Note: Descriptions are shown in the official language in which they were submitted.
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BIOTINYLATED-CHEMOKINE ANTIBODY COMPLEXES
Field of the Invention
This invention relates to biotinylated compounds that, optionally, are
complexed with an anti-biotin antibody or fragment thereof. More specifically,
the
invention relates to complexes comprising biotinylated-chemokines and an
antibody or
fragment thereof selectively bound to the biotin. The biotinylated-chemokines
and
complexes containing same are useful for modulating the mechanism underlying a
variety of disease states, including a chemokine-mediated cellular response,
and for
1 o the selective delivery of agents to sites of disease activity.
Background of the Invention
Chemokines are a class of cytokine molecules that are involved in cell
recruitment and activation in inflammation. These chemokines have been
classified
into four subgroups, depending on the nature of the spacing of two highly-
conserved
15 cysteine amino acids that are located near the amino terminus of the
polypeptide. The
first chemokine subgroup is referred to as "CXC"; the second subgroup is
referred to
as "CC"; the third chemokine subgroup is referred to as "CX3C"; and the fourth
chemokine subgroup is referred to as "C". Within these subgroups, the
chemokines
are further divided into related families that are based upon amino acid
sequence
2o homology. The CXC chemokine families include the IP-10 and Mig family; the
GROa, GROG, and GROG family; the interleukin-8 (IL-8) family; and the PF4
family.
The CC chemokine families include the monocyte chemoattractant protein (MCP)
family; the family including macrophage inhibitory protein-la (MIP-la),
macrophage
inhibitory protein-1 (3 (MIP-1 (3), and regulated on activation normal T cell
expressed
25 (RANTES). The stromal cell-derived factor 1 a (SDF-1 a) and stromal cell-
derived
factor 1 (3 (SDF-1 (3) represent a chemokine family that is approximately
equally related
by amino acid sequence homology to the CXC and CC chemokine subgroups. The
CX3C chemokine family includes fractalkine; The C chemokine family includes
lymphotactin. In general, the CXC chemokines are bound by members of the CXCR
3o class of receptors; the CC chemokines are bound by the CCR class of
receptors; the
CX3C chemokines are bound by the CX3CR class of receptors; and the C
chemokines
are bound by the CR class of receptors.
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Cells which express chemokine receptors include migratory cells such as
lymphocytes, granulocytes, and antigen-presenting cells (APCs) that are
believed to
participate in immune responses or that may release other factors to mediate
other
cellular processes in vivo. The presence of a chemokine gradient serves to
attract
migratory cells which express the chemokine receptors. For example, migratory
cells
can be attracted by a chemokine gradient to a particular site of inflammation,
at which
location they play a role in further modifying the immune response. Chemokine
receptors also are involved in interacting with viral proteins. In particular,
CXCR4(fusin), CCRS, and other chemokine receptors have been identified as co-
to receptors for HIV-1 and HIV-2. In addition, chemokine receptors are
expressed on a
variety of non-motile cells such as neurons, microglia, epithelial cells and
fibroblasts.
Chemokines are also known to affect a variety of non-migratory cell functions-
such as
granule release, cytokine release, angiogenesis, growth and differentiation.
However.
the half life for chemokines in vivo is relatively short. (See, e.g., D.
Hechtman, et al.,
J. Immunol. 147(3):883-892 (1991) which reports a decline to preinjection
levels of
IL-8 in 30 minutes).
Various approaches also have been tried to extend the half life of injected
chemokines in vivo, as well as to accomplish the targeted delivery of
chemokines to
cell populations to establish a chemokine gradient. For example, International
Application No. PCT/US98/04002, (Publication No. WO 98/38212, inventors S.
Herrmann and S. Swanberg), entitled "Chimeric Polypeptides Containing
Chemokine
Domains," reports a chimeric DNA molecule comprising a sequence encoding a
chemokine polypeptide covalently attached to a heterologous polypeptide such
as the
binding domain of an antibody. Similarly, International Application No.
PCT/US98/01785, (Publication No. WO 98/33914, inventors J. Rosenblatt, et
al.,),
entitled "Chimeric Antibody Fusion Proteins for the Recruitment and
Stimulation of
an Antitumor Immune Response," reports chimeric molecules which include a
binding
region which specifically binds to a tumor-specific antigen and a chemokine
and/or
co-stimulatory ligand. United States Patent No. 5,645,835, issued to H. Fell.
Jr. and
M. Gayle, entitled "Therapeutic Antibody based Fusion Proteins," also reports
an
antibody-based fusion protein which includes an immunoglobulin portion coupled
to a
biologically active lymphokine. Unfortunately, each of the foregoing methods
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requires the creation of a chemokine fusion protein, thereby necessitating the
development of tailored methods for the generation of each different chemokine
construct. Thus, despite the innovations advocated in connection with fusion
protein
technology, there exists no universal approach for the targeted delivery of
chemokines
to cells which express the cognate chemokine receptor. Accordingly, a need
still
exists for a generally applicable method of chemokine delivery to cells in
vivo or in
vitro. Preferably, the universal method would be one which is easily
reversible in vivo
or in vitro.
Summary of the Invention
1 o The present invention overcomes these and other obstacles by providing a
biotinylated-chemokine antibody complex that can be reversibly dissociated in
the
presence of free biotin. Accordingly, the invention provides a method by which
the
concentration of biotinylated-chemokine antibody complex can be adjusted by
exposing the complex to varying amounts of free biotin in vivo or in vitro.
The
15 complexes of the invention are useful for a variety of applications,
including
modulating an immune response such as one which is mediated by the chemokine-
induced recruitment of migratory cells to a site of inflammation, as well as
for the
targeted delivery of agents to cells which express a chemokine receptor. In a
broader
aspect, the invention embraces biotinylated peptide ligands that bind to G-
protein
2o coupled receptors. Thus, the invention permits the targeted delivery of
agents
(diagnostic and therapeutic agents) to pre-selected cells which express such
receptors
and, advantageously, both avoids the necessity for the synthesis of individual
fusion
proteins to accomplish these objectives and provides a mechanism for rapid
down
regulation of activity.
25 According to one aspect of the invention, a composition is provided which
includes at least one type of each of the following components: (a) a biotin
conjugate
(also referred to as a "biotinylated agent"), including: (i) a biotin
covalently coupled to
(ii) a pharmacologically active agent; and (b) an anti-biotin antibody. The
anti-biotin
antibody is selectively bound to the biotin to form a complex of the
invention. The
3o composition optionally further includes a pharmaceutically acceptable
carrier and may
be formulated for a variety of modes of delivery in vivo, including
intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, and controlled-
release
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delivery. The preferred pharmacologically active agents are chemokines,
although
other peptide ligands which bind to G-protein coupled receptors also are
embraced
within the broader aspects of the invention.
Biotin is a naturally occurring enzyme cofactor in its L isomeric form.
Although it is preferred that L biotin be used for forming the biotin
conjugates of the
invention, the invention also embraces the D isomeric form of biotin, as well
as other
biotin derivatives. In general, the biotin is conjugated to the
pharmacologically active
agent (e.g., chemokine) by way of a linker molecule to form a "biotin
conjugate".
Thus, as used herein, the phrase "a biotin" embraces the D and L isomeric
forms of
1 o biotin and biotin derivatives in which biotin is coupled to a linker
molecule. The
linker molecule is selected to have a structure and to provide a sufficient
distance
between the biotin and the pharmacologically active agent to ensure that the
presence
of the biotin and/or the linker molecule does not adversely affect the
pharmacological
activity of the agent. Such linker molecules are well known in the art for
coupling
15 peptides and proteins to one another and for the attachment of other
functional
molecules to proteins.
According to certain preferred embodiments, the biotinylated
pharmacologically active agents are peptides which selectively bind to G-
protein
coupled receptors. Exemplary peptide ligands which bind to G-protein coupled
2o receptors include Angiotensin; Bradykinin; Bombesin/Neuromedin; C3a; CSa;
Calcitonin; Calcitonin Gene Related Peptide; Chemokine; Cholecystokinin;
Conopressin; Corticotropin Releasing Factor (CRF); CD55 - Decay Accelerating
Factor (DAF); Diuretic Hormone Receptors; Endothelin; MLP; FSH Glycoprotein
Hormone; Galanin; Growth Hormone Releasing Hormone (GHRH); Growth Hormone
25 Secretagogue (GHS); Gastric Inhibitory Peptide; Gastric Inhibitory Peptide;
Glucagon-like Peptide; Glucagon; Gonadotropin Releasing Hormone; LH
Glycoprotein Hormone; Melanocortin Receptors; Neuropeptide Y; Neurotensin;
Opioid; Oxytocin; Thrombin and Protease Activated; Pituitary Adenylyl Cyclase
Activating Peptide; PTH/PTHrP; Secretin; Somatostatin; Tachykinin; Thyrotropin
3o Releasing Hormone; TSH Glycoprotein Hormone: Vasopressin; Vasotocin;
Vasoactive Intestinal Peptide (VIP). In the preferred embodiments, the
pharmacologically active agents are chemokines. Exemplary chemokines are
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provided in Table 1 for use in accordance with the methods of the invention.
The
Tables are located immediately preceding the claims. In general, biotin is
coupled to
the pharmacologically active peptide via a linker molecule. More specifically,
for
peptides which are chemokines, biotin is coupled via a linker to the carboxyl
terminus
of the chemokine. The biotinylated-chemokines of the invention embrace biotin
coupled to the complete sequence of the chemokine, as well as biotin coupled
to
truncated or elongated versions of such peptides (e.g., a chemokine peptide
which
lacks a portion of its amino terminal or carboxyl terminal sequences).
Although not
wishing to be bound by any particular mechanism or theory, it is believed that
1o interaction of the chemokine highly basic carboxyl terminus with the
negatively
charged glucosaminoglycans mediates the cellular uptake of chemokines and that
truncation or deletion of the highly basic carboxyl terminus of chemokines can
be used
to create novel chemokine agonists having improved half life characteristics.
The
invention also embraces biotinylated truncated or elongated chemokine
peptides, in
15 particular, at the amino terminus, for use as chemokine antagonists to
inhibit the
normal pharmacological activity of the chemokine. (See, e.g., D. Hechtman, et
al., J.
Immunol. 147(3):883-892 (1991) which reports an IL-8-like inhibitor of
polymorphonuclear leukocyte accumulation at sites of acute inflammation).
The complexes of the invention further include an anti-biotin antibody which
20 selectively binds to biotin in the biotin conjugate. As used herein, the
term "antibody''
embraces intact antibodies as well as antibody fragments, e.g., Fab'2
fragments, CDR3
regions. Preferably, the anti-biotin antibody is a human, humanized, or
primatized
antibody and is non-antigenic in humans. The anti-biotin antibodies of the
invention
have a biotin binding domain that selectively binds to biotin alone or coupled
to a
25 linker molecule. One distinguishing feature of the anti-biotin antibodies
of the
invention, when complexed to a biotin conjugate, is a half life of the complex
which is
significantly greater than the half life of the free chemokine (not conjugated
or
associated in a complex) or biotinylated chemokine. Thus, in contrast to free
chemokine which exhibits a short half life in vivo (see, D. Hechtman, et al.,
supra.) the
30 complexes of the invention have a half life on the order of one day to one
month
(more preferably, from about one week to about two weeks). Moreover, the
effective
half life of the complex can be attenuated by contacting the complex with
biotin (in
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vivo or in vitro) to shift the equilibrium in the direction of complex
dissociation.
Thus, the anti-biotin antibodies of the invention are selected which permit
the
dissociation of the antibody from the biotin conjugate only in the presence of
supra
physiological levels of free biotin (alone or coupled to the linker molecule),
i.e., the
complex will not become dissociated under the conditions of physiologic biotin
concentrations. Physiological concentrations of biotin in blood are
approximately 0.5
nMol/liter or 122 ng/L (122 pg/ml). As used herein, superphysiological
concentrations
are defined as at least ten-fold, more preferably at least 100-fold and, most
preferably,
at least 1000-fold greater than the physiological concentration of the
particularly agent
l0 being considered. Thus, in contrast to the fusion proteins of the prior
art, the invention
provides a mechanism by which the antibody can be selectively dissociated from
the
biotinylated chemokine, e.g., by contacting the complex with
superphysiological
concentrations of an exogenous source of free biotin under conditions which
shift the
equilibrium reaction in favor of complex dissociation. Accordingly, the
antibodies of
the invention are selected, in part, based upon their ability to dissociate
from the
biotinylated pharmacologically active agent in the presence of exogenous
biotin.
Preferably, the antibodies of the invention, when complexed, exhibit a half
life
of the complex that is significantly shorter than the half life of an avidin-
biotin
complex in the presence of a supra physiological level of free biotin. The
half life of
2o the complex in the presence of a supra physiological level of free biotin
can be
determined in accordance with routine procedures known to those of ordinary
skill in
the art. Thus, in contrast to the avidin-biotin complex, the half life for the
complexes
of the invention are at least one-tenth, preferably one-one hundredth, and,
more
preferably, one-one thousandth of the half life of the avidin-biotin complex
in the
presence of a supra physiological level of free biotin. More particularly, the
half life
for a preferred complex of the invention in the presence of a supra
physiological level
of free biotin, with respect to the dissociation of biotin from the anti-
biotin antibody, is
less than about one hour, more preferably less than about 0.5 hours and, most
preferably, less than about 15 minutes. Exemplary anti-biotin antibodies that
are
3o publicly available and that can be tested in screening assays to determine
whether the
antibody exhibits an acceptable dissociation rate constant and/or affinity
constant for
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use (or further modification for use) in accordance with present invention are
identified below.
Optionally, the antibodies of the invention further include a diagnostic or
therapeutic agent for targeted delivery to a cell which expresses a receptor
for the
pharmacological agent (e.g., chemokine receptor). Exemplary therapeutic and
diagnostic agents are described below.
In certain preferred aspects of the invention, the anti-biotin antibody
exhibits a
mufti-specificity, preferably, a dual specificity. By this it is meant that
the antibody
includes a first binding domain which selectively binds to biotin and a second
binding
domain which selectively binds to at least one other molecule. For example,
the
antibody can exhibit a second specificity for a second biotin molecule, a
tumor cell
associated antigen, or a viral associated antigen. Exemplary tumor associated
antigens
are those from the following types of tumor cells: breast cancer cells,
ovarian cancer
cells, lung cancer cells, prostate cancer cells, as well as other, for
example, her2-neu
expressing cancer cells. In general, the tumor cell associated antigens are
cell-surface
antigens. More specific examples of tumor associated antigens include
carcinoembryonic antigen (tumors of epithelial origin, such as colon, lung,
and breast
and their metastases), EGF-R (bladder and breast cancer), prostate specific
membrane
antigen (prostate cancer), GD2 (neuroblastoma), membrane immunoglobulins
(lymphomas), and T-cell receptors (T-cell lymphoma). Exemplary viral
associated
antigens include: gp120 of HIV, HbsAg, immediate and early genes of hepatitis
C
virus, CMV, Epstein Barr virus, and respiratory syncytial virus.
In certain preferred embodiments, the complex includes a biotin conjugate
containing a chemokine that is involved in the recruitment of migratory cells
which
mediate a TH1 response, and the antibodies of the invention selectively bind
to a
tumor cell associated antigen and to the biotinylated-chemokine. According to
this
embodiment, the complex of the invention is delivered to tumor cells which
express
the tumor cell antigen and, thereby, mediates recruitment of TH1-type cells to
the
location of the tumor cells to enhance a localized TH 1 immune response. In
yet other
3o embodiments, the antibodies of the invention exhibit a dual specificity to
permit
recruitment of pre-selected TH2-type cells to a location to treat a localized
site of
inflammation.
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According to yet another aspect of the invention, a biotin composition
comprising a therapeutically effective amount of a biotin of the invention and
a
pharmaceutically acceptable carrier is provided. The biotin composition is
useful for
administration to a patient who has or will receive a biotinylated-
pharmacologically
active agent antibody complex of the invention. Administration of the biotin
composition facilitates dissociation of the complex in vivo and, thereby,
allows one of
ordinary skill in the art to further adjust the half life of the complex in
vivo. In
general, the half life of a biotinylated-chemokine of the invention is longer
when the
biotin conjugate is complexed with an anti-biotin antibody of the invention
and is
1o shorter when the biotin conjugate is present "free" (uncomplexed) ih vivo.
Accordingly, dissociation of the complex in vivo results in hastening the
natural
degradation of the biotin conjugate in vivo. The biotin composition also can
be used to
adjust the concentration in vitro.
The biotin composition can be administered in a variety of methods and,
preferably, is administered in an oral form. In general, the therapeutically
effective
amount of biotin is significantly greater for the oral biotin composition of
the
invention compared to dietary biotin supplements. Typically, the oral biotin
compositions of the invention are at least 10-fold, preferably 100-fold, and,
more
preferably, at least 1,000-fold greater than the concentration of biotin in
dietary
2o supplements. In the preferred embodiments, the therapeutically effective
amount of
biotin that is present in the biotin composition is from about 100 p,g to
about 100 mg.
More preferably, the effective amount of biotin is from about 100 g,g to about
10 mg
and, most preferably, the effective amount of biotin is from about 1 mg to
about 10
mg.
According to yet another aspect of the invention, a composition comprising a
mixture of biotin conjugates is provided. The composition includes: (a) a
first biotin
conjugate, including (i) a first biotin covalently coupled to (ii) a first
agent having a
first pharmacological activity; and (b) a second biotin conjugate, including
(i) a second
biotin covalently coupled to (ii) a second agent having a second
pharmacological
3o activity. The first biotin and the second biotin may be the same or
different. For
example, the first biotin may include a linker which differs from that
included in the
second biotin. Regardless of the nature of the biotin, the biotins are coupled
to the
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first and second agents, respectively, in a manner which does not adversely
affect
(diminish to a significant extent) the first and second pharmacological
activities.
Preferably, the first agent and the second agent represent different
chemokines which
bind to different receptors expressed on the same or different cell types. The
composition, optionally, further includes an anti-biotin antibody that binds
to the first
biotin conjugate and/or the second biotin conjugate. Thus, the composition
also
provides a means of reversibly attaching two or more chemokines by way of an
anti-
biotin antibody. Accordingly, the complexes of the invention can be used to
target
different categories of pre-selected chemokine receptors, e.g., CCR2 and CXCR3
l0 receptors, without requiring de novo fusion protein construction. In yet
other
embodiments, the anti-biotin antibody has a dual specificity for binding to
biotin and
to an antigen expressed on the surface of a pre-selected cell. For example, a
first anti-
biotin antibody can be used to target a receptor that is expressed on a
cytotoxic T-cell
(e.g., CD8), and a second anti-biotin antibody can be used to target a
receptor that is
expressed on a monocyte or a virus-infected cell. In this manner, the
complexes of
the invention containing the same or different chemokines can be delivered to
the
same or different cell types.
According to yet another aspect of the invention, a biotin conjugate
composition is provided. The composition includes the above-described biotin
2o conjugate of the invention and a pharmaceutically acceptable carrier. The
biotin
conjugate includes a biotin covalently coupled to an agent having a
pharmacological
activity (e.g., a chemokine). Preferably, the pharmaceutically acceptable
carrier is one
which is tailored for in vivo use, particularly parenteral use. Exemplary
pharmaceutically acceptable carriers are disclosed below.
According to still another aspect of the invention, a method for treating
inflammation in a subject is provided. As used herein, the word treating
embraces
preventing, inhibiting, and ameliorating the symptoms of the particular
condition
which is being treated. The method of treatment involves: administering to a
subject
in need of such treatment a therapeutically effective amount of a biotin
conjugate or of
a complex comprising: (a) a biotin conjugate and an anti-biotin antibody. The
biotin
conjugate includes: (i) a biotin covalently coupled to (ii) an agent that
selectively binds
to a receptor expressed by a pre-selected cell associated with inflammation
(e.g., a
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migratory cell). Administration of the biotin conjugate or the complex
prevents or
reduces inflammation in the subject. In certain embodiments, the biotin
conjugates)
and the anti-biotin antibody are simultaneously or sequentially administered
to the
subject. In the most preferred embodiments, the biotin conjugate is a
biotinylated
chemokine. Exemplary chemokines that can be used as the agent to which biotin
is
coupled in this aspect of the invention are provided in Table 1. In certain
preferred
embodiments, the pre-selected cell is a migratory cell, such as a T-cell or
leukocyte,
and the administration of a complex enhances recruitment of the migratory cell
to a
site of tumor cells in the subject.
l0 According to yet another aspect of the invention, a method for delivering a
diagnostic or a therapeutic agent to a pre-selected cell is provided. The pre-
selected
cells express on their surface a receptor which selectively binds to a biotin
conjugate
of the invention. According to this aspect of the invention, the method
involves
contacting a population of cells containing a pre-selected cell (e.g., a
leukocyte) with
15 an effective amount of a complex of the invention under conditions to
deliver the
complex to the pre-selected cell. The complex includes: (a) a biotin conjugate
including (i) a biotin covalently coupled to (ii) an agent that selectively
binds to a
receptor expressed by the pre-selected cell; and (b) an anti-biotin antibody
selectively
bound to the biotin. The anti-biotin antibody further includes a diagnostic
and/or
20 therapeutic agent. Exemplary therapeutic agents include cytotoxic agents
such as
those described elsewhere in this application. Exemplary diagnostic agents
include
detectable labels such as those described below. The diagnostic and/or
therapeutic
agents of the invention are attached to the anti-biotin antibodies in a manner
which
does not adversely affect the ability of the antibody to selectively bind to
biotin.
25 According to yet another aspect of the invention, a method for modulating
(up
regulating or down regulating/desensitizing) a pre-selected chemotactic
response is
provided. The method involves administering to a subject in need of such
treatment a
therapeutically effective amount of a biotinylated chemokine agonist or
chemokine
antagonist to modulate the chemotactic or other chemokine mediated
proinflammatory
3o response. The method for modulating a chemokine response can be used to
evaluate
the role of specific chemokine receptors in various animal models of human
disease.
The method for modulating a chemokine response also can be used to prevent
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recruitment and/or activation of resident inflammatory cells, e.g., by
administering to a
subject in need of such treatment a biotinylated chemokine antagonist of the
invention,
particularly a biotinylated CXC chemokine antagonist. Additionally, the method
can
be used to identify specific leukocyte populations which express receptors for
chemokines, diagnostic imaging, reagent screening, and creating new animal
models
of human disease (e.g., by disrupting the normal chemotactic response and
observing a
change in symptoms in the animal model).
These and other aspects of the invention, as well as various advantages and
utilities, will be more apparent with reference to the detailed description of
the
1 o preferred embodiments and to the accompanying drawings.
All documents referenced in this application are incorporated in their
entirety
herein by reference.
Brief Description of the Drawings
Fig. 1 illustrates an exemplary starting material for synthesis of the
biotinylated
chemokines of the invention.
Fig. 2 shows that C-terminally biotinylated ITAC is equivalent to ITAC in
binding to CXCR3.
Fig. 3 shows that biotinylated ITAC is equivalent to ITAC in chemotaxis of
CXCR3 expressing RBL cells.
2o Fig. 4. shows that significant recruitment of eosinophils to the peritoneum
does
not begin until 8 hours post challenge in an ovalbumin model for eosinophil
recruitment into the peritoneum.
Fig. 5 shows that eosinophil recruitment to the peritoneum of ova-sensitized
mice is selectively inhibited by low doses of biotinylated eotaxin complexed
with
marine anti-biotin antibody at 48 hours post challenge.
Fig. 6 shows that the complex of biotinylated eotaxin and mouse anti-biotin
does not inhibit neutrophil recruitment to the peritoneum in ova-sensitized
mice at 6
hours post challenge.
Fig. 7 shows that lymphocyte recruitment to the peritoneum of ova-sensitized
3o and challenged mice is selectively inhibited by biotinylated ITAC but not
eotaxin
complexed with marine anti-biotin antibody at 72 hours post challenge.
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Fig. 8 shows that soluble eotaxin administered 24 hours following ova
challenge renders the recruited peritoneal eosinophils refractory to further
chemotactic
stimuli for at least 24 hours.
Detailed Description of the Invention
The present invention provides a biotinylated-chemokine antibody complex
that can be reversibly dissociated in the presence of free biotin.
Accordingly, the
invention provides a method by which the concentration of a biotinylated-
chemokine
antibody complex can be adjusted by exposing the complex to varying amounts of
free
biotin in vivo or in vitro. The complexes of the invention are useful for a
variety of
applications, including modulating an immune response such as one which is
mediated
by the chemokine-induced recruitment of migratory cells to a site of
inflammation, as
well as for the targeted delivery of agents to cells which express a chemokine
receptor.
As used herein, a pharmacologically active agent refers to a peptide that
binds
to a G-protein coupled receptor. The G protein-coupled receptor superfamily
(GPCR)
is a large group of receptor proteins which share a common structural
homology.
Exemplary GPCR include the calcium sensing receptor (CSR) and the metabotropic
glutamate receptors (mGluRs) (Tanage, Y., et al., Neuron, 1992, 8:169-179;
Brown,
E., et al., Nature, 1993, 366:575-580); several receptors for glycoprotein
hormones
(Segaloff, D., et al., Oxf. Rev. Reprod. Biol., 1992, 14:141-168), pheromones
2o receptors (Ors and VNRs), which are also GPCRs (Buck, L., et al., Cell,
1991,
51:127-133; Dulac, C., et al., Cell, 1995, 83:159-206. Other exemplary peptide
ligands that bind to G-protein coupled receptors include Angiotensin;
Bradykinin;
Bombesin/Neuromedin; C3a; CSa; Calcitonin; Calcitonin Gene Related Peptide;
Chemokine; Cholecystokinin; Conopressin; Corticotropin Releasing Factor (CRF);
CD55 - Decay Accelerating Factor (DAF); Diuretic Hormone Receptors;
Endothelin;
MLP; FSH Glycoprotein Hormone; Galanin; Growth Hormone Releasing Hormone
(GHRH); Growth Hormone Secretagogue (GHS); Gastric Inhibitory Peptide; Gastric
Inhibitory Peptide; Glucagon-like Peptide; Glucagon; Gonadotropin Releasing
Hormone; LH Glycoprotein Hormone; Melanocortin Receptors; Neuropeptide Y;
3o Neurotensin; Opioid; Oxytocin; Thrombin and Protease Activated; Pituitary
Adenylyl
Cyclase Activating Peptide; PTH/PTHrP; Secretin; Somatostatin; Tachykinin;
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Thyrotropin Releasing Hormone; TSH Glycoprotein Hormone; Vasopressin;
Vasotocin; and Vasoactive Intestinal Peptide (VIP).
The pharmacologically active agents are covalently coupled to a biotin in a
manner which does not adversely affect the pharmacological activity of the
pharmacologically active agent. The binding of a peptide to its cognate G-
protein
coupled receptor is accompanied by G-protein signal transduction, an event
which can
be measured using conventional screening assays, such as assays that measure
changes
in the intracellular concentrations of calcium and/or cyclic nucleotides (see,
e.g., PCT
Publication No. WO 94/18959, entitled "Calcium Receptor-Active Molecules,"
1o inventors E. Nemeth et al.). The chemokines are a preferred class of
pharmacologically active agents. Exemplary and preferred chemokines that are
useful
in the compositions and methods of the invention include those which are
depicted in
Table 1.
The Tables are located immediately preceding the claims.
15 For ease of discussion, the following detailed description of the invention
is
directed primarily to compositions and methods that include a biotinylated-
chemokine
as an exemplary biotinylated pharmacologically active agent. It is to be
understood
that other peptides which bind to G-protein coupled receptors can be
substituted for
the chemokines described herein to make and use additional biotinylated
2o pharmacologically active agents and complexes of the invention.
According to one aspect of the invention, a composition is provided which
includes the following components: (a) a biotin conjugate, including: (i) a
biotin
covalently coupled to (ii) a pharmacologically active agent; and (b) an anti-
biotin
antibody. The anti-biotin antibody is selectively bound to the biotin to form
a complex
25 of the invention. The composition optionally further includes a
pharmaceutically
acceptable carrier and may be formulated for a variety of modes of delivery in
vivo,
including intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and
controlled-release delivery. The preferred pharmacologically active agents are
chemokines.
30 Biotin is a naturally occurring enzyme cofactor in its L isomeric form.
Although it is preferred that L biotin be used for forming the biotin
conjugates of the
invention, the invention also embraces the D isomeric form of biotin, as well
as other
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biotin derivatives. In general, the biotin is conjugated to the
pharmacologically active
agent (e.g., chemokine) by way of a linker molecule to form a "biotin
conjugate". The
linker molecule is selected to have a structure and to provide a sufficient
distance
between the biotin and the pharmacologically active agent to ensure that the
presence
of the biotin and/or the linker molecule do not adversely affect the
pharmacological
activity of the agent. Such linker molecules are well known in the art for
coupling
peptides and proteins to one another and for the attachment of other
functional
molecules to proteins. As used herein, a "biotin" refers to the naturally
occurring
metabolic biotin which is in its L-isomeric form (Sigma Chemical Co., St.
Louis, MO;
1 o Pierce Chemical Co., Rockford, IL), as well as the D-isomeric form and
modifications
of the foregoing biotin molecules. By "modified biotin," it is meant a biotin
(either L-
or D-isomer) to which a further molecule is coupled in a manner to facilitate
the
covalent attachment of biotin to the pharmacologically active agent.
Typically,
modified biotins will refer to a biotin to which a linker molecule is
attached.
15 In general, biotin is coupled to the pharmacologically active peptide via a
linker molecule. More specifically, for peptides which are chemokines, biotin
is
coupled via a linker to the carboxyl terminus of the chemokine. The
biotinylated-
chemokines of the invention embrace biotin coupled to the complete sequence of
the
chemokine, as well as biotin coupled to truncated or elongated versions of
such
20 peptides (e.g., a chemokine peptide which lacks a portion of its amino
terminal or
carboxyl terminal sequences). Although not wishing to be bound by any
particular
mechanism or theory, it is believed that interaction of the chemokine highly
basic
carboxyl terminus with the negatively charged glucosaminoglycans mediates the
cellular uptake of chemokines and that truncation or deletion of the highly
basic
25 carboxyl terminus of chemokines can be used to create novel chemokine
agonists
having improved half life characteristics. The invention also embraces
biotinylated
truncated or elongated chemokine peptides, in particular, at the amino
terminus, for
use as chemokine antagonists to inhibit the normal pharmacological activity of
the
chemokine. (See, e.g., D. Hechtman, et al., J. Immunol. 147(3):883-892 (1991)
which
30 reports an IL-8-like inhibitor of polymorphonuclear leukocyte accumulation
at sites of
acute inflammation). Such biotinylated truncated chemokine peptides can be
used as
antagonists to inhibit the normal pharmacological activity of the chemokine.
Thus, for
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example, the biotinylated truncated chemokines of the invention include biotin
that is
covalently coupled to the carboxyl terminus of a chemokine peptide which lacks
a
portion of its amino terminal sequence such as the portion N-terminal to the
CXC or
CC sequence in the CXC and CC families of chemokines, respectively. See, for
example, J. Gog, et al., J. Exp. Med. 186:131-137 (1997) which reports a
truncated
human MCP-1 that functions as an antagonist in a mouse lupus-like disease
model.
Linker molecules are used to covalently attach the biotin to the
pharmacologically active agent of the invention. Such molecules are discussed
in
numerous books and catalogues, e.g., Pierce Catalog and Handbook, Rockford,
Ill.
1o Typically, these reagents are used to assist in the determination of near-
neighbor
relationships in proteins, three-dimensional structures of proteins, enzyme-
substrate
orientation, solid-phase immobilization, hapten-carrier protein conjugation
and
molecular associations in cell membranes. They also are useful for preparing
antibody-enzyme conjugates, immunotoxins and other labeled protein reagents.
15 For use in accordance with the present invention, it is necessary to
maintain the
native structure of the pharmacologically active agent. Accordingly, the
linker
molecules are selected which contain functional groups that couple to amino
acid side
chains of peptides. Bifunctional reagents (capable of crosslinking biotin to a
pharmacologically active agent) are classified on the basis of the following:
20 1. Functional groups and chemical specificity
2. Length of cross-bridge
3. Whether the cross-linking groups are similar (homobifunctional) or
different (heterobifunctional)
4. Whether the groups react chemically or photochemically
25 5. Whether the reagent is cleavable
6. Whether the reagent can be radio-labeled or tagged with another label.
Reactive groups that can be targeted using a linker molecule include primary
amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids. In
addition, any
reactive group can be coupled nonselectively using a linker molecule such as
3o photoreactive phenyl azides.
Linker molecules are available with varying lengths of spacer arms or bridges.
These bridges connect the two reactive ends. The linker molecules for use in
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accordance with the instant invention must be selected to minimally inhibit
the
pharmacological activity of the pharmacologically active agent and the ability
of biotin
to bind to the anti-biotin antibody. Because steric effects dictate the
distance between
potential reaction sites for cross-linking, different lengths of bridges are
required for
the interaction. Intermolecular cross-linking is favored with a cross-linker
containing
a longer space arm (about three to about fifteen atoms).
Conformational changes of proteins associated with a particular interaction
may also be analyzed by performing cross-linking studies before and after the
interaction. A comparison is made by using different arm-length cross-linkers
and
l0 analyzing the success of conjugation, e.g., in screening assays that
measure the
pharmacological activity of the conjugated agent or that measure the ability
of the
conjugated agent to bind to its cognate receptor. The use of cross-linkers
with
different reactive groups and/or spacer arms may be desirable when the
conformation
of the protein changes such that hindered amino acids become available for
cross-
15 linking.
Conjugation reagents contain at least two reactive groups. Homobifunctional
cross-linkers can contain at least two identical reactive groups, and
heterobifunctional
reagents contain two or more different reactive groups. Homobifunctional and
heterobifunctional cross-linkers that couple through amines, sulfhydryls or
react non-
2o specifically are available from many commercial sources. Exemplary linker
molecules
that are available from Pierce Co., Rockford, IL are shown in Tables 2,3, 4,
and 5.
The tables also identify the group that the linker molecule is reactive
toward, e.g.,
sulfhydryl, amino, etc.
In general, linker molecules are covalently attached to biotin using standard
25 protein coupling techniques which involve coupling a linker molecule to a
free amino
group, a free sulfhydryl group, or a free carboxyl group. Similarly, the
second
functional portion of the linker molecule is attached to the chemokine using
standard
protein coupling chemistry techniques. In general, the linker molecule is
attached to
the carboxyl terminus of the chemokine. The preferred linker molecules for
30 attachment of a biotin to a chemokine of the invention are those provided
in the
examples. In general, linker molecules are selected so that attachment of
biotin via the
linker molecule to the chemokine does not inhibit the ability of the chemokine
to bind
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to its respective receptors as determined in. for example, binding assays such
as those
disclosed in the examples. Accordingly. the invention also provides a method
for
selecting linker molecules for use in accordance with the method of the
invention,
namely, by coupling biotin to a chemokine via a "test" linker molecule and,
thereafter,
subjecting the biotinylated-chemokine to a pharmacological activity assay or
binding
assay to determine whether the chemokine has substantially retained its
pharmacological activity and/or its ability to bind to its respective
receptor.
The biotinylated chemokines of the invention can exhibit an agonist activity
or
an antagonist activity. By "agonist" is meant a molecule or compound which
activates
the signaling pathway in question. By "antagonist" is meant a molecule or
compound
which inhibits the signaling pathway in question. As used herein, a chemokine
agonist
activity refers to the ability of a chemokine to bind to its cognate receptor
and activate
the receptor, e.g., by triggering of an intracellular signal. As used herein,
a chemokine
antagonist activity refers to the ability of a chemokine of the invention to
bind to its
I5 cognate receptor without activating the receptor and triggering the
intracellular
signaling events. Accordingly, the chemokine antagonist of the invention can
be used
to competitively inhibit the binding and activity of a chemokine to its
respective
receptor in vivo or in vitro and, thereby, inhibit intracellular signaling.
Additionally or
alternatively, the chemokine agonists of the invention can be used to render
cells
refractory to further stimulation by circulating chemokines. By "rendering
cells
refractory," it is meant that the cells are no longer responsive in
chemotaxis, Ca2+ flux
or degranulation assays.
According to one aspect of the invention, a composition ("complex
composition") containing a biotin conjugate non-covalently coupled to an anti-
biotin
antibody to form a complex of the invention is provided. The biotin conjugate
includes a biotin covalently coupled to a pharmacologically active agent, and
the anti-
biotin antibody selectively binds to the biotin to form the complex. The
complex
compositions of the invention are useful, for example, for modulating an
immune
response and, in particular, are useful for treating inflammation, enhancing
an anti-
3o tumor response, and treating viral infections.
As used herein, an anti-biotin antibody refers to an antibody or antibody
fragment that selectively binds to biotin alone, coupled to a linker molecule,
or
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coupled to a pharmacologically active agent of the invention. The anti-biotin
antibodies of the invention are selected to bind to biotin and, when
complexed, exhibit
a half life of the complex that is significantly shorter than the half life of
an avidin-
biotin complex. The half life of an avidin-biotin complex is on the order of
about 200
days in vitro. Thus, in contrast to the avidin-biotin complex, the half life
for the
complexes of the invention are at least one-tenth, preferably one-one
hundredth, and,
more preferably, one-one thousandth of the half life of the avidin-biotin
complex.
More particularly, the half life for a preferred complex of the invention with
respect to
the dissociation of biotin from the anti-biotin antibody is less than about
one hour,
1 o more preferably less than about 0.5 hours and, most preferably, less than
about 15
minutes. Exemplary anti-biotin antibodies that are publicly available and that
can be
tested in screening assays to determine whether the antibody exhibits an
acceptable
dissociation rate constant and/or affinity constant for use (or further
modification for
use) in accordance with present invention are described in H. Bagci, et al.,
FEBS
322(1):47-50 (1993). See also, F. Kohen, et al., Meth. in Enzymol. 279:451-463
(1997); Vincent, P., and Samuel, D., J. Immunol. Meth. 165:177-182 (1993); and
K.
Dakshinamurti, et al., Meth. in Enzymol. 184:111-119 (1990).
Dissociation and association rate constants can be determined using known
methods. For example, such rate constants can be measured using the Biacore~
2o systems instrument (Biacore AB, Uppsala, Sweden). According to this method,
a
ligand, such as a biotinylated chemokine of the invention, is immobilized onto
a gold
film and a binding protein, such as an anti-biotin antibody, is contacted with
the gold
film under conditions to allow the binding protein to bind to the ligand. The
Biacore~
instrument employs a laser to measure the refractive index at the surface of
the gold
film and to provide real time measurements of the association and dissociation
of the
binding protein to the immobilized ligand. The instrument is used according to
manufacturer's directions to determine the on-rate constant, the off rate
constant, and
the affinity constant (the ratio of the on-rate constant to the off rate
constant).
The antibodies of the invention also can be characterized in terms of their
3o affinity constants which can be determined according to conventional
methods such as
those identified above. In general, the antibodies of the invention have an
affinity
constant ranging from about 1.0 to about 100.0 nanomolar. Preferably, the anti-
biotin
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antibodies of the invention have an affinity constant ranging from about 1 to
about 50
nanomolar and, more preferably, the antibodies have an affinity constant
ranging from
about 1 to about 10 nanomolar. In contrast, the affinity constant for an
avidin-biotin
complex (approximately 1 O15M) is several logs higher than those of the
antibodies of
the invention.
The antibodies of the invention are selected in screening assays which, in
part,
measure the dissociation rate constant of a biotin-anti-biotin antibody
complex. In
general, antibody selection is accomplished by performing an ELISA assay that
measures the ability of a soluble biotin molecule (e.g., L-or D-biotin, biotin
coupled to
l0 a linker molecule, or biotin coupled directly or indirectly to a
pharmacologically active
agent of the invention) to displace or compete with the biotin to which the
anti-biotin
antibody is bound in the complex. Although the antibodies of the invention are
selective for binding to biotin, the specific epitope region to which the anti-
biotin
antibody binds may include portions of biotin and the linker molecule.
Accordingly,
15 the antibodies disclosed herein allow for the competitive binding of
soluble biotin to
displace or compete with the biotin conjugates that are complexed with the
antibodies
of the invention in vivo or in vitro. As a result, the complexes of the
invention can be
dissociated in vivo or in vitro by contacting the complexes with a biotin
molecule (e.g.,
L-biotin) at a concentration that is effective to displace the equilibrium of
the complex
2o to allow dissociation of the complex. Antibodies with extremely high
affinities for
biotin (e.g., greater than about I nM to 0.1 nM) are to be avoided for
applications
which require a reversible reaction, but are preferred for those applications
where an
extended half life is desirable as in chronic inflammatory diseases and
cancer. An
exemplary screening assay for selecting an antibody, which when complexed, has
the
25 appropriate half life constant and the ability to permit complex
dissociation is
provided in the examples. Thus, the invention provides for the selection of
anti-biotin
antibodies having particular structural characteristics which allow the
antibodies to be
used in accordance with the methods of the invention. The invention also
permits the
selection of linker molecules having the appropriate spacer length to minimize
3o interference with the pharmacological activity of the chemokine to which
the biotin is
coupled, as well as to select linker molecule primary structures which, when
coupled
to biotin, can be used to generate monoclonal antibodies that selectively bind
to biotin
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as well as to a portion of the linker molecule. The examples also provide
assays for
evaluating complex stability in vivo by, for example. measuring the half life
of the
complex in vivo, as well as its tissue localization.
Throughout this application, the term "antibody" has been used in its broadest
sense to embrace full-length ("intact") antibody molecules, as well as
functionally
active fragments thereof (e.g., Fab, Fab'2, Fd, scFv, and antibody fragments
which
include a CDR3 region which binds selectively to a biotin). Antibodies include
polyclonal and monoclonal antibodies, prepared according to conventional
methodology. Preferably, the antibodies for human therapeutic applications are
1 o human antibodies.
As is well-known in the art, only a small portion of an antibody molecule, the
paratope, is involved in the binding of the antibody to its epitope (see, in
general,
Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley &
Sons, Inc., New York; Roitt, I. ( 1991 ) Essential Immunology, 7th Ed.,
Blackwell
15 Scientific Publications, Oxford). The pFc' and Fc regions, for example, are
effectors
of the complement cascade but are not involved in antigen binding. An antibody
from
which the pFc' region has been enzymatically cleaved, or which has been
produced
without the pFc' region, designated an Fab'2 fragment, retains both of the
antigen
binding sites of an intact antibody. Similarly, an antibody from which the Fc
region
2o has been enzymatically cleaved, or which has been produced without the Fc
region,
designated an Fab fragment, retains one of the antigen binding sites of an
intact
antibody molecule. Fab fragments consist of a covalently bound antibody light
chain
and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the
major
determinant of antibody specificity (a single Fd fragment may be associated
with up to
25 ten different light chains without altering antibody specificity) and Fd
fragments retain
epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the
art,
there are complementarily determining regions (CDRs), which directly interact
with
the epitope of the antigen, and framework regions (FRs), which maintain the
tertiary
30 structure of the paratope (see, in general, Clark, 1986; Roitt, 1991 ). In
both the heavy
chain Fd fragment and the light chain of IgG immunoglobulins, there are four
framework regions (FR1 through FR4) separated respectively by three
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complementarily determining regions (CDR1 through CDR3). The CDRs, and in
particular the CDR3 regions, and more particularly the heavy chain CDR3, are
largely
responsible for antibody specificity.
It is now well-established in the art that the non-CDR regions of a mammalian
antibody may be replaced with similar regions of nonspecific or heterospecific
antibodies while retaining the epitopic specificity of the original antibody.
This is
most clearly manifested in the development and use of "humanized" antibodies
in
which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions
to
produce a functional antibody. Thus, for example, PCT International
Publication
to Number WO 92/04381 teaches the production and use of humanized murine RSV
antibodies in which at least a portion of the murine FR regions have been
replaced by
FR regions of human origin. Such antibodies, including fragments of intact
antibodies
with antigen-binding ability, are often referred to as "chimeric" antibodies.
Thus, as will be apparent to one of ordinary skill in the art, the present
15 invention also provides for Fab'2, Fab, Fv and Fd fragments; chimeric
antibodies (e.g.,
based on the commercially available anti-biotin antibodies) in which the Fc
and/or FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies
in
which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
2o replaced by homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3
regions have been replaced by homologous human or non-human sequences; and
chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2
regions
have been replaced by homologous human or non-human sequences. The present
25 invention also includes so-called single chain antibodies, as well as human
antibodies
derived from libraries such as totally synthetic V gene libraries. In such
instances, the
biotin or biotin conjugates of the invention, or a fragment thereof, or
complexes of
biotin can be used to screen peptide libraries, including phage display
libraries. to
identify and select peptide binding polypeptides that selectively bind to the
biotin or
30 biotin conjugates of the invention. Such molecules can be used, as
described. for
screening assays, for purification protocols, for use in the complexes of the
invention
to treat inflammation, deliver a diagnostic or therapeutic agent, or modulate
a
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chemotaxis response in vivo or in vitro, and for other purposes that will be
apparent to
those of ordinary skill in the art.
For treatment where longer half lives are desirable, the antibodies of the
present invention are preferably intact antibody molecules including the Fc
region.
Such intact antibodies will have longer half lives than smaller fragment
antibodies
(e.g., Fab) and are more suitable for intravenous, intraperitoneal,
intramuscular,
intracavity, subcutaneous, or transdermal administration.
Fab fragments, including chimeric Fab fragments, are preferred in methods in
which the peptides of the invention are administered directly to a local
tissue
environment. For example, the Fab fragments are preferred when the peptide of
the
invention is administered directly to the site of the tumor or infection. Fabs
offer
several advantages over Fab'2 and whole immunoglobulin molecules for this
therapeutic modality. First, because Fabs have only one binding site for their
cognate
antigen, the formation of immune complexes is precluded whereas such complexes
can be generated when bivalent Fab'2 and whole immunoglobulin molecules
encounter
their target antigen. This is of some importance because immune complex
deposition
in tissues can produce adverse inflammatory reactions in certain instances.
Second,
because Fabs lack an Fc region they cannot trigger adverse inflammatory
reactions
that are activated by Fc, such as activation of the complement cascade. Third,
the
tissue penetration of the small Fab molecule is likely to be much better than
that of the
larger whole antibody. Fourth, Fabs can be produced easily and inexpensively
in
bacteria, such as E. coli, whereas whole immunoglobulin antibody molecules
require
mammalian cells for their production in useful amounts. Production of Fabs in
E. coli
makes it possible to produce these antibody fragments in large fermenters
which are
less expensive than cell culture-derived products.
Thus, the invention involves binding polypeptides of numerous size and type
that bind selectively to biotin. and conjugates (e.g., with linker molecule
and/or with
pharmacologically active agent) containing biotin ("biotin conjugate"). These
binding
polypeptides also may be derived from sources other than antibody technology.
For
example. such polypeptide binding agents can be provided by degenerate peptide
libraries which can be readily prepared in solution, in immobilized form, as
bacterial
flagella peptide display libraries or as phage display libraries.
Combinatorial libraries
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also can be synthesized of peptides containing one or more amino acids.
Libraries
further can be synthesized of peptides and non-peptide synthetic moieties.
Alternatively, anti-biotin antibodies of the invention can be selected to
enhance
clearance, e.g., for applications in which it is desirable to minimize the
half life of a
complex in vivo. Depending on the desire to mediate cell clearance versus
passive
blockade/desensitization of receptors, anti-biotin antibodies of suitable
isotypes can be
prepared. To achieve this objective, antibody fragments are administered or
intact
antibodies of a subclass selected from the group consisting o~ IgGl, IgG2a,
IgG2b,
IgG3, IgA, and IgM.
1o According to yet another embodiment, it may be desirable to select an anti-
biotin antibody that is capable of mediating the cross-linking of two
receptors on the
same or different cell surface. For such applications, it is desirable to
employ an anti-
biotin antibody which has a greater hinge region flexibility, such as an IgG3
antibody
subtype.
15 For applications which are directed to human therapeutics, it is desirable
to
have antibodies which are non-antigenic in humans, e.g., human monoclonal
antibodies or fragments thereof. For applications which are directed to
treating other
mammals, (e.g., domestic animals (e.g., dogs, cats) and livestock (e.g., cows,
sheep,
and horses), the biotinylated-chemokine and antibody components of the complex
20 should be selected to be non-antigenic in the species that is being
treated.
The antibodies of the invention can be selected for targeted delivery to a pre-
selected cell. This selection can be accomplished by selecting an antibody of
a
particular subgroup (e.g., targeted delivery of the antibody to Fc receptor-
bearing cells
by virtue of the nature of the Fc domain of the antibody) or by the
specificity of an
25 antibody binding domain. For example, the anti-biotin antibodies of the
invention
optionally include a binding domain which selectively binds to a tumor
associated
antigen or a viral associated antigen.
As used herein, a "tumor cell associated antigen" is a term of art as used in,
for
example, International Application No. PCT/US98/01785, (Publication No. WO
3o 98/33914), entitled "Chimeric Antibody Fusion Proteins for the Recruitment
and
Stimulation of an Anti-Tumor Immune Response," inventors J. Rosenblatt, et al.
Antibodies of the invention which have a dual specificity preferably bind to a
tumor
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cell associated antigen from tumor cells which are breast cancer cells,
ovarian cancer
cells, lung cancer cells, prostate cancer cells, or other cells which express
on their
surface a protein which is associated with cancer cell growth. Exemplary tumor
cell
associated antigens include: carcinoembryonic antigen (tumors of epithelial
origin,
such as colon, lung, and breast and their metastases), EGF-R (bladder and
breast
cancer), prostate specific membrane antigen (prostate cancer), GD2
(neuroblastoma),
membrane immunoglobulins (lymphomas), and T-cell receptors (T-cell lymphoma).
Further, specific examples of tumor cell associated antigens include: proteins
such as
Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma,
to Pmel-17 (gp100) of melanoma, MART-1 (Melan-A) of melanoma, p15 protein of
melanoma, tyrosinase of melanoma, MAGE l, 2 and 3 of melanoma, thyroid
medullary, small cell lung cancer, colon and/or bronchial squamous cell
cancer,
BAGE of bladder, melanoma, breast, and squamous cell carcinoma, gp75 of
melanoma, oncofetal antigen of melanoma; carbohydrate/lipids such as muc 1
mucin of
breast, pancreas, and ovarian cancer, GM2 and GD2 gangliosides of melanoma;
oncogenes such as mutant p53 of carcinoma, mutant ras of colon cancer and HER-
2/neu proto-oncogene of breast carcinoma; viral products such as human
papilloma
virus proteins of squamous cell cancers of cervix and esophagus. See, also,
Morioka,
et al., J. Immunol. 153:5650 (1994), for additional tumor antigens (e.g., P1A,
Connexin 37, MAGE-l, MAGE-3, MART 1/Aa, gp100, Tyrosinase) and/or
information relating to the tissue distribution of selected tumor antigens.
For
example, CD 19 on B cells also can be used to target certain leukemias and
lymphomas. Additional Melanoma tumor antigen sequences are those reported by
Slingluff et al., in Curr. Opin. in Immunol. 6:733-740 (1994); Additional
tumor cell
antigens that are peptides of the mutated APC gene product are those reported
by
Townsend et al., in Nature 371:662 (1994)).
In yet other embodiments, the anti-biotin antibodies of the invention have a
dual specificity for binding to a viral associated antigen. Exemplary viral
associated
antigens include: gp120 of HIV, HbsAg, immediate and early genes of hepatitis
C
virus, CMV, Epstein Barr virus, and respiratory syncytial virus.
The antibodies with dual specificity are useful for the targeted delivery of
chemokines to a cell expressing an antigen to which the anti-biotin antibody
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selectively binds. In this manner, a biotinylated chemokine and, optionally, a
further
therapeutic or diagnostic agent can be selectively delivered to, for example,
cells
expressing tumor cell associated antigens or cells/viruses expressing virus
associated
antigens. In a preferred embodiment of the invention, the antibodies with dual
specificity are used to enhance a localized immune response at the site of the
tumor
cells or virus particles/virus infected cells. For example, the complexes of
the
invention can be used to enhance a localized THl response for treating a
cancer by
administering a complex of the invention, including a biotinylated-chemokine
and an
anti-biotin antibody having a binding domain that selectively binds to a tumor
cell
1 o associated antigen. Targeted delivery of the complex to the tumor cell
results in the
selective recruitment of THl-type cells which express receptors for the
chemokine.
Exemplary chemokines that bind to receptors expressed on THl-type cells
include: IP-
10, MIG, RANTES, and ITAC.
The anti-biotin antibodies that are useful in the complex for treating cancer
have a binding domain that is selective for binding to biotin and at least one
other
binding domain that is selective for binding to a tumor cell associated
antigen.
Administration of the complex to a subject having a cancer that is associated
with the
tumor cell associated antigen is performed to allow targeting of the complex
to the
tumor cell and, thereby, result in a localized concentration of chemokine at
the site of
2o tumor cells. Although not wishing to be bound by any particular theory or
mechanism, it is believed that the presence of the elevated chemokine
concentration at
the site of the tumor cells results in a chemokine gradient which mediates the
recruitment of THl cells to the site and enhancement of a TH1 immune response
to
inhibit or prevent further tumor cell growth and/or proliferation. In an
analogous
manner, other conditions or diseases which flourish as a result of a lack of a
TH 1
response (e.g., lupus, arthritis) can be treated in this manner. Similarly,
diseases or
conditions which flourish, in whole or in part, due to a lack of a sufficient
TH2
response (e.g., leprosy, asthma, inflammatory bowel diseases such as Crohn's
disease
and ulcerative colitis, diseases associated with fibrosis such as emphysema
and hepatic
3o fibrosis) can be treated in an analogous manner by administering complexes
which
include a anti-biotin antibody of dual specificity (to target the complex to
the diseased
cell) and a biotinylated-chemokine which is effective in recruiting and/or
binding to
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receptors located on TH2-type cells. Exemplary chemokines that recruit and/or
bind
to TH2-type cells include: eotaxin, TARO, and MDC.
The anti-biotin antibodies of the invention optionally include a therapeutic
or
diagnostic agent attached to the antibody in a manner which does not adversely
affect
the ability of the antibody to bind to biotin or to a further antigen, if
applicable. As
used herein, "therapeutic agents" include any therapeutic molecule which
desirably is
targeted selectively to a cell expressing a tumor cell associated antigen or
other
infection-associated antigen. Therapeutic agents include antineoplastic
agents, radio
iodinated compounds, toxins, other cytostatic or cytolytic drugs, and so
forth.
to Antineoplastic therapeutics are well known and include: aminoglutethimide,
azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,
cisplatin,
cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,
daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon-a,
lomustine,
mercaptopurine, methotrexate, mitotane, procarbazine HCI, thioguanine,
vinblastine
15 sulfate and vincristine sulfate. Additional antineoplastic agents include
those
disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.
Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's
"The
Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill,
Inc.
(Health Professions Division). Toxins can be proteins such as, for example,
pokeweed
2o anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin,
diphtheria
exotoxin, or Pseudomonas exotoxin. Toxin moieties can also be high energy-
emitting
radio nuclides such as cobalt-60. Cytotoxic agents also include, for example,
the so-
called "suicide" enzymes such as thymidine kinase (TK) and its "suicide"
substrate,
gangcyclovir, DAB389 EGF (Pickering et. al., J. Clin. Invest. 91:724-9
(1993)), and
25 allylamine (Hysmith et al., Toxicology 38:141-50 (1986)).
Alternatively, or additionally, the anti-biotin antibodies of the invention
further
include a diagnostic agent, such as a detectable label. The antibodies may be
labeled
using radio labels, fluorescent labels, enzyme labels, or free radical labels,
using
techniques known to the art. The antibodies may be coupled to specific
diagnostic
30 labeling agents for imaging of cells and tissues, in vivo or in vitro, that
express cancer
associated antigens and/or to the above-noted therapeutic agents according to
standard
coupling procedures. Diagnostic agents include, but are not limited to, barium
sulfate,
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iocetamic acid. iopanoic acid, ipodate calcium, diatrizoate sodium,
diatrizoate
meglumine, metrizamide, tyropanoate sodium and radio diagnostics including
positron
emitters such as fluorine-18 and carbon-1 l, gamma emitters such as iodine-
123,
technetium-99m, iodine-131 and indium-111, nuclides for nuclear magnetic
resonance
such as fluorine and gadolinium. The antibodies and complexes of the invention
can
be used in vivo or in vitro. Accordingly, the detectable labels can include
more
traditional in vita°o labels such as those described herein. Typical
fluorescent labels
include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, and fluorescamine. Typical chemiluminescent compounds include
l0 luminol, isoluminol, aromatic acridinium esters, imidazoles, and the
oxalate esters.
Typical bioluminescent compounds include luciferin, and luciferase. Typical
enzymes
include alkaline phosphatase, 13-galactosidase, glucose-6-phosphate
dehydrogenase,
maleate dehydrogenase, glucose oxidase, and peroxidase. Other diagnostic
agents
useful in the invention will be apparent to one of ordinary skill in the art.
According to yet another aspect of the invention, a biotin composition is
provided which includes a therapeutically effective amount of a biotin and a
pharmaceutically acceptable carrier. The biotin composition is useful for
reducing the
amount of complex in vivo or in vitro. Although not wishing to be bound by any
particular theory or mechanism, it is believed that administration of a biotin
2o composition to a subject who has received a complex of the invention
results in the
displacement of the biotinylated-chemokine from the complex, thereby reducing
the
effective complex concentration in vivo. Thus, by dissociating the
biotinylated-
chemokine from the anti-biotin antibody, it is possible to alter the half life
of the
biotinylated-chemokine. Accordingly, the biotin compositions are useful for
adjusting
the concentration of the complex in vivo or in vitro and as a pharmaceutical
antidote to
terminate therapy, if warranted.
According to yet another aspect of the invention, a composition comprising a
therapeutically effective amount of a biotin in a pharmaceutical acceptable
carrier is
provided. The therapeutically effective amount of biotin is that amount needed
to
3o counteract the effect of a complex of the invention. Thus, the biotin
compositions
disclosed herein are intended for use for adjusting the effective
concentration of the
complexes of the invention in vivo or in vitro. By administering the biotin
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compositions to a subject who has received a complex of the invention, the
equilibrium equation of the complex, free biotinylated pharmacologically
active agent
and free antibody can be adjusted. In this manner. administration of free
biotin can be
used to shift the equilibrium to favor dissociation of the complex.
Preferably, the therapeutically effective amount of biotin in the biotin
composition is from about 100 ~g to about 100 mg. More preferably, the
concentration of biotin in the biotin composition is from about 100 p.g to
about 10 mg
and, most preferably, the concentration of biotin is from about 1 mg to about
10 mg.
In general, the concentration of biotin that is administered as a dietary
supplement is
the U.S. recommended daily allowance of 0.3 mg. In contrast, the
concentrations of
biotin in the claimed biotin composition are at least 10 fold and, more
preferably, 100
fold greater than those which are administered for dietary supplement
applications.
According to yet another aspect of the invention, a composition including a
mixture of complexes of the invention is provided. The composition contains:
(a) a
first biotin conjugate comprising (i) a first biotin covalently coupled to
(ii) a first agent
having a first pharmacological activity; and (b) a second biotin conjugate
comprising
(i) a second biotin covalently coupled to (ii) a second agent having a second
pharmacological activity. The first biotin and the second biotin may be the
same or
different from one another. For example, the first biotin may be the L isomer
and the
2o second biotin may be the D isomer.
The identity of the first and the second agents is dependent upon the
particular
use to which the mixture of complexes is to be applied. Thus, the mixture of
biotin
conjugates can further include one or more anti-biotin antibodies to form the
complexes of the invention and, optionally, to effect the targeted delivery of
the
complex to a particular cell population in vivo or in vitro. For example, the
anti-biotin
antibody can have a dual specificity for biotin and a tumor cell associated
antigen. In
this example, the first and the second agent can be two different chemokines
that
mediate the recruitment of different immune system cells to the site of the
tumor cell
location to enhance a TH1 response to the tumor. Similarly, the mixture of
complexes
of the invention can be used to effect the targeted delivery of different
chemokines to a
virus infected cell. In certain preferred embodiments, the composition further
includes
an anti-biotin antibody which has a dual specificity for binding to biotin and
to an
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antigen expressed on the surface of a pre-selected cell. For example, a first
anti-biotin
antibody can be used to target a receptor that is expressed on a cytotoxic T-
cell (e.g.,
CD8), and a second anti-biotin antibody can be used to target a receptor that
is
expressed on a monocyte or a virus-infected cell. In this manner, complexes of
the
invention containing the same or different chemokines can be delivered to the
same or
different cell types.
According to yet another aspect of the invention, a pharmaceutical composition
containing a biotin conjugate of the invention and a pharmaceutically
acceptable
carrier is provided. As used in reference to this particular embodiment, a
l0 "pharmaceutically acceptable carrier" is defined below in reference to the
pharmaceutical compositions of the invention, preferably, with the further
limitation
that the pharmaceutically acceptable carriers containing the biotin conjugates
exclude
buffer preparations which are commonly used in in vitro assays (e.g.,
phosphate
buffered saline). More preferably, the pharmaceutically acceptable carrier for
the
15 biotin conjugates of the invention is selected to be used for an
intravenous,
intraperitoneal, or subcutaneous mode of administration. Accordingly, the
therapeutically effective amount of the biotin conjugate also is selected to
be suitable
for in vivo administration. In general, the therapeutically effective amount
of the
biotin conjugate is at least 10 fold, more preferably 100 fold, and most
preferably 1000
20 fold greater than the concentration of a biotin conjugate that would be
used in
connection with an in vitro assay, such as an ELISA assay.
According to yet another aspect of the invention, a therapeutic method which
employs the biotinylated chemokines alone or complexed with anti-biotin
antibody is
provided. The therapeutic method is useful for treating inflammation in a
subject in
25 need of such treatment. The method involves administering to a subject a
therapeutically effective amount of a complex comprising: (a) a biotin
conjugate
comprising (i) a biotin covalently coupled to (ii) an agent that selectively
binds to a
receptor expressed by a pre-selected cell (e.g., a migratory cell); and (b) an
anti-biotin
antibody selectively bound to the biotin to form a complex. Administration of
the
3o complex prevents or reduces inflammation in the subject. Although not
wishing to be
bound to any particular theory or mechanism, in particular embodiments in
which the
pre-selected cell is a migratory cell, administration of the complex is
believed to result
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in the inhibition of recruitment of the migratory cell to the site of
inflammation. The
biotin conjugate and the anti-biotin antibody can be administered
simultaneously or
sequentially to the subject. The method optionally involves further
administering a
pharmaceutically acceptable composition of biotin to modulate the effective
concentration of the complex in vivo or in vitro (by shifting the equilibrium
toward
dissociation of the complex). Examples of chemokines, their respective
receptors and
the migratory cells which express these receptors are provided in Table 1.
According to yet another aspect of the invention, a method to deliver
cytotoxic
agents to eliminate a specific pre-selected cell (e.g., a leukocyte or virus-
infected cell)
population is provided. The method can be performed in vivo or i~ vitro. For
example, the method can be used to deliver a cytotoxic agent to a pre-selected
cell
population prior to infusion of the cell population into a patient. The method
also can
be used in vitro to desensitize chemokine receptors on pre-selected cells
prior to cell
reinfusion, e.g., by contacting the cells with a biotin conjugate or complex
of the
invention at one hour, 37 C or under other conditions sufficient to
desensitize the cell.
In this manner, the susceptibility of the treated pre-selected cells to a
chemokine
gradient in vivo can be inhibited.
The method for delivering a therapeutic agent (e.g., a cytostatic or cytotoxic
agent) to a pre-selected cell involves: contacting a population of cells
containing a
pre-selected cell (e.g., a virus infected cell) with an effective amount of a
complex
comprising the therapeutic agent under conditions to deliver the therapeutic
agent to
the pre-selected cell. The complex includes: (a) a biotin conjugate comprising
(i) a
biotin covalently coupled to (ii) an agent that selectively binds to a
receptor expressed
by the pre-selected cell; and (b) an anti-biotin antibody selectively bound to
the biotin
to form the complex. The anti-biotin antibody further comprises the
therapeutic agent.
The method is performed under conditions whereby contacting the population of
cells
with the complex allows delivery of the therapeutic agent to the pre-selected
cell.
According to a still further aspect of the invention, a method for modulating
(up regulating or down regulating/desensitizing) a pre-selected chemotactic
response is
provided. The method involves administering to a subject in need of such
treatment a
therapeutically effective amount of a biotinylated chemokine agonist or a
biotinylated
chemokine antagonist to modulate the chemotactic response. The method is
useful for
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evaluating the role of specific chemokine receptors in various animal models
of human
disease. Preferably, the complexes include an anti-biotin antibody that has a
dual
specificity to permit the targeted delivery of the biotinylated chemokine
agonist or
chemokine antagonist to cells expressing the chemokine receptor.
According to yet another aspect of the invention, a method for identifying
specific leukocyte populations which express receptors for chemokines is
provided.
This method can be used to identify the cognate receptors (and cells
expressing the
cognate receptors) for chemokines for which receptors have not previously been
identified. The method involves contacting the cells suspected of expressing a
cognate
1 o receptor with a labeled complex of the invention and identifying the
receptor and/or
cell expressing the receptor to which the complex binds. The receptor can be
identified either by immunoprecipitating the cognate receptor or using the
responding
cells to construct a cDNA library from which to expression clone the receptor
from
host cells transfectants expressing smaller and smaller pools of cDNAs.
The compositions of the invention are administered in effective amounts. An
effective amount is a dosage of the biotinylated pharmacologically active
agent or
complex of the invention sufficient to provide a medically desirable result.
In general,
the therapeutically effective amount of a complex of the invention is less
than about
10 mg/kg body weight and, more preferably, is from about 1 mg/kg to about 5
mg/kg
2o body weight. However, the effective amount will vary with the particular
condition
being treated, the age and physical condition of the subject being treated,
the severity
of the condition, the duration of the treatment, the nature of the concurrent
therapy (if
any), the specific route of administration and like factors within the
knowledge and
expertise of the health practitioner. For example, in connection with cancer
treatment,
an effective amount is that amount which slows or inhibits the growth and/or
proliferation of tumor cells that are associated with the cancer. Likewise, an
effective
amount for treating a viral infection would be an amount sufficient to lessen
or inhibit
altogether the growth and/or proliferation of infected cells so as to slow or
halt the
development of or the progression of the infection. Thus, it will be
understood that the
compositions of the invention can be used to treat the above-noted conditions
prophylactically in subjects at risk of developing the foregoing conditions.
As used in
the claims, "inhibit" embraces all of the foregoing. It is preferred generally
that a
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maximum dose be used, that is, the highest safe dose according to sound
medical
j udgment.
The isolated biotin conjugates and biotin complexes may be administered alone
or in combination with alternative (complementary) drug therapies, by any
conventional route, including injection or by gradual infusion over time. The
administration may, for example, be intravenous, intraperitoneal,
intramuscular, intra-
cavity, subcutaneous, or transdermal. When using the isolated biotin
conjugates and
biotin complexes of the invention, direct administration to the tumor or
infected cell
site is preferred.
The drug therapies are administered in amounts which are effective to achieve
the physiological goals (e.g:, to prevent or reduce the physiological
consequences of
cancer, infection, or an aberrant immune response), in combination with the
biotin
conjugates and biotin complexes of the invention. Thus, it is contemplated
that the
drug therapies may be administered in amounts which are not capable of
preventing or
reducing the physiological consequences of the condition being treated when
the drug
therapies are administered alone but which are capable of preventing or
reducing the
physiological consequences of the condition being treated when administered in
combination with the biotin conjugates and biotin complexes of the invention.
The biotin conjugates and biotin complexes may be administered alone or in
2o combination with the above-described drug therapies as part of a
pharmaceutical
composition. Such a pharmaceutical composition may include the isolated biotin
conjugates and/or biotin complexes in combination with any standard
physiologically
and/or pharmaceutically acceptable carriers which are known in the art. The
compositions should be sterile and contain a therapeutically effective amount
of the
isolated biotin conjugates and biotin complexes in a unit of weight or volume
suitable
for administration to a patient. The term "pharmaceutically-acceptable
carrier" as
used herein means one or more compatible solid or liquid filler, diluents or
encapsulating substances which are suitable for administration into a human or
other
animal. The term "carrier" denotes an organic or inorganic ingredient, natural
or
synthetic, with which the active ingredient is combined to facilitate the
application.
The components of the pharmaceutical compositions also are capable of being co-
mingled with the molecules of the present invention, and with each other, in a
manner
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such that there is no interaction which would substantially impair the desired
pharmaceutical efficacy. Pharmaceutically acceptable further means a non-toxic
material that is compatible with a biological system such as a cell, cell
culture, tissue,
or organism. The characteristics of the carrier will depend on the route of
administration. Physiologically and pharmaceutically acceptable carriers
include
diluents, fillers, salts, buffers, stabilizers, solubilizers, and other
materials which are
well known in the art.
Compositions suitable for parenteral administration conveniently comprise a
sterile aqueous preparation of the biotin conjugates and/or biotin complexes,
which is
preferably isotonic with the blood of the recipient. This aqueous preparation
may be
formulated according to known methods using suitable dispersing or wetting
agents
and suspending agents. The sterile injectable preparation also may be a
sterile
injectable solution or suspension in a non-toxic parenterally-acceptable
diluent or
solvent, for example, as a solution in 1,3-butane diol. Among the acceptable
vehicles
and solvents that may be employed are water, Ringer's solution, and isotonic
sodium
chloride solution. In addition, sterile, fixed oils are conventionally
employed as a
solvent or suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or di-glycerides. In addition, fatty acids
such as
oleic acid may be used in the preparation of injectables. Carrier formulations
suitable
for oral, subcutaneous, intravenous, intramuscular, etc. administrations can
be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
Preservatives and other additives may also be present such as, for example,
antimicrobials, anti-oxidants. chelating agents, and inert gases and the like.
A variety of administration routes are available. The particular mode selected
will depend, of course, upon the particular drug selected, the severity of the
condition
being treated, and the dosage required for therapeutic efficacy. The methods
of the
invention, generally speaking, may be practiced using any mode of
administration that
is medically acceptable, meaning any mode that produces effective levels of
the active
compounds without causing clinically unacceptable adverse effects. Such modes
of
administration include rectal, topical, interdermal, or parenteral routes. The
term
"parenteral" includes subcutaneous, intravenous, intramuscular, or infusion.
Intravenous or intramuscular routes are not particularly suitable for long-
term therapy
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and prophylaxis. They could, however, be preferred in emergency situations.
Oral
and nasal administration are optional, although less preferred, methods.
The pharmaceutical compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well-known in the art of
pharmacy. All methods include the step of bringing the biotin conjugates and
biotin
complexes into association with a carrier which constitutes one or more
accessory
ingredients. In general, the compositions are prepared by uniformly and
intimately
bringing the biotin conjugates and biotin complexes into association with a
liquid
carrier, a finely divided solid carrier, or both, and then, if necessary,
shaping the
l0 product.
Other delivery systems can include time-release, delayed release or sustained
release delivery systems. Such systems can avoid repeated administrations of
the
biotin conjugates and biotin complexes described above, increasing convenience
to the
subject and the physician. Many types of release delivery systems are
available and
known to those of ordinary skill in the art. They include the above-described
polymeric systems, as well as polymer base systems such as poly(lactide-
glycolide),
copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing
polymers containing drugs are described in, for example, U.S. Patent
5,075,109.
Delivery systems also include non-polymer systems that are: lipids including
sterols
such as cholesterol, cholesterol esters and fatty acids or neutral fats such
as mono- di-
and tri-glycerides; hydrogel release systems; sylastic systems; peptide based
systems;
wax coatings; compressed tablets using conventional binders and excipients;
partially
fused implants; and the like. Specific examples include, but are not limited
to: (a)
erosional systems in which an agent of the invention is contained in a form
within a
matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and
5,736,152, and (b) diffusional systems in which an active component permeates
at a
controlled rate from a polymer such as described in U.S. Patent Nos.
3,854,480,
5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can
be
3o used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for
treatment of chronic conditions. Long-term release, are used herein, means
that the
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implant is constructed and arranged to deliver therapeutic levels of the
active
ingredient for at least 30 days, and preferably 60 days. Long-term sustained
release
implants are well-known to those of ordinary skill in the art and include some
of the
release systems described above.
The invention will be more fully understood by reference to the following
examples. These examples, however, are merely intended to illustrate the
embodiments of the invention and are not to be construed to limit the scope of
the
invention.
EXAMPLES
1o Example 1. Preparation of a representative Biotinylated Chemokine
In general, the biotinylated pharmacologically active agents of the invention
("biotin conjugates") are prepared in accordance with routine procedures known
to
those of ordinary skill in the art or are commercially available. Commercially-
available biotinylated chemokines include: biotinylated human MCP-l, human MIP-
1
15 alpha, and human MIP-1 beta (R&D Systems, Minneapolis, MN).
Specific protocols are provided below as exemplary methods for preparing a
representative biotin conjugate; however, it is to be understood that
alternative linker
molecules and pharmacologically active agents can be substituted for the
particular
linker molecules and pharmacologically active agents described in the Examples
to
2o make a wide variety of biotin conjugates using no more than routine
experimentation.
In addition, the biotin conjugates embraced within the scope of the invention
can be
tested in high throughput screening assays, e.g., ELISA and other colorimetric
assays,
to select biotin conjugates having the optimum properties (e.g., retention of
chemotactic binding activity, optimum binding affinities with anti-biotin
antibodies)
25 for use in accordance with the methods of the invention.
(a) Preparation of Biotinylated Chemokines:
Biotinylated chemokines and chemokines in general were synthesized by
automated
solid phase f moc chemistry (t-boc chemistry can also be used) using a 433
automated
peptide synthesizer (PE Biosystems: Foster City, CA). See, e.g., Fig. 1 for an
3o exemplary starting material for synthesis of the biotinylated chemokines of
the
invention. Special Fsat-moc HBTU cycles were used for the synthesis. The
protein
was then deprotected and cleaved from the resin by TFA, extracted and purified
by
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preparative reverse-phase HPLC. After purification, the protein was folded and
repurified as previously described (Clark-Lewis, L, et al., 1991. Chemical
synthesis,
purification, and characterization of two inflammatory proteins, neutrophil
activating
peptide 1 (interleukin-8) and neutrophil activating peptide-2 Biochemistry,
30:3128-
3135). Biotinylated chemokines were synthesized by incorporating during
synthesis
an extra C-terminal Lysine derivatized at the s-NH2 group with aminocaproic
acid-
biotin (f moc-Lys (aminocaproil-biotin) is commercially available from
Calbiochem-
Novabiochem, UK). The chemokines described herein were made with the biotin-
aminocaproyl-Lys amino acid being the second one starting from the C-terminus.
l0 However, this modification can be introduced into any other lysine or
lysines in the
sequence. By using orthogonally protected Lys residues (e.g., fmoc-Lys(Dde)),
any
other spacer groups (see tables 2-5) having a carboxyl and an amino
functionalities at
each end can be used. In general, this synthesis reaction involves a 2-step
coupling
reaction, in which the spacer group is added to the lysine residue first and
then the
biotin is coupled to the spacer. In addition, by synthesizing chemokines
containing an
extra C-terminal cysteine, modifications of this cysteine can be made with any
biotin-
crosslinker reagents containing sulfhydryl reactive groups (see tables 2-5)
for
examples of crosslinking reagents). This latter modification can be performed
after
synthesis, cleavage and folding and purification of the protein.
2o Example 2. Preparation of an Anti-biotin Antibody
In general, the anti-biotin antibodies of the invention are prepared in
accordance with routine procedures known to those of ordinary skill in the art
or are
commercially available. An exemplary protocol for preparing a monoclonal
antibody
that selectively binds to biotin with a relatively high affinity (KA ~ 109 M-
~) is
provided in H. Bagci, et al., FEBS 322(1):47-50 (1993). See also, F. Kohen, et
al.,
Meth. in Enzymol. 279:451-463 (1997); Vincent, P., and Samuel, D., J. Immunol.
Meth. 165:177-182 (1993); K. Dakshinamurti, et al., Meth. in Enzymol. 184:111-
119
(199U); and Sigma Chemical Co. (St. Louis, MO, cat. no. #B753, Murine IgGI a-
biotin clone BD-34). In addition, mice transgenic for human Vh and Vl genes
are
3o commercially available from Medarex, Annandale, NJ, or from phage display
libraries
of human Vh and Vl genes, prepared according to the procedure described in
M.D.
Sheets, et al., PNAS (USA) 95(11):617-62 (1998), entitled, "Efficient
Construction
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of a Large Nonimmune Phage Antibody Library: the production of high-affinity
human single-chain antibodies to protein antigens".
Protocols are provided below as exemplary methods for preparing a
representative anti-biotin antibody; however, it is to be understood that
alternative
antigenic compositions (containing biotin alone or coupled to linker molecules
of
various structures and lengths) can be substituted for the particular
antigenic
compositions described in the Examples to make anti-biotin antibodies having a
range
of pre-selected kinetic characteristics (e.g., half life and affinity
constants) using no
more than routine experimentation.
l0 (a) Identification of Biotin-Specific Monoclonal Antibodies
To produce murine monoclonal antibodies specific for biotin that possess the
binding parameters required for in vivo efficacy, we immunize BALB/c mice 3-4X
(at
two week intervals) with an emulsion of 10-100q,g of immunogen in Freund's
Adjuvant. Complete Freund's Adjuvant was used for the first immunization and
15 Incomplete Freund's Adjuvant was used for all subsequent immunizations. The
immunogen consists of a soluble chemokine conjugated via an aminocaproic acid
linker covalently bound to the s-amino group of a lysine residue added at the
carboxyl
terminus. Three weeks after the last immunization, the mice are boosted with
an
intravenous injection of about 10-100g.g of soluble biotinylated chemokine.
Four days
20 later the mice are euthanized and the spleen cells fused with an
appropriate murine
fusion partner in a ratio of about 5-10:1 (spleen:myeloma). The entire fusion
is plated
in 10 96-well plates and incubated for 8-10 days until the macroscopic
appearance of
clones. The supernatants are then analyzed according to the following
protocol.
(b) Primary Screen
25 The purpose of the primary screen is to identify all wells containing
antibodies
that bind conjugated biotin. This is accomplished using an ELISA with
biotinylated
KLH or BSA coated wells. The biotinylated proteins are coated at
concentrations
between 2-Sq,g/ml in carbonate buffer overnight at 4°C. The wells are
blocked for 1-2
hours at 37°C with a 2% solution of BSA in PBS, then washed in an
automated plate
30 washer. Supernatants from each well are incubated for 1 hour at 37°C
then washed in
the plate washer. The murine biotin-specific antibodies are detected using a
horseradish peroxidase conjugated affinity purified secondary antibody
specific for
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murine IgG. The secondary antibody is incubated for 30 minutes at 37°C,
then
unbound antibody is washed out in the plate washer. The assay is developed
using
OPD as a substrate. Cells from wells that are positive in the ELISA are
expanded into
24-well plates and the supernatants reassayed in the secondary screen.
(c) Secondary Screen
To identify antibodies with a broad range of binding affinities for conjugated
biotin vs. free biotin, each supernatant is assayed in the presence and
absence of 0.5
nM biotin to approximate the concentration of biotin in peripheral blood.
Supernatants
where binding activity is not affected by the presence of soluble biotin are
reassayed in
1o the presence of increasing concentrations of a biotinylated chemokine,
preferably other
than the immunogen, (and/or a lysine-aminocaproic acid biotin) vs. free
biotin. The
assay is developed as in the primary assay. The relative binding ratio is
calculated
from the concentration of free biotin that results in a 50% reduction in
antibody
binding/concentration of conjugated biotin that results in a 50% reduction in
binding.
15 Clones are grouped according to ratio and representatives from each group
are
subcloned by limiting dilution. The subclones are re-tested, production is
scaled up,
and the antibodies purified. The purified antibodies are then assessed for
their relative
ability to bind conjugated biotin in the presence of free biotin in an ELISA
with an
immobilized biotinylated chemokine (1 biotin moiety/molecule protein) using
the
20 same mass amount of each antibody and increasing concentrations of free
biotin. In
general, the selection criteria for selecting antibodies for use in accordance
with the
methods of the invention are as follows. Antibodies useful for therapeutic
intervention
preferably have an affinity for conjugated biotin 1-4 orders of magnitude
greater than
the affinity for free biotin. Preferably, the antibody also would be
dissociable from the
25 conjugated biotin in the presence of free biotin at higher levels than
found in blood.
Thus, an increased dietary intake of biotin can be employed as an antidote to
dissociate
the biotinylated chemokine-MAb complexes when medically necessary. To make
full
use of this invention, it is desirable to identify antibodies that have
varying degrees of
affinity for free biotin to tailor the complexes to the individual patients
and clinical
disease states.
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Example 3. Screening Assa for Binding to a Chemokine Receptor
A general protocol for a screening assay is presented below (see Fig. 2 for
the
results of a representative screening assay).
(a) Ligand Binding Assays.
Competition binding assays were performed with 125I-chemokine as the hot
ligand and biotinylated or unbiotinylated chemokine as the cold competitor
ligands.
The binding reaction was performed with 500,000 receptor cell transfectants
and 0.1
nM radiolabeled chemokine in binding buffer (SOmM HEPES< pH 7.5, 1 mM CaCi2,
mM MgCl2, 0.5% BSA and 0.05% azide) for 1 hour at room temperature in the
l0 presence or absence of cold chemokine in a range of 8 different
concentrations from
0.0008-20 nM. Non-specific binding is determined by residual binding obtained
in the
presence of 250 nM cold ligand. Cells are then washed with binding buffer
containing
0.5 M NaCI and cell associated radioactivity counted with a top count
radioactivity
counter. Specific binding was calculated by subtracting non-specific binding
from the
total binding observed at each concentration and IC50 values were calculated
by
KaleidGraph software.
Example 4. Screening Assay for Chemotaxis Activity in vitro
A general protocol for a screening assay for chemotaxis activity is presented
below (see Fig. 3 for the results of a representative screening assay). This
assay is
2o predictive of the results obtained in the in vivo chemotaxis assay
described below.
(1) Assessment of Chemotactic Activity of Biotinylated Chemokines i~
vitro
To confirm that the presence of the biotin moiety attached to a c-terminal
lysine
residue in the chemokine did not interfere with biological activity, a
chemotaxis assay
was performed in a 24 well transwell plate using L 1.2 transfectants
expressing the
murine or human form of the cognate chemokine receptor. To perform the assay,
increasing concentrations of the unmodified chemokine or the biotinylated form
of the
chemokine were added to the bottom chamber of appropriate wells in the
transwell
plate while 1,000,000 chemokine-receptor transfectants were placed in the top
3o chamber. The cells were allowed to migrate for 3-5 hours at 37°C.
the filters were
then removed, and the cells which migrated into the bottom chamber were
resuspended and quantitated by flow cytometric analysis.
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(2) Assessment of Chemotactic Activity of Biotinylated Chemokines in
VIVO
Example 4a. A Model of Leukocyte Recruitment
To assess the biological activity of the biotinylated chemokine-antibody
complexes, the inventors used a model of antigen-induced recruitment of
leukocytes to
the peritoneum of mice. In this model, mice of the BALB/c or C57B 1. /6 strain
were
sensitized by subcutaneous administration on days 0 and 7 with 100 gg of
ovalbumin
mixed with an adjuvant consisting of 2 mg of aluminum hydroxide. On day 14 the
mice were challenged by intraperitoneal administration of 10 ug of soluble
ovalbumin.
1 o Following the i.p. challenge leukocytes infiltrating into the peritoneum
were harvested
by peritoneal lavage and the number of each type of leukocyte was determined
by
differential cell count. As seen in the figure (Fig.4), three types of
leukocytes
(eosinophils, neutrophils, lymphocytes) infiltrated into the peritoneum in
response to
ovalbumin challenge. Neutrophil recruitment occurs early in the response to
ovalbumin and peaks between 4-6 hours post challenge. Significant eosinophil
recruitment does not begin until approximately 10 hours post challenge and
reaches
maximum levels 48 hours post challenge. Lymphocyte recruitment is delayed the
longest and is maximal by 72 hours post challenge.
Example 4b. Complexes of Biotinylated Eotaxin with Biotin-Specific Antibody
2o Inhibit Eosinophil Recruitment to the Peritoneum
Complexes of biotinylated chemokine with the biotin-specific monoclonal
antibody are formed ex vivo by mixing 2 ug or 0.2 ug of chemokine with 75 ug
of
antibody in a volume of 200 ~,l of saline, PBS or HBSS for 1 hour at
37°C. This
represents a molar ratio of antibody to chemokine of 2:1 and 20:1
respectively. The
complexes are then administered subcutaneously to ovalbumin sensitized mice 15
minutes prior to i.p. challenge. The effect of biotinylated chemokine/MAb
complexes
on the recruitment of eosinophils to the peritoneum was assessed at 48 hours
post
challenge. As seen in the figure (Fig.S), the biotinylated eotaxin alone had
no
significant effect on eosinophil recruitment when administered at the high
dose (2 ug)
or the low dose (0.2 ug). In contrast the complex of biotinylated eotaxin with
the
biotin-specific monoclonal antibody significantly inhibited eosinophil
recruitment.
This was true for both the high dose (2 ug) as well as the low dose (0.2 ug)
of eotaxin.
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As a control, complexes of biotinylated ITAC with antibody, produced and
administered under identical conditions, had no significant effect on
eosinophil
recruitment.
Example 4c. Neutrophil Recruitment of the Peritoneum in this Model is
Unaffected by Complexes of Biotinylated Eotaxin or ITAC with Biotin-Specific
Monoclonal Antibodies
The specificity of this invention was confirmed by evaluating the activity of
complexes of eotaxin or ITAC/MAb on neutrophil recruitment to the peritoneum
at 6
hours post challenge. Murine neutrophils do not express receptors for eotaxin
or
1o ITAC and thus should not be inhibited by either complex. As seen in the
figure
(Fig.6), neither complex is capable of inhibiting neutrophil recruitment to
the
peritoneum in this model.
Example 4d. Lymphocyte Recruitment to the Peritoneum Is Inhibited by
Complexes with Biotin fated ITAC but not Eotaxin
15 The specificity and broad applicability of this invention is demonstrated
in the
ability to dissect the role of different chemokine receptors in modulating the
recruitment of specific leukocyte populations. As demonstrated in the figure
(Fig. 7),
complexes of biotinylated eotaxin with Mab had no significant effect on
lymphocyte
recruitment to the peritoneum at 72 hours. In contrast, complexes of
biotinylated
2o ITAC with Mab significantly inhibited lymphocyte recruitment to the
peritoneum at 72
hours.
Example 4e. Administration of a Soluble Chemokine In Vivo Can Desensitize
Leukocytes, Bearing the Cognate Receptor, That Are Localized in a Tissue
Desensitization as a mechanism by which complexes of biotinylated
25 chemokine complexed with biotin-specific monoclonal antibodies inhibit
recruitment
of leukocytes to tissue was supported by the results of the experiment shown
in the
figure (Fig. 8). Eosinophils were allowed to infiltrate the peritoneum of
ovalbumin
sensitized and challenged mice for a period of 24 hours. Soluble eotaxin was
then
administered 2x, 3 hours apart, either i.p. or s.c. 24 hours post treatment
(48 hours post
3o challenge), the eosinophils were harvested from the peritoneum and assessed
for their
chemotactic potential in vitro. As seen in the figure (Fig. 8), eosinophils
from mice
treated with PBS had significant and robust chemotaxis to eotaxin compared to
media
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alone. In contrast, eosinophils from mice treated with eotaxin either s.c. or
i.p. failed
to chemotax or eotaxin suggesting that exposure to eotaxin in vivo had
desensitized the
eosinophils, rendering them refractory to further stimulation by eotaxin ex
vivo.
Conclusions:
In view of the foregoing, we have reached the following conclusions.
S.C. administration of complexes of biotinylated chemokine with anti-biotin
Mab can
inhibit the recruitment of receptor-bearing leukocytes to tissue following
antigenic
challenge. The inhibition is specific for leukocyte populations that express
the
relevant receptor. Preliminary evidence suggests that s.c. administration of a
soluble
chemokine can result in desensitization in vivo. This experimental approach
may have
broad applications for investigating the role of a variety of chemokine
receptors in
inflammatory processes.
Example 5. Evaluation of Complex Stability In Yivo
There are several approaches to measuring the relative amount of complexed
15 chemokine to free chemokine in the blood following administration. One
approach is
to use an ELISA in which a non-competing chemokine specific monoclonal
antibody
is immobilized in the well. This is used to capture chemokine from samples of
peripheral blood. The amount of antibody still complexed to the chemokine can
be
assessed using an HRP-conjugated secondary antibody specific of the heavy
chain
2o isotype of the biotin-specific monoclonal. Using this technique only
biotinylated
chemokine complexed to the biotin specific monoclonal antibody will be
identified.
The stability of the complex can be assessed by following the disappearance of
the
chemokine-complexed antibody over time. The stability of the complex in animal
models can be assessed by forming the biotinylated chemokine-MAb complexes
with
25 radiolabeled chemokine. Following the collection of blood samples at
various times
post administration, the immunoglobulin fraction of the blood is separated
from small
molecular weight species such as biotin by precipitation with ammonium sulfate
(or by
binding to protein G-sepharose beads). The ratio of labeled material in the
supernatant
relative to the immunoglobulin-containing precipitate (or protein G-sepharose
bead
30 associated) is a function of the dissociation rate of the chemokine from
the antibody.
It should be understood that the preceding is merely a detailed description of
certain preferred embodiments. It therefore should be apparent to those
skilled in the
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art that various modifications and equivalents can be made without departing
from the
spirit and scope of the invention. It is intended to encompass all such
modifications
within the scope of the appended claims.
All references, patents and patent publications that are recited in this
application are incorporated in their entirety herein by reference.
The Tables are presented below and are followed by what is claimed:
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CC Chemokines
TABLE 1 - CHEMOKINES
Ligand AKA Receptor Active On Availability
RANTES CCR1, CCR3,mono, T, Pep, R&D,
CCRS NK, eos, LKS
baso
MIP-la CCRI, CCRSmono, T, Pep, R&D
NK
MIP-lb CCRS mono, T, Pep, R&D
NK
MCP-1 CCR2 mono, T, Pep, R&D
NK, baso
MCP-2 CCR2 mono, T, Pep, R&D
NK, baso
MCP-3 CCR1, CCR2,mono, T, Pep, R&D
CCR3 NK, eos,
baso
MCP-4 NCC-1, CKb-10CCR2, CCR3mono, T, Pep, R&D
eos, baso
Eotaxin CCR3 eos, baso Pep, R&D,
LKS
Eotaxin-2 MPIF-2, CCR3 eos, brio Pep, R&D
CKb6
*TARC dendrokine CCR4 T Pep, R&D
*MDC STOP-1 CCR4 act T, mono,Pep
den,
NK
MIP-3a LARC, exodus-1,CCR6 den, T, lymphPep, R&D
CKb4
MIP-3b ELC, exodus-3,CCR7 act B, act Pep, R&D
CKb-1 I T
I-309 TCA3 CCR8 mono, thy Pep, R&D
HCC-1 HCC-3, NCC-2,CCRI mono, CD34+ Pep, R&D
Ckbetal, BM
MIP-
1 gamma
HCC-2 NCC-3, MIP-S,? neu, mono,
Lkn-1, MIP-ld lymph
MIP-3 MPIF-I,
CK68
MIP-4 DC-CK1, ? T Pep
PARC,
AMAC-1
SLC 6Ckine, CCR7 thy, act R&D
exodus- T
2, TC, A4,
PAN
EC2
TECK ?
macro, den,
thy
LEC NCC-4, LEC,?
ILINCK,
HCC4
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Ligand AKA Receptor Active On Availability
CKb-15
PTEC
CXC Chemokines
IL-8 CXCR1 neu, NK, Pep, LKS,
T LKS
GROa MGSA-a CXCR1, neu, NK Pep, R&D
CXCR2
GROb MGSA-b, CXCR1, neu, NK Pep, R&D
MIP- CXCR2
2a
GROg MIP-2b CXCR1, neu, NK Pep, R&D
CXCR2
PF4 ? ? pep
NAP-2 CXCR2 neu, NK Pep
ENA78 CXCR2 neu, NK Pep, R&D
GCP2 CXCR2 neu, NK Pep, R&D
**IP-10 CXCR3 act T Pep, R&D,
LKS
**MIG CXCR3 act T Pep, R&D
**ITAC H174, b-RI CXCR3 act T LKS
MIP-2 0 0
CKa2 ? '? LKS
ADEC BLC, BCA-1 CXCRS LKS
SDF CXCR4 lymph Pep, R&D
CX3C Chemokines
FractakineNeurotactinCX3CR T, mono, Pep. R&D,
eos LKS
C Chemokines
Lympholactin GPR57 lymph Pep
T i rtt cnemoxmes
**TH1 chemokines
KEY FOR TABLENK = natural act T = activated
I killer T cells
mono =monocytesNeu = neutrophilact B = activated
B cells
eos =eosinophilsAct = activatedlymph = lymphocytes
baso =basophilsDen =dendritic macro = macrophages
cell
T =T cells Thy =thymocytes
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WO 00/50088 PCT/US00/04312
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0
0
x
0
T
x
0
0.
z
a
C~ ~ X
a~
W .~ ~ ~ x ~ ~
a v
a
xo
d o
~
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a
Hy U
N ~C ~C
O
~
W _
z~
a
V
U
w
O ~ x
E~ o
U
U
b b
x x
c~3 x
~
N
O
z x x x x x x x x x
y
Q
~~ o
a
d x z ~ ~ coa ~ M x o z d a.
._
~
~ z ~ ~ 0 0
O Ga Ca
G~ ~ q
CA 02371849 2001-08-17
WO 00/50088 PCT/US00/04312
-47-
C_
O
X
O
4
.
b
:n
x
o x
z
a ~,
0
U ~ x X X x x X X
U H
a
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o~
~,, ~,
v
a
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'U
U ~ X X X ~ X x
o
>
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O
U
w
O .~; X X X
x
O
U
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.d X X X 7CX ?C
x~
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a H
z' ~ x X X x X x X X X X X X X x
~
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T p" ~ ~ ~ L1 cnN ~ d ~ c. G1
vW -~G7 ~ U v~~ d ~ paU ~
~
~ ~ te a a
x ~ ~ ~ z .. tnc
A /W
CA 02371849 2001-08-17
WO 00/50088 PCT/US00/04312
-48-
0
o x
b
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rig ~ ~ x
a ,~ H x x
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x x x x x x x x x xx x x x x x x x
Q
'~' o. d
o H
W U W o~0.'O CaW ~ ~ ~ p cn ~
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~ d ~ ~ ~ a ~ ~ 0.lww o ".~w z ~,.o
o
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'~ c o ~ co ~ o ..
d n ~ ~
~o cn ~ o
U w cncn ~ ~ w ~ w z
V7 ~ p cn
a
CA 02371849 2001-08-17
WO 00/50088 PCT/US00/04312
-49-
c
0
>,
x
0
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.
b
x
b
0
w
z
,_,
a
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U = ~
o > X ?G 5G
o
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