); LaTour et al., Mol.
Endocrinol. 4:1806-1814 (1990); and Shimasaki, et al., Mol.
Endoc~inol. 4:1451-1458 (1990).
IGFBP-1 has been isolated from various sources
including amniotic fluid, placental membranes, decidua and HEP
G2 hepatoma cells. The N-terminal amino acid sequences of the
IGFBP-1 proteins isolated from these different sources have
been found to be identical. The cloning and complete sequence
of cDNA encoding IGFBP-1 from HEP G2, human uterus and human
placental cDNA libraries have been reported. IGFBP-2 has been
purified from conditioned medium collected from rat liver cells
(BRL-3A) and from Madin-Darby bovine kidney cells. The gene
encoding IGFBP-2 has been cloned from BRL-3A and human fetal
liver cDNA libraries. IGFBP-3 is found in serum as a 150
kilodalton ternary complex between IGF-I or IGF-II, an acid
labile glycoprotei~ of about 85 kilodaltons, and the IGFBP-3
molecule, which is an acid stable glycoprotein of 53
kilodaltons. IGFBP-3 has been purified to homogeneity from
human serum and the cloning and sequencing of the cDNA encoding
IGFBP-3 has been reported. IGFBP-4 was originally purified
from human bone cell conditioned medium as inhibitory IGFBP and
from rat serum. The cloning and sequencing of IGFBP-4 cDNA
clone isolated from human bone cell cDNA library and liver cDNA
library have been recently reported. In addition to these four
classes of IGFBPs, Martin et al., J. Biol. Chem., 265:4124-4130
(1990), Roghani et al., FEBS Lett., 255:253-258 (1989), and
WO92/18154 ~ ~ 7 ~ a PCT/US92/03122
Zapf et al., J. Biol. Chem., 265:14892-14898 (lsso)~ report the
partial purification of IGFBP from human cerebrospinal fluid,
from culture medium conditioned by AG 2804 transformed
fibroblasts and from hypoglycemic serum respectively, which
exhibit strong affinity for IGF-II over IGF-I.
Thus the art describes a variety of IGFBPs that are
produced by different sources and that exhibit disparate
binding properties to IGFs as well as different biological
functions. For example, IGFBP-1, IGFBP-3 and IGFBP-4 bind both
IGF-I and IGF-II with nearly equal affinity, while IGFBP-2 and
IGFBP purified from amniotic fluid, fibroblast cells and human
serum bind IGF-II with higher affinity than IGF-I. With regard
to functions, IGFBP-l has been shown to both inhibit and
potentiate the proliferative action of IGFBP-l in
choriocarcinoma cells and in human fibroblasts (Elgin, et al.,
J. Biol. Chem., 84:3254-3258 (1987) and Ritvos, et al.,
~n~ocr~nology, 122:2150-2157 (1988)). IGFBP-3 has been shown
to inhibit or stimulate IGF-I actions depending on culture
conditions in fibroblasts (De Mellow, et al., Biochem. Biophys.
Res. Comm., 156:199-204 (1988)). In contrast to IGFBP-l and
IGFBP-3, IGFBP-4 has only been shown to inhibit IGF-I and IGF-
II actions in bone cells (Mohan, et al., ~roc. Natl. Acad. Sci.
US~, 86:8338-8342 (1989)). The art also suggests that the
production of different IGFBPs are modulated disparately in a
tissue specific manner. For example, IGFBP-l production is
modulated by insulin while IGFBP-3 production is modulated by
growth hormone (Baxter, et al., P~syL,5a wth Factor ~s., 1:49-
68 (1989)). These findings suggest that the multiplicity of
different IGFBPs in conjunction with their unique regulation,
may cause IGF activities to be modulated in a localized, tissue
specific manner.
There remains a need in the art to relate IGF-I and
IGF-II functions in bone metabolism via IGFBPs, and thus there
is a need to identify the IGFBPs produced by bone cells and
present in human bone matrix. Such IGF8Ps are likely to be
involved in regulating bone metabolism and can be used in
clinical assays to provide information in the diagnosis of
defects in bone metabolism. IGFBPs which potentiate the IGF-
W O 92/18154 2 1 0 7 4 7 5 4 P~r/US92~0312~.
dependent growth of bone would be particularly useful intherapeutic applications for treatment of metabolic bone
diseases such as osteoporosis. Quite surprisingly, the present
invention fulfills these and other related needs.
Summary of the Invention
The present invention provides methods and
compositions for clinical diagnosis and treatment of metabolic
disorders related to IGF-mediated bone formation and cell
proliferation. More particularly, the invention provides human
bone derived IGF binding protein (hBD-IGFBP). The hBD-IGFBP
acts synergistically with IGF-II to potentiate IGF-II mediated
cell proliferation under conditions where both IGF-II and IGFBP
are administered simultaneously. The purified hBD-IGFBP also
binds to hydroxyapatite with strong affinity and thus provides
a reagent to target molecules specifically to bone, such as
IGF-II and/or IGF-I, other growth factors or drugs which may
affect bone resorption or formation, and thus is useful in
treatment of, e.g., bone fractures and bone diseases such as
osteoporosis or osteosarcoma. hBD-IGFBP may also be used in
the treatment of wound healing and in skin repair. hBD-IGFBP
may be used diagnostically as a marker of bone formation rate,
such as during treatment of bone disorders with therapeutic
agents. hBD-IGFBP may also be used as a reagent for clinical
evaluation of IGF levels in samples from patients with bone
metabolism and other disorders.
Brief ~Descr~ptiQn of_thç Drawi~ss
Fig. 1. Comparison of N-terminal amino acid sequence
of hBD-IGFBP with that of other known IGFBPs. The sequences
are aligned to give maximum identity.
Fig. 2. Competitive binding curves of hBD-IGFBP. The
sample was assayed for binding protein activity using labeled
IGF-II in the presence or absence of unlabeled IGF-I and IGF-
II.
,
.
.
WO92/181S4 ~,~rl~rl~ PCT/US92/03122
Fig. 3. Protein profile ~A) and IGFBP activity
profile (B) of the human bone extract in the FPLC Mono Q
chromatography step. Three 50 ml aliquots of HA bound fraction
pools were applied to the IGF-II affinity column. The
resulting three affinity bound fractions were pooled and run on
the Mono Q anion-exchange column. Protein profile is monitored
by absorbance at 280 nm. Two ml, 2 min fractions were
collected. Aliquots of the fractions were diluted l0 fold and
assayed for binding pro~ein activity. IGFBP activity is
expressed in amounts of specifically bound labeled IGF-II.
Fig. 4. Ligand blot analysis of hBD-IGFBP at
different stages of purification. 50 ~l of the sample were run
on SDS-PAGE (3-27% gradient), transferred to nitrocellulose
membranes, blotted with labeled IGF-II, and autoradiographed.
Lane a, HA bound fraction of human bone extract; Lane b, IGF-II
affinity bound fraction; Lane c, Mono Q IGFBP peak A; Lane d,
Mono Q IGFBP peak B; Lane e, Mono Q IGFBP peak C and Lane f,
Mono Q IGFBP peak D.
Description_of the Specific Embodiments
The invention provides purified and isolated human
bone derived IGFBP which binds specifically to IGF-II with
greater affinity than to IGF-I. The protein may be purified to
homogeneity as desired from proteins extracted from, e.g.,
human bone preparations, human bone cell conditioned medium, or
human serum. Substantially pure hBD-IGFBP of at least about
50% is preferred, at least about 70-80% more preferred, and
95-99% or more homogeneity most preferred, particularly for
pharmaceutical uses. Once purified, partially or to
homogeneity, as desired, the hBD-IGFBP may then be used
diagnostically, as an immunogen, therapeutically, etc.
The hBD-IGFBP produced according to the present
invention may be purified by hydroxyapatite apatite
chromatography followed by affinity chromatography on a column
WO92/181S4 21 ~ ~ ll 7 5 6 PCT/US92/0312
with IGF-II and finally for additional purity by Mono Q anion
exchange chromatography using an FPLC system. Apparent
homogeneity of the purified protein is demonstrated by, e.g.,
its migration as a single band on SDS-PAGE and by the
5 production of a single amino acid sequence upon N-terminal
sequence analysis. Affinity chromatography on an antibody
column using antibodies specifically directed against hBD-IGFBP
can also be used in a purification scheme. Additional
purification may be achieved by conventional chemical
purification means, such as liquid chromatography, gradient
centrifugation, and gel electrophoresis, among others. Methods
of protein purification are known in the art (see generally,
Scopes, R., Protein Purification, Springer-Verlag, NY (1982),
which is incorporated herein by reference) and may be applied
to the purification of the hBD-IGFBP described herein.
Specific binding of hBD-IGF8P to IGF-II and IGF-I is
demonstrated by a polyethylene glycol precipitation assay. In
this aspect the invention provides a purified protein which is
useful in structure-function 6tudies of the determinants of
IGFs which allow binding to specific receptors a6 well as to
hBD-IGFBP. Further utility of the purified hBD-IGFBP of the
invention is disclosed in the descriptions of other aspects of
the invention below.
The purified IGFBP of the invention is unique and
distinct from all previously identified IGFBPs. Human IGFBP-l,
IGFBP-2, IGFBP-3 and IGFBP-4 are characterized by amino acid
sequences which have been published and which are distinct from
the amino acid sequence of hBD-IGFBP. The N-terminal amino
acid sequence reported for the IGFBP purified from
cerebrospinàl fluid, fibroblast cell conditioned medium and
human serum is also distinct from that of hBD-IGFBP.
The homogeneous human IGFBP of the invention (hBD-
IGFBP) is characterized by an N-terminal amino acid sequence
identical to, or substantially identical to that shown in Fig.
l. For purposes of invention, an N-terminal amino acid
sequence substantially identical to that shown in Fig. l is
understood to mean an amino acid sequence identical to that of
Fig. l except for the presence of conservative amino acid
~ ' .
: ' '
WO92/181~ 2 ~ ~ 7 l1 7 5 PCT/US92/03122
substitutions or other amino acid substitutions, insertions or
deletions which do not materially affect the binding of the
substantially identical protein to IGF-I or IGF-II, or
otherwise materially alter its function in the applications set
forth below.
To produce hBD-IGFBP by recombinant routes, the gene
which encodes the hBD-IGFBP of the present invention is cloned
and expressed by insertion in a suitable expression vector
which in turn is used to trans,orm or transfect appropriate
host cells for expression of recombinant hBD-IGFBP polypeptide.
one or more synthetic oligonucleotide probes reflecting at
least a portion of the amino terminal sequence of purified hBD-
IGFBP, typically from about 14 to about 25 nucleotides are used
to screen a human bone cell cDNA library. The positive clones
containing the longest insert is sequenced according to
standard procedures. The deduced amino acid sequence is
compared with the N-terminal amino acid sequence of the
purified protein, and the predicted molecular weight and amino
acid composition based on the deduced amino acid sequence are
compared with those observed for purified hBD-IGFBP. The clone
is then used to produce recombinant hBD-IGFBP by using standard
procedures, as generally described in, e.g., Sambrook et al.,
oleculax Clonina. A LabQ~ory Manu~l, 1989 Cold Spring Harbor
Press, NY, which is incorporated herein by reference.
In another aspect, the invention concerns
polypeptides and fragments of hBD-IGFBP. Polypeptides and
fragments of hBD-IGFBP may be isolated from recombinant
expression systems or may be synthesized by the solid phase
method of Merrifield, Fed. Proc. 21:412 (1962), Merrifield, J.
Am. Chem. Soc. 85:2149 (1963), or Barany and Merrifield, in ~h~
Pep ides, vol. 2, pp. 1-284 (1979) Academic Press, NY, each of
which are incorporated herein by reference, or by use of an
automated peptide synthesizer. By "polypeptides" is meant a
sequence of at least about 3 amino acids, typically 6 or more,
up to 100-200 amino acids or more, including entire proteins.
For example, the portion(s) of hBD-IGFBP protein which bind
hydroxyapatite and/or IGF-II may be identified by a variety of
methods, such as by treating purified hBD-IGFBP with a protease
WO92/18154 PCT/US92/03122
21, 07~7~ 8
or a chemical agent to fragment it and determine which fragment
is able to bind to labeled IGF-II or hydroxyapatits.
Polypeptides may then be synthesized and used as antigen, to
inhibit IGF-II or hydroxyapatite-hBD-IGFBP interaction, etc.
It should be understood that as used herein, reference to hBD-
IGFBP is meant to include the proteins, polypeptides, and
fragments thereof unless the context indicates otherwise.
In another aspect, the invention provides means for
regulating aspects of the hydroxyapatite/hBD-IGFBP/IGF-II
interaction, and thus treating, therapeutically and/or
prophylactically, a disorder which can be linked directly or
indirectly to hBD-IGFBP or to its ligands, such as IFG-II. By
virtue of having the binding protein of the invention, agonists
or antagonists may be identified which stimulate or inhibit the
lS interaction of IGF-II, hydroxyapatite or other ligand with a
hBD-IGFBP. With either agonists or antagonists the metabolism
and reactivity of cells in response to hBD-IGFBP or IGF-II are
controlled, thereby providing a means to abate or in some
instances prevent the disease of interest.
Thus, the invention provides screening procedures for
identifying agonists or antagonists of events mediated by the
ligand/hBD-IGFBP interaction. Such screening assay6 may employ
a wide variety of formats, depending to some extent on which
aspect of the ligand/binding protein interaction is targeted.
For example, such assays may be designed to identify compounds
which bind to the binding protein and thereby block or inhibit
interaction with the IGF-II or hydroxyapatite. Other assays
can be designed to identify compounds w~ich can substitute for
hBD-IGFBP. Yet other assays can be used to identify compounds
which inhibit or facilitate the ascociation of IGF to hBD-IGFBP
and thereby mediate the cellular response to IGF.
In another aspect the invention provides a protein
which binds IGF-II with selective affinity over IGF-I. Fig. 2
shows the competitive binding curve of the hBD-IGFBP [l25I~IGF-
II as ligand and IGF-I and IGF-II as competitors. To displace
50% of bound [l25I]IGF-II from the IGFBP, about lO ng/ml of
IGF-I and l ng/ml of IGF-II was needed, indicating that IGF-II
was lO times more potent than IGF-I in displacing tracer. When
WO92/18~54 ~1 a, ~ ~ ~ PCT/USg2/03122
[125I]IGF-I was used as tracer, IGF-II was still more potent (4
times) than IGF-I in displacing tracer. These results suggest
that the IGFBP binds IGF-II with greater affinity than IGF-I.
Typically, the binding affinity of hBD-IGFBP for IGF-II will
range from about 10 9 M up to about lo~12 M or more, and more
likely in the range of at least about lo~10 to lo~ll M, whereas
the binding affinity of hBD-IGFBP for IGF-I ranges about 10
times less, i.e., from about 10 8 M up to about 10 10 M. The
selective affinity of hBD-IGFBP for IGF-II over IGF-I could
explain why IGF-II is 10-15 times more abundant than IGF-I in
human bone.
The invention provides therapeutic and pharmaceutical
compositions of hBD-IGFBP which take advantage of hBD-IGFBP's
strong binding affinity to hydroxyapatite, which is at least
about 10 9 M up to about 10 11 M or more. The hBD-IGFBP of the
invention binds to hydroxyapatite even in the presence of
strong denaturing agents, such as 4M guanidine HCl, while
purified IGF-II does not bind to hydroxyapatite. Thus, the
hBD-IGFBP provides a means or vehicle to fix or target IGF-II
in or to bone. IGF-II can be chemically coupled to hBD-IGFBP
via conjugation means that will be readily apparent to those of
skill in the art, but should not substantially diminish the
desired activity of either protein.
The linkage of hBD-IGFBP to another molecule which is
to be targeted to bone tissue or cells, such as the IGFs or
other molecules as set forth hereinbelow, can be produced by
chemical conjugation using well known laboratory procedures,
such as by employing cross-linking reagents. By chemically
linked is meant that the protein molecules are linked,
typically one to another, typically by covalent bonds. A
preferred method of conjugation is the formation of at least
one covalent bond between the hBD-IGFBP/IGF-II molecules.
The linkage may be direct, which includes linkages containing a
synthetic linking group, or indirect, by which is meant a link
having an intervening moiety, such as a protein or peptide,
e.g., plasma albumin, or other spacer molecule. For example,
the linkage may be by way of heterobifunctional or
homobifunctional cross-linkers, e.g., carbodiimide,
WO92/181~ 2 ~ ~ 7 4 7 5 lo PCT/US92/03122-
glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate
(SPDP) and derivatives, bis-maleimide, 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (SMCC), cross-linking
without exogenous cross-linkers by means of groups reactive
with the individual molecules, such as carbohydrate, disulfide,
carboxyl or amino groups via oxidation or reduction of the
native protein, or treatment with an enzyme or the like.
Methods for chemically cross-linking protein molecules are
generally known in the art, and a number of hetero- and
homobifunctional agents are described in, e.g., U.S. Pat. Nos.
4,355,023, 4,657,853, 4,676,980, 4,925,921, and 4,970,156, and
ImmunoTechnology Catalogue and Handbook, Pierce Chemical Co.
(1989), each of which is incorporated herein by reference. In
general, such cro6s-linking should not 6ubstantially affect the
desired function(s) of IGF-II or hBD-IGFBP.
Hybrid, chimeric or fusion protein molecules of IGF-
II and hBD-IGFBP or portion~ thereof can also be prepared by
recombinant DNA techniques, as described in, for example, U.S.
Patent 4,859,609, and Sambrook et al., supra, incorporated
herein by reference.
The deposition of the IGF-II/hBD-IGFBP complex in
bone allows, during bone resorption and the dissolution of
hydroxyapatite, the complex to be released and initiate new
bone formation by stimulating the proliferation of osteoblasts
in the vicinity of the resorption site. Based on the high
affinity of hBD-IGFBP towards both IGF-II and hydroxyapatite,
this invention thus provides therapeutic agents and
compositions which are useful to, inter alia, target IGF-II
and/or IGF-I specifically to bone.
The novel hBD-IGFBP, hBD-IGFBP/IGF-II and other
conjugates, antibodies to hBD-IGFBP and antagonists thereof,
and pharmaceutical compositions prepared therefrom are
particularly useful for administration for treatment of a wide
variety of hBD-IGFBP and IGF-II related disease. Preferably,
the pharmaceutical compositions can be administered
parenterally, i.e., subcutaneously, intramuscularly or
intravenously, or topically, orally, via aerosol, intranasal
delivery and the like. Thus, this invention provides
WO92/18154 2 ~ ~ 7 4 7 ~ PCT/US92/03122
compositions for parenteral administration which comprise a
solution of the hBD-IGFBP, hBD-IGFBP/IGF-II and other
conjugates, antibodies to hBD-IGFBP and antagonists thereof or
a cocktail of hBD-IGFBP and IGF-II dissolved in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers can be used, e.g., water, buffered water, 0.4% saline,
0.3% glycine and the like. These compositions may be
sterilized by conventional, well known sterilization
techniques. The compositions may contain pharmaceutically
acceptable auxiliary sub6tance6 as required to approximate
physiological conditions such as pH adjusting and buffering
agents, toxicity adjusting agents and the like, for example,
sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, etc. The concentration of the
desired hBD-IGFBP, hBD-IGFBP/IGF-II, or antibodies to hBD-IGFBP
or other antagonists thereof in these formulations can vary
widely, i.e., from less than about 0.00001%, u6ually at or at
least about 0.001%, to as much as about 0.05 to 0.1% by weight
and will be selected primarily by fluid volumes, viscosities,
etc., in accordance with the particular mode of administration
selected, the condition being treated, e.g., fracture repair,
06teoporosis, surgical or traumatic wound repair, tumors such
as osteosarcoma or breast carcinomas, etc., and the subject
being treated, i.e., an adult, child or neonate.
Thus, a typical pharmaceutical composition for
intravenous infusion to treat an adult suffering from moderate
06teodegenerative disea6e could be made up to contain 250 ml of
sterile Ringer's solution, and about 50 mg to 5 grams of hBD-
IGFBP or hBD-IGFBP/IGF-II. Actual methods for preparing
parenterally or orally administrable compounds will be known or
apparent to those skilled in the art and are described in more
detail in for example, Reminaton's Pharmaceutical Science, 16th
ed., Mack Publishing Company, Easton, PA (1982), which is
incorporated herein by reference.
The compositions containing the present hBD-IGFBP or
hBD-IGFBP/IGF-II or cocktails thereof can be administered for
prophylactic and/or therapeutic treatments. In therapeutic
applications, compositions are administered to a patient
WO92/18154 2 ~ ~ 7 ~ 7 ~ 12 PCT/US92/0312~
already suffering from an hBD-IGFBP or IGF-II related disease,
in an amount sufficient to cure or at least partially arrest
the disease and its complications. An amount adequate to
accomplish this is defined as a "therapeutically effective
dose." Amounts effective for this use will depend on the
disease, i.e., bone degeneration as in osteoporosis, fracture,
wound, tumor, etc., and its severity, the age of the patient
and the general state of the patient's health. Generally, the
amounts will range from about l.0 to about 500 ~g of hBD-IGFBP
or hBD-IGFBP/IFG-II per kilogram of body weight per hour of
infusion, with dosages of from lO to 50 ~g of hBD-IGFBP or hBD-
IGFBP/IFG-II per kilogram per hour of infusion being more
commonly used. As the materials of the present invention may
be employed in serious disease states, in view of the
minimization of extraneous substances and the absence of
foreign substance responses, it is possible and may be felt
desirable by the treating physician to administer substantial
excesses of these pharmaceutical compositions.
In prophylactic applications, compositions
containing the present hBD-IGFBP or hBD-IGFBP/IPG-II or
cocktails thereof are administered to a patient not already in
a disease state to enhance the patient's resistance to the
disease. Such an amount is defined to be a "prophylactically
effective dose." In this use, the precise amounts again depend
on the patient's state of health, etc. but generally range from
l to 500 ~g per kilogram per hour of infusion, especially lO to
50 ~g per kilogram per hour. A preferred prophylactic use is
for treatment of patients at ris~ of severe osteodegenerative
diseases.
Single or multiple administrations of the
compositions can be carried out with dose levels and pattern
being selected by the treating physician. In any event, the
pharmaceutical formulations should provide a quantity of hBD-
IGFBP or hBD-IGFBP/IGF-II, for example, sufficient to treat the
patient.
In yet another aspect, the invention provides an
agent which is effective in stimulating bone cell proliferation
in response to IGF-II. Exogenous addition of IGF-II to bone
2 ~ 3 Yl '1 ~ ~
WO92/18154 13 PCT/US92/03122
cells in serum free conditions increases their proliferation.
This proliferative effect of IGF-II is potentiated by the
addition of hBD-IGFBP in conjunction with IGF-II. This
synergistic action of the combination of hBD-IGFBP and IGF-II,
that is, greater than the additive effect achieved by combining
the results obtained with either agent individually, has not
been reported for other IGF~Ps in any cell type. Thus the
invention provides a therapeutic agent for the treatment of
bone disordars (e.g. osteoporosis) where bone formation is
impaired. The pharmaceutical compositions will comprise the
hBD-IGFBP and, if desired, IGF, with physiologically acceptable
carriers and or excipients.
This invention also provides an agent that can be
used in general wound healing and in skin repair to increase
the potency and half life of IGFs, as hBD-IGFBP may increase
the half-life of IGFs by protecting them from proteases, act to
target IGFs specifically to bone, and/or potentiate the
proliferative action of IGFs.
Complexes of hBD-IGFBP + IGF can be prepared in a
variety of ways, e.g., by incubating concentrations of purified
hBD-IGFBP and IGF at neutral pH overnight prior to adminis-
tration. The concentrations of hBD-IGFBP and IGF in the
composition can vary widely, but preferably are approximately
equimolar. Other formulations will be apparent to the skilled
artisan from the context of the present disclosure. When
formulated separately, the compositions of hBD-IGFBP can be
administered separately or simultaneously with the IGF-II
compositions. When administered separately, typically the hBD-
IGFBP will be administered first, followed by the IGF-II. Such
compositions can be administered in specific areas to stimulate
local bone formation (e.g., fracture repair) or administered
systemically to increase general bone formation, as in the
treatment of bone disorders such as osteoporosis.
In other embodiments, the present invention provides
for the preparation of more potent IGF molecules. Structure-
function analysis of IGFs and hBD-IGFBP can identify region(s)
of IGFs that is involved in binding to hBD-IGFBP. It is
possible to produce modified IGF molecules by amino acid
~o ~ ~75
W092/t81s4 ~ PCT/US92/0312~-
14
substitutions, insertions or deletions such that the modified
molecules bind to IGF receptors and the hBD-IGFBP with high
affinity but not to inhibitory IGFBPs, such as IGFBP-4 and
IGFBP-3. Such modified IGF molecules can serve as potent
anabolic agents in promoting general wound healing and in skin
repair. In other embodiments fragments of the hBD-IGFBP are
produced. The fragments will typically have a desired
function, such as the ability to bind IGF-II or hydroxyapatite,
while eliminating other portions of the molecule which are not
essential for this function. Fragments may be used
individually or joined together.
In addition to the IGFs, the hBD-IGFBP of the
invention are useful in targeting other molecules of interest
to the bone. The molecules may affect the formation or
resorption of bone directly or indirectly. As will be apparent
to those of skill in the art, a wide variety of agents can be
targeted to bone tissue in this manner. Representative
examples include those which stimulate bone formation, such as
bone morphogenic protein (BMP), TGF~, fibroblast growth factor
(FGF), platelet-derived growth factor ~PDGF), agents which
decrease bone formation as may be desired in certain cancers,
such as glucocorticoid or l, 25-dihydroxyvitamin D3, those
compounds which increase bone resorption, such as macrophage
colony stimulating factor (M-CSF) and interleukins, and
. 25 compounds which decrease bone resorption, such as bis-
phosphonate and calcitonin, for example. The compounds can be
joined to the hBD-IGFBP of the invention in a variety of ways,
including conjugation means set forth above as well as fusion
and chimeric proteins, as appropriate. Typically dosages of
the above mentioned targeted compounds such as growth factors
will be delivered to the surface of the bone tissue at
concentrations ranging from about lO pg/ml up to about 50 ng/ml
of bone tissue.
In another aspect, the invention provides a
diagnostic marker to evaluate bone formation in clinical
samples taken from patients who have metabolic bone disease or
bone neoplasia. Based on the findings that hBD-IGFBP is an
important modulator of IGF-II actions and that IGF-II is an
WO92/18154 ~1 V 7 4 7 j PCT/US92/03122
important human bone growth factor, levels of hBD-IGFBP can be
used to monitor disorders of bone metabolism, where aberrant
levels of hBD-IGFBP represent present of the disorder. Thus
the invention also provides reagents for a clinical diagnostic
bone formation marker to monitor bone formation during
treatment of bone disorders with therapeutic agents.
The compositions of hBD-IGFBP or antibodies thereto can be used
for the detection and quantitation of hBD-IGFBP in a biological
fluid, such as human plasma, serum or urine.
As will be recognized by those s~illed in the art,
numerous types of immunoassays are available for use in the
present invention. For instance, direct and indirect binding
assays, competitive assays, sandwich assays, and the like, are
generally described in, e.g., U.S. Pat. Nos. 4,642,285;
4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752;
3,839,153; 3,791,932; and Harlow and Lane, An~ibodies. A
Laborato~Y Manua~, Cold Spring Harbor Publications, N.Y.
(1988), each incorporated by reference herein. In one assay
format hBD-IGFBP is quantified directly by measuring the
binding of antibodies to hBD-IGFBP, which antibodies are then
detected by, e.g., labeled anti-IgG, IgM and/or IgA human
antibodies. In another format, a patient's hBD-IGFBP can be
measured by competing with labeled or unlabeled hBD-IGFBP, for
binding. A wide variety of labels may be employed, such as
radionuclides, particles (e.g., gold, ferritin, magnetic
particles, red blood cells), fluors, enzymes, enzyme
substrates, enzyme cofactors enzyme inhibitors, ligands
(particularly haptens), chemiluminescers, etc., but preferably
radionuclides.
Thus, hBD-IGFBP or antibodies thereto for use in such
assays can be attached to an insoluble or solid support, such
as an ELISA microtiter well, microbeads, filter membrane,
insoluble or precipitable soluble polymer, etc. to function as
an affinity resin. The antisera or monoclonal antibodies to
hBD-IGFBP are typically non-human in origin, such as rabbit,
goat, mouse, etc. Kits can also be supplied for use in
detecting the hBD-IGFBP, where the hBD-IGFBP and/or antibodies
thereto may be provided, usually in lyophilized form, in a
w092/18154 2~7 ~7~ : 16 PCT~US92/0312~
container, either alone or in conjunction with additional
reagents, labels, and/or anti-antibodies, and the like. The
hBD-IGFBP polypeptide and antibodies, which may be conjugated
to a label, or unconjugated, and are included in the kits with
buffers, such as Tris, phosphate, carbonate, etc., stabilizers,
biocides, inert proteins, e.g., serum, albumin, or the like.
Frequently it will be desirable to include an inert extender or
excipient to dilute the active ingredients, where the excipient
may be present in from about l to 99~ of the total composition.
Antibodies for diagnostic or therapeutic uses which
bind hBD-IGFBP polypeptides of the invention can be produced by
a variety of means. The production of non-human monoclonal
antibodies, e.g., murine, is well known and may be accomplished
by, for example, immunizing the animal with a recombinant or
synthetic hBD-IGFBP molecule or a selected portion thereof
(e.g., a peptide). For example, by selected screening one can
identify a region of the h8D-IGFBP molecule such as that
predominantly responsible for recognition by IGF, if desired.
Antibody producing cells obtained from the immunized animals
are immortalized and screened, or screened first for, e.g., the
production of antibody which binds the hBD-IGFBP, and then
immortalized.
The following examples are offered by way of
illustration, not limitation.
EXAMPLE I
Preparation Of h~E~n bone e~tract for
~urification of hBD-IGFBP
Human femoral heads obtained during total hip
replacement surgery were stored frozen at -20C until used.
Bones were cut using a band saw and ground to fine particles in
a Wiley mill for hBD-IGFBP extraction. Bone proteins were
extracted by demineralization of the femoral head bone powder
with 10% ethylenediaminetetraacetate (EDTA) in the presence of
4M guanidine HCl and protease inhibitors (Guanidine EDTA
extract) after an initial extraction with water and 4M
guanidine HCl as described in Mohan, et al., (Biochem. Biophys.
Acta, 884:234-242 ~1986)). Guanidine EDTA extract was then
,WO92/18154 ~ ~ 0 7 4 7 ~ PCT/US92~03122
concentrated in an Amicon using YM5 (5 kilodaltons molecular
weight cut-off) membrane and used for the purification of hBD-
IGFBP .
EXAMPLE I I
Purification,,,and çharacterization of
hBD-IGFBP from human bone extract
Human bone extract was subjected to hydroxyapatite
(HA) chromatography in 4 M guanidine HCl as described in Mohan,
et al., ibid. An IGF-II affinity column was constructed by
coupling 250 ~g of IGF-II purified from human bone to cyanogen
bromide-activated Sepharose 4B beads. A pool of HA bound
fractions was concentrated in an Amicon cell using a YM5 mem-
brane to about 300 ml and dialyzed against 20 times volume of
potassium phosphate buffer (lO mM potassium phosphate, pH 6.0)
containing protease inhibitors (lO0 mM epsilon a~inocaproic
acid, 5 mM benzamidine, l mM phenylmethylsulfonyl fluoride).
The IGF-II affinity column was equilibrated with the potassium
phosphate buffer after which a 50 ml aliquot (approximately 3-4
mg total protein/ml) of the dialyzed HA bound fraction pool of
the human bone extract was loaded into the column. The column
was then extensively washed with potassium phosphate buffer to
completely remove the unbound proteins. The bound proteins
were eluted with 20-25 ml of 30 mM tris-acetate (pH 7.2)/4 M
guanidine-HCl. The affinity bound fraction was concentrated in
an Amicon cell using a YM 5 membrane and then dialyzed against
20 mM tris-HCl (pH 8.0) buffer. The dialyzed affinity bound
fraction was applied to a Pharmacia FPLC Mono Q anion exchange
column previously equilibrated with the tris-HCl buffer. The
bound proteins were eluted with a linear gradient from 0-l M
NaCl in 20 mM tris-HCl buffer in lO0 min.
hBD-IGFBP activity was determined by a polyethylene
glycol precipitation method. Briefly, 50 ~l of sample to be
assayed was incubated with 25,000 to 50,000 cpm of l25I-labeled
IGF-I or IGF-II for 60 minutes at room temperature in 250 ~l of
O.lM HEPES/0.1% bovine serum albumin/0.1% Triton XlO0/44 mM
Na2C03/0.02% NaN3, pH 6Ø To this mixture was added lO0 ~l of
2% immune serum globulin and 500 microliters of 25%
~' :
' . , ' . '.
. .
. . .
WO92J18154 2 ~ ~ 7 ~ ~ ~ 18 PCT/US92/0312~-
polyethylene glycol, followed by centrifugation. Under these
conditions the polyethylene glycol precipitated the larger
complex between IGF-I or IGF-II and hBD-IGFBP, but did not
precipitate unbound IGF-I or IGF-II. The amount of 125I-IGF-II
in the PEG precipitate was then counted. Non-specific binding
was determined by carrying out the assay in the presence of
excess unlabeled IGF-I or IGF-II and the amount of 125I-IGF-I
or 125I-IGF-II precipitated was subtracted from the value
obtained above.
To determine the apparent molecular weight of hBD-
IGFBP, ligand blot analysis was carried out using 125I-IGF-II
as a tracer. In this procedure, 50 ~1 of ~amples was
electrophoresed under non-reducing conditions on precast 3-27%
SDS polyacrylamide slab gels. After transfer of the samples to
nitrocellulose by electroblotting, the nitrocellulose membrane
was incubated with radiolabeled IGF-II. After washing the
unbound radiolabeled IGF-II, the membrane was subjected to
autoradiography as described in Hossenloop, et al., (Anal.
Biochem., 154:138-143 (1986)).
Amino acid compositions of samples were analyzed with
an Applied Biosystems model 420 analyzer and N-terminal
sequences were determined with an Applied Biosystems model 470A
vapor phase protein sequencer (Mohan, et al., Biochi~ Biophys.
~ç~, 966:44-55 (1988)).
Fig. 3 shows the protein profile and IGFBP activity
profile of the affinity-bound fraction pool in the Mono Q
chromatography step. There was neither a protein absorbance
peak nor a IGFBP activity peak in the region where authentic
I5FBP-4 elutes (Fractions 9-15, O.lM NaCl), thus suggesting
that the bone derived IGFBP is not IGFBP-4. However, there
were four protein absorbance peaks eluting at different NaCl
concentrations. Of the four protein peaks, the first two peaks
(A & B) contained significant IGFBP activity while the last two
protein peaks (C & D) had little IGFBP activity.
To examine the apparent molecular weights of the
IGFBPs present in human bone extract, ligand blotting and
~125~]IGF-II affinity labeling were used. Fig. 4 shows ligand
blots in which the HA bound, IGF-II bound and Mono Q protein
2~a747~
W O 92/18154 19 PC~r/US92/03122
peaks were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, transferred to nitrocellulose, and probed -
with [125I]IGF-II tracer. The major IGFBP present in HA bound
and IGF-II bound fractions had an apparent molecular weight of
29 kDa. In addition, these fractions also exhibited a broad,
- less intense band between the 68 and 43 kDa molecular weight
markers. The major 29 kDa IGFBP was separated from the higher
molecular weight IGFBP by Mono Q chromatography. Mono Q peak A
showed a major band at 29 kDa and a minor band at 24 kDa. Mono
Q peak B showed a broad diffuse band between the 68 and 43 kDa
markers and a minor band at 29 kDa. Mono Q peak C (represents
the major protein absorbance peak) and D showed only a weak
band at 29 kDa. These data suggest that the major IGFBP in
human bone extract is a 29 kDa IGFBP.
Both the amino acid composition and the N-terminal
amino acid sequence of the 29 kDa IGFBP in Mono Q peak A
appeared to be unique, having limited sequence similarity to
other known IGFBPs ~Tables 1, 2; Fig. 1).
: , . . .:
. .
-
- . '
., ' . ~ "
W O 92/18154 2 ~07 ~ ~ 20 PC~r/US92/0312~
Table 1. Amino acid compositions of hBD-IGFBP and the
known IGFBPs
. _
s Peak A IGFBP-l IGFBP-2 IGFBP-3 IGFBP-4
asx 8.1 6.8 6.6 6.8 8.0
glx 5.9 13.2 13.9 10.2 12.2
lo ser 9.3 9.0 3.5 10.2 5.9
gly 9.0 7.3 11.8 8.7 9.7
his 5.1 2.6 3.8 2.7 4.6
arg 6.3 4.3 6.9 7.2 8.0
thr 6.3 3.8 3.8 3.4 2.5
ala 7.9 11.1 7.3 6.8 6.8
pro 5.8 7.7 9.3 8.7 8.9
tyr 3.7 2.6 1.7 3.4 1.3
val 6.6 3.8 5.5 5.3 3.8
met 3.5 1.3 3.1 0.8 1.7
cys 5.5 7.7 6.6 6.8 8.4
ile 3.3 3.8 1.4 2.3 2.5
leu 6.8 7.3 9.0 7.2 8.0
phe 2.9 1.7 1.0 1.9 2.1
ly6 4.0 3.8 4.5 7.2 5.1
trp 2.1 0.3 0.4 0.4
_
10 mm diameter immobilon membrane cut-outs were soaked in
methanol, placed into Millipore filtration units, and
held in place with rubber O-rings. After rinsing the
immobilon membrane with water, the BP in Peak A was fixed
into the membrane by filtering through 1 ml of Peak A.
One-half of the membrane was used for amino acid
composition studies.
2 i ~ 7 ~ ~ ~
~092/18154 PCT/US92/03122
21
Table 2. Aminoterminal sequence of hBD-IGFBP in MonoQ
Peak A.
Residue Amino acid (pmoles)
1) L=59.4
2) G=50.1
3) F=34.8
4) F=48.0
5) V=49.1
6) X
~) V=27.l
8) E=20.6
9) P=22.2
lO) D=15.2
ll) D=18.7
12) K=13.4
13) A=22.8
14) A=32.8
15) L=28.5
. _
50 pmoles of hBD-IGFBP was used for N-terminal amino acid
sequence analysis. Combined average repetitive yield was
86.3%. X = not known.
Therefore, the 29 kDa IGFBP has been designated human
bone derived IGFBP (hBD-IGFBP). In addition to the major
sequence (Leu-Gly-Phe-Phe-Val-X-Val-Glu-Pro-Asp-Asp-Lys-
Ala-Ala-Leu), there was evidence for the presence of an
additional sequence in Mono Q peak A which lacked one or
two amino acids at the N-terminus. The purified hBD-
IGFBP appeared to be susceptible to proteolytic cleavage
since storage of purified 29 kDa IGFBP at 5C for
overnight led to the disappearance of 29 kDa band and
appearance of a major band at 24 kDa and several low
molecular weight minor bands. Upon N-terminal sequence
analysis of the 24 kDa band, the sequence obtained (Leu-
Gly-Phe-X-Val-X-X-Glu-Pro-X-X-Lys) was similar to the
sequence of 29 kDa IGFBP. Further storage of the 24 kDa
IGFBP led to its disappearance and appearance of several
low molecular weight protein bands which appeared to
contain very little IGFBP activity as determined by
. .
. . , .- : . .. ..
::, . .
., ~ , .
2 i 0 7 ~7 5 22 PCT/US92/0312~--
ligand blot analysis. Consistent with these results,
proteases associated with IGFBPs have been recently
reported.
Mono Q peak B appeared to have a different
amino acid composition and did not potentiate the action
of IGF-II on bone cells. our attempts to sequence peak B
proved unsuccessful. The fiequence determination of the
first few cycles of the major protein peak (Fraction 45,
peak C), yielded multiple amino acids with no readable
sequence. Thus, Mono Q peaks C and D which contained
very little IGFBP activity may represent the degradation
products of the 29 kDa IGFBP.
Since N-terminal sequence of Mono Q purified
hBD-IGFBP peak yielded in addition to the major sequence,
additional sequences which lacked one or two amino acids
(perhaps due to co-purification of IGFBP protease which
degrades this IGFBP) and since the cysteine residues were
not derivatized, the valine at position 7 of the present
hBD-IGFBP sequence and the a6partic acid at position 10
may be cysteines (cysteine residues have been preserved
among different members of IGFBP family). Storage of
Mono Q purified human bone derived IGFBP at 5C led to
disappearance of the 29 kDa IGFBP and appearance of
smaller molecular weight IGFBPs by SDS-PAGE. When the
small molecular weight IGFBP was sequenced, the valine
and aspartic acid at position 7 and 10 respectively were
not found, in that there were no signals at these
positions. These findings suggest that hBD-IGFBP may
have the sequence of L-G-F-F-V-X-C-E-P-C-D-K-A-A-L in one
embodiment. An alternative sequence for hBD-IGFBP which
also takes into account the foregoing considerations is:
L-G-S-F-V-H-C-E-P-C-D-E-X-A-L, which sequence is similar
to the BP-5 sequence of Kiefer et al., Biochem. Biophys.
Re$~am. 176:219-225 (1991), Shimasaki et al., J. Biol.
Chel~ 266:10646-10653 (1991), and as disclosed in Drop,
En~QçLiLsL,. 130:1736-1737 (1992). The sequence may be
subject to possible variations based on native or
introduced substitutions, additions or deletions, which
.
.
~ W092/18~ 2 1 0 7 ll 7 ~ PCT/US92/03122
variations may be allelic variants or produced through
particular sequence techniques employed herein. It will
be recognized that using the methods described herein,
the protein may be isolated and purified and the sequence
determined by a variety of well known methods. Further,
the N-terminal sequence allows for the construction of
degenerate oligonucleotide probes for the cloning of the
gene which encodes the hBD-IGFBP of the invention.
EXAMPLE III
Use of hBD-IGFBP in bo~_cell proliferation assays
An assay for IGF-mediated proliferation of bone
cells in serum-free culture medium, which is described in
Mohan, et al., (Br~oc~im. Biophys. Acta, 884:234-242
(1986)), hereby incorporated by reference, measures the
incorporation of [3H]thymidine into trichloroacetic acid
precipitable cellular material. This as6ay was performed
using the mouse osteoblastic cell line MC3T3-E1.
Approximately 10,000 cells were plated per well in serum-
free Dulb¢cco's modified Eagle's medium into 4B-well
culture dishes and used for the ~3H~thymidine assay as
generally described in Mohan et al., ibid.
In this assay hBD-IGFBP by itself had little
mitogenic activity, as determined by the incorporation of
t3H]thymidine into trichloroacetic acid insoluble
macromolecules ~Table 3, below). However, when hBD-IGFBP
was added along with submaximal concentrations of IGF-II
to serum-free cultures of mouse bone cells, hBD-IGFBP
potentiated the proliferative action of IGF-II. IGFBP-3
has been shown to increase IGF-I action only when added
to cultures several hours prior to the addition of IGF-I,
(Mohan, et al., Proc. ~atl. AÇ~- ~cL. USA, 86:8338-8342
(1989) and De Mellow, et al., Biochem. Biophvs. Res.
Co~m., 156:199-204 (1988)) and no other IGFBPs are
believed to have been shown to potentiate the actions of
IGFs under conditions where both an IGF and an IGFBP are
wo 92~18-~ 2 10 7 ~ 7 ~ 24 PCT/US92/03122~--
added simultaneously. These data suggest that the hBD-
IGFBP is not merely a passive carrier for the IGFs but
also positively regulates the action of the IGFs.
Although the mechanism(s) by which hBD-IGFBP potentiates
IGF-II stimulated t3H]thymidine incorporation is not
known, several mechanisms are offered by way of possible
explanation but not limitation. For example, the hBD-
IGFBP targets the IGF-II to the cell membrane for easy
access of the IGF-II to its receptor (perhaps through an
RGD sequence as in the case of IGFBP-l). Or the hBD-
IGFBP may increase the affinity of IGF-II to its receptor
by virtue of its binding to IGF-II, and/or increase the
half-life of IGF-II by protecting it from proteases.
~1~ 7i 7~
W092/18l54 25 PCT/USg2/03122
Table 3
Potentiating effect of hBD-IGFBP on IGF-II induced bone
cell proliferation
[ H]thymidine incorporation (% of control)
Experiment Experiment Experiment
Treatment 1 2 3
BSA control 100 + 15 100 + 22 100 + 12
hBD-IGFBP128 + 18 213 + 29 116 + 9
IGF-II 138 + 18 265 + 43 214 + 24
hBD-IGFBP
+ IGF-II*195 + 32 672 + 74 320 + 40
Se~rum free cultures of mouse osteoblastic cell line,
MC3T3-El were incubated fo~ 18 hrs with the effectors
prior to the addition of [-~H]thymidine. The final
concentrations of IGF-II and hBD-IGFBP were 3 and 10
ng/ml re3spectively. Values are Mean + SD of 6 replicate
wells. [ H~thymidine incorporation in bovine serum
albumin (BSA) treated control cultures were 1404 + 217,
237 + 53 and 730 + 91 respectively in the three
experiments.
* The interaction term between hBD-IGFBP and IGF-II was
highly significant (P<0.00001) by three way analysis
between experiment, hBD-IGFBP and IGF-II using CSS
computer program.
EXAMPLE IV
Purif~cation of hBD-IGFBP from bone cell
conditioned medium
Since bone cells in culture produce hBD-IGFBP,
serum free conditioned medium collected from bone cell
cultures can also be used for the purification of hBD-
IGFBP. Briefly, bone cell conditioned medium is
concentrated in an Amicon using YM5 (5 kilodalton
molecular weight cut-off), acidified with acetic acid to
a final concentration of lM and subjected to Sephadex G-
100 gel filtration to separate IGFs from IGFBPs. The
proteins are eluted with lM acetic acid. The fractions
containing IGFBPs are pooled, lyophilized, reconstituted
WO92/18154 2 ~ ~ 7 '~ 7 ~ 26 PCT/US92/0312?~
with phosphate buffered saline and subjected to an IGF-II
affinity column. The affinity bound proteins are then
subjected to FPLC Mono Q anion exchange chromatography to
separate hBD-IGFBP from other IGFBPs.
EXAMPLE V
Ouantitative diagnosti assay ~or hBD-IGFBP
hBD-IGFBP purified from human bone or expressed
by recombinant means is used for polyclonal and/or
monoclonal antibody production, which antibodies are then
used in quantitative as~ays for hBD-IGFBP. Briefly, hBD-
IGFBP is mixed with complete Freund' 6 adjuvant and
injected into rabbits, guinea pigs, rats or mice
following established protocols for antibody production.
Animals are subsequently injected with hBD-IGFBP mixed
with incomplete Freund's adjuvant every 3-4 weeks. For
polyclonal antisera, the animals are bled after 3-4
injections and the antibody titer to hBD-IGFBP determined
using radioimmunoassay or other mean6. Monoclonal
antibodies are produced by immortalizing antibody
producing cells obtained from the immunized animals using
well known techniques. Purified hBD-IGFBP is
radiolabeled and used as the signal-producing tracer.
The monoclonal antibody or antiserum with high titer is
then used for development of hBD-IGFBP radioimmunoassay
for measurement of hBD-IGFBP levels in the serum urine
and other biological fluids.
In general, the production of hBD-IGFBP is
increased by treatment of bone cells with agents which
increase bone cell proliferation. Thus, hBD-IGPBP can be
used as diagnostic marker for disease states associated
with bone cell proliferation, such as osteoporosis.
Accordingly, low serum hBD-IGFBP indicates osteoporosis
associated with low bone formation. Since hBD-IGFBP
potentiates IGF-II action, a high serum hBD-IGFBP may
also be associated with some cancers.
2~7~7~
WO92/181~ 27 PCT/US92/03122
. ~
EXAMPLE VI
Ouantitative diagnostic assay for IGFs using hBD-IGFBP
As described in this Example, recombinant or
purified hBD-IGFBP can also be used to quantify levels of
S IGFs in a biological sample, such as serum. 2-5 ng of
purified hBD-IGFBP was incubated with 40,000 cpm of 125I-
IGF in the presence or absence of unlabeled competitor.
IGF standards or sample containing unknown amounts of IGF
were used as competitors. After 60 minutes of incubation
at room temperature, hBD-IGFBP-IGF complex was
precipitated by adding polyethylene glycol in the
presence of bovine gamma globulin. After a 30 minute
centrifugation at 118S X g, an aliquot of the supernatant
was counted in a gamma counter. A standard curve was set
lS up with different concentrations of unlabeled IGF and the
amount of IGF in the unknown sample was calculated using
the standard curve. Thus the amount of biologically
active free IGFs was determined by this assay utilizing
purified hBD-IGFBP that has high affinity for IGFs.
EXAMPLE VII
hBD-IGFBp Fixes IGF-II in bone
2S Human bone contains a relatively large amount
of IGF-II. However, as shown in Table 4, 125I labeled
IGF-II by itself does not specifically bind to
hydroxyapatite (the Table shows only non-specific binding
which is less than 10% of total counts added) or to
collagen. In contrast to IGF-II, labeled hBD-IGFBP
exhibited specific binding to hydroxyapatite. The
binding of hBD-IGFBP to hydroxyapatite was specific since
the major serum binding protein, i.e., IGFBP-3 had no
similar activity and since hBD-IGFBP did not bind to
collagen, the other major constituent of bone.
Furthermore, the binding of hBD-IGFBP to hydroxyapatite
was strong, in that the hBD-IGFBP-hydroxyapatite complex
could not be dissociated with 4M guanidine HCl (4M
WO92/18154 2 ~ 0 7 ~ 7 ~ 28 PCT/US92/0312,~-~
guanidine HCl has been shown to dissociate interactions
between antibody-antigen complex). Preincubation of
labeled IGF-II with hBD-IGFBP prior to the addition to
hydroxyapatite column significantly increased IGF-II
binding to hydroxyapatite column. This activity af hBD-
IGFBP to facilitate the binding of IGF-II to
hydroxyapatite was specific in that IGFBP-3 had no such
activity. These findings are consistent with a
conclusion that under normal conditions IGF-II is fixed
in bone by means of hBD-IGFBP.
Table 4: hBD-IGFBP Facilitates the Binding of IGF-II to
Hy~roxyapatite
15 Ligand % Tracer Bound to
_ _Hydroxyapatite Type I Collaaen
[ I]IGF-II <l0 <l0
20 [l25I~hBD-IGFBp60 <l0
tl25I]IGF-II + hBD-IGFBP 45 <l0
[ 125I ] IGFBP-3 <10 . <10
[l25I]IGF-II + IGFBP-3<l0 <l0
. _ .
EXAMPLE VIII
Requlation of IGFBP-5 production in
human bone cells in vitro
To determine if human bone cells produce hBD-IGFBP
}n vitro, Northern blots of total RNA extracted from
serum free cultures of MG63, TE85, TE89, SaOs2 and U2
40 human osteosarcoma cells were hybridized using an
oligonucleotide probe against the N-terminal sequence of
hBD-IGFBP and using a human IGFBP-5 cDNA probe (Dr.
Shimasaki, La Jolla, CA). These studies revealed that
all the cell lines tested expressed hBD-IGFBP mRNA. By
W092tl81~ ~ PCT/US92/03122
!
Western ligand blot analysis, IGF-I and IGF-II increased
production of hBD-IGFBP by several fold in U2 cells.
Biological characterization of hBD-IGFBP revealed that
this protein potentiated the proliferative action of IGF-
II in bone cells.
The proliferation of human osteoblasts and the
production of IGP-II stimulated by progesterone n vitro
has been reported. In the present experiment the effect
of progestsrone on the other components in the IGF
regulatory 6ystem (IGF-I, IGF8Ps and IGF receptors) was
studied in the human osteoblast-like osteosarcoma cell
line MG63. In all experiments, MG63 cells were plated at
a density of 7500 cells/cm2 in DMEM containing 1% calf
serum. After overnight incubation, the medium was
changed to 6erum-free before progesterone or vehicle
(ethanol) was added. In a time course study, cells were
incubated with 100 nM progesterone for 0.5 hours, 2
hours, 4 hours and 6 hours. Northern blot analyse6
demonstrated increases in mRNA levels for IGF-II, IGF-I,
hBD-IGFBP and type-l and type-2 IGF receptor form 30
minutes to 6 hours compared to respective control6. mRNA
levels of the inhibitory IGFBP-4, however, were decreased
as early as 30 minutes after progesterone addition.
There were no pronounced changes in IGFBP-3 mRNA levels.
Thus, progesterone, a steroid hormone which has been
6hown to stimulate human bone cell proliferation,
increases production of IGFBP-5 in human bone cells.
The stimulatory effect of progesterone on bone cell
proliferation could be mediated not only by increased IGF
production, but also by an increase in IGF receptor
expression, an increase in hBD-IGFBP (potentiates IGF
action), and a decreased production of the inhibitory
IGFBP-4.
