U.S. patent application number 09/302863 was filed with the patent office on 2003-01-30 for methods of use of the taci/taci-l interaction.
Invention is credited to DIN, WANWAN S., GOODWIN, RAYMOND G..
Application Number | 20030022233 09/302863 |
Document ID | / |
Family ID | 23169532 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022233 |
Kind Code |
A1 |
GOODWIN, RAYMOND G. ; et
al. |
January 30, 2003 |
METHODS OF USE OF THE TACI/TACI-L INTERACTION
Abstract
The invention discloses a novel interaction between a TNF
receptor (TACI) and its interacting ligand (TACI-L). Also disclosed
are methods of screening candidate molecules to determine potential
antagonists and agonists of the TACI/TACI-L interaction. The use of
the antagonists and agonists as therapeutics to treat autoimmune
diseases, inflammation, and to inhibit graft vs. host rejections is
further disclosed.
Inventors: |
GOODWIN, RAYMOND G.;
(SEATTLE, WA) ; DIN, WANWAN S.; (ISSAQUAH,
WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
LAW DEPARTMENT
51 UNIVERSITY STREET
SEATTLE
WA
98101
|
Family ID: |
23169532 |
Appl. No.: |
09/302863 |
Filed: |
April 30, 1999 |
Current U.S.
Class: |
435/7.1 ;
435/184 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 19/02 20180101; G01N 2333/70578 20130101; Y10S 930/14
20130101; A61P 25/00 20180101; G01N 2500/20 20130101; G01N 2333/525
20130101; G01N 33/74 20130101; G01N 33/6863 20130101 |
Class at
Publication: |
435/7.1 ;
435/184 |
International
Class: |
G01N 033/53; C12N
009/99 |
Claims
What is claimed:
1. A method of screening a candidate molecule to identify its
ability to inhibit or prevent the dissociation of a TACI/TACI-L
complex, said method comprising the steps of: a. forming said
TACI/TACI-L complex or a TACI/TACI-L fragment complex in the
absence of said candidate molecule; b. adding said candidate
molecule to a medium containing said TACI/TACI-L complex or said
TACI/TACI-L fragment complex; c. changing the conditions of said
medium so that, but for the presence of said candidate molecule,
said TACI or TACI fragments, would be released from the complex; d.
measuring the concentration of free or bound said TACI, TACI-L or
fragments thereof; and e. determining the dissociation constant of
said TACI/TACI-L complex or said TACI/TACI-L fragment complex and
comparing said constant to a dissociation constant of a TACI/TACI-L
complex or TACI/TACI-L fragment complex measured in a medium not
containing the candidate molecule.
2. A method of screening a candidate molecule to identify its
ability to inhibit a TACI/TACI-L complex, said method comprising
the steps of: a. adding TACI to a medium containing TACI-L and said
candidate molecule, wherein one of either said TACI or said TACI-L
is labeled and the other is bound; b. measuring the level of signal
produced; and c. comparing the level of signal produced in step (b)
to the level of signal produced by a TACI/TACI-L complex or
TACI/TACI-L fragment complex formed with said labeled TACI or
TACI-L in the absence of said candidate molecule; wherein
diminished levels of signal produced in step (b) indicate that said
candidate molecule inhibited said TACI/TACI-L complex.
3. A method of screening a candidate molecule to identify its
ability to mimic the biological activity of the TACI/TACI-L
complex, said method comprising the steps of: a. determining if
said candidate molecule binds to TACI, TACI-L or fragments thereof;
b. adding said candidate molecule to a biological assay to
determine its biological effects; and c. comparing said biological
effects of said candidate molecule with the biological effects of
said TACI/TACI-L complex or a TACI/TACI-L fragment complex.
4. A method of screening a candidate molecule to identify its
ability to be useful in the treatment of diseases modulated by the
TACI/TACI-L complex, said method comprising the steps of: a.
forming said TACI/TACI-L complex or a TACI/TACI-L fragment complex
in the absence of said candidate molecule; b. adding said candidate
molecule to a medium containing said TACI/TACI-L complex or said
TACI/TACI-L fragment complex; c. changing the conditions of said
medium so that, but for the presence of said candidate molecule,
said TACI or TACI fragments, would be released from said
TACI/TACI-L complex or said TACI/TACI-L fragment complex; d.
measuring the concentration of free or bound said TACI, TACI-L or
fragments thereof; and e. determining the dissociation constant of
said TACI/TACI-L complex or said TACI/TACI-L fragment complex and
comparing said constant to a dissociation constant of a TACI/TACI-L
complex or TACI/TACI-L fragment complex measured in a medium not
containing the candidate molecule.
5. A method of screening a candidate molecule to identify its
ability to be useful in the treatment of diseases modulated by the
TACI/TACI-L complex, said method comprising the steps of: a. adding
TACI to a medium containing TACI-L and said candidate molecule,
wherein one of either said TACI or said TACI-L is labeled and the
other is bound; b. measuring the level of signal produced; and c.
comparing the level of signal produced in step (b) to the level of
signal produced by a TACI/TACI-L complex or TACI/TACI-L fragment
complex formed with said labeled TACI or TACI-L in the absence of
said candidate molecule.
6. A method of screening a candidate molecule to identify its
ability to be useful in the treatment of diseases modulated by the
TACI/TACI-L complex, said method comprising the steps of: a.
determining if said candidate molecule binds to TACI or TACI-L; b.
adding said candidate molecule to a biological assay to determine
its biological effects; and c. comparing the biological effects of
said candidate molecule with the biological effects of said
TACI/TACI-L complex or a TACI/TACI-L fragment complex.
7. The method of any one of claims 1 through 6 in which the
candidate molecule is selected from a group consisting of a small
molecule, antibody, or peptide.
8. The method of claims 1, 3, 4 or 6, in which either TACI or
TACI-L, or fragments thereof, is labeled.
9. The method of any one of claims 1 through 6, in which at least
one fragment of said TACI/TACI-L fragment complex is soluble.
10. The method of any one of claims 1 through 6, in which said
TACI/TACI-L complex is comprised of the sequence of SEQ. ID. NO.:2
and the sequence of SEQ. ID. NO.:4.
11. A method of screening a candidate molecule to identify its
ability to inhibit (antagonize) or agonize a TACI/TACI-L complex,
said method comprising the steps of: (a) adding said candidate
molecule to a medium which contains cells expressing TACI and cells
expressing TACI-L; (b) changing the conditions of said medium so
that, but for the presence of said candidate molecule, said
TACI/TACI-L complex and/or a TACI/TACI-L fragment complex would be
formed; (c) determining the level of biological activity of said
TACI/TACI-L complex and/or said TACI/TACI-fragment complex formed
in said medium; and (d) comparing the level of biological activity
of step (c) with the level of biological activity that occurs in
said medium in the absence of said candidate molecule.
12. An antagonist as identified by the method of claim 11.
13. An agonist as identified by the method of claim 11.
14. A method of modulating an intracellular signaling cascade
mediated by the TACI/TACI-L complex in a mammal comprising
administering to such a mammal an effective amount of an agonist or
an antagonist of the TACI/TACI-L complex.
Description
FIELD OF INVENTION
[0001] This invention relates generally to the interaction between
the transmembrane activator and CAML-interactor (TACI) protein and
its ligand, TACI ligand (TACI-L), use of the interaction in
screening assays thereof, and related kits.
BACKGROUND OF THE INVENTION
[0002] Cellular change is often triggered by the binding of an
extrinsic element, such as a ligand, to the extracellular domain of
a cell surface membrane receptor protein. This binding can result
in cellular changes by activating and controlling intracellular
signaling cascades and cell regulatory mechanisms. As such,
understanding the initial binding interaction between the ligand
and its receptor protein can be of great interest to the scientific
community. A greater understanding of this interaction would enable
one skilled in the art to modulate the resulting signaling cascade
governed by the ligand/receptor interaction by selecting agents for
co-stimulation or inhibition of the binding of the ligand to its
receptor.
[0003] The tumor necrosis factor (TNF) receptor family is a class
of mammalian signaling molecules that play an important role in
protection against infection and immune inflammatory responses such
as cellular signal transduction, stimulation of cells to secrete
cytokines, cytotoxic T cell proliferation, general cellular
proliferation, lymph node formation, bone formation, and bone
degradation. TNF-mediated cellular signaling often involves a
molecular activation cascade, during which a receptor triggers a
ligand-receptor mediated signal. Alterations in TNF activation can
have profound effects on a multitude of cellular processes, such as
the activation or inhibition of cell-specific responses, cell
proliferation, inflammatory reactions, and cell death.
[0004] The interactions between TNF ligands and receptors may
result in one-directional signaling (the interaction of the TNF
receptor/ligand triggers a signaling cascade in the receptor only)
or may result in bi-directional or reverse signaling. In the
instances of bi-directional or reverse signaling, the interaction
would not only activate the signaling cascade of the TNF receptor
but would also trigger a signaling cascade in a cell bearing the
TNF ligand. (S. Wiley et al., Jour. of Immun., 3235-39 (1996).)
Thus, understanding the interaction between a TNF receptor and
ligand may result in therapeutic treatments involving the
inhibition or enhancement of either one or both of the TNF receptor
activity or TNF ligand activity.
[0005] One member of the TNF receptor family is the transmembrane
activator and CAML-interactor (TACI), a cell surface protein. The
TACI protein has been isolated and is described in WO 98/39361.
When activated, TACI stimulates the influx of calcium in
lymphocytes and initiates the activation of a transcription factor
through a combination of a Ca.sup.2+-dependent pathway and a
Ca.sup.2+-independent pathway. Functions of TACI include
controlling the response of lymphocytes to cancer and to foreign
antigens in infections, graft rejection, and graft-vs.-host disease
(GVHD). Furthermore, activation of lymphocyte signaling allows the
positive selection of functional lymphocytes and negative selection
against self-reactive clones. (WO 98/39361 at 15.)
[0006] TACI modulated signals are often activated by a
extracellular ligand/receptor interaction, which then triggers an
intracellular protein/protein interaction. One of the intracellular
proteins which bind with the TACI protein has been identified. TACI
interacts with the calcium-signal modulating cyclophilin ligand
(CAML), a protein associated with the calcium pathway in
lymphocytes. According to WO 98/39361, after the binding of the
extracellular domain of TACI to an extracellular ligand, the
cytoplasmic domain of TACI binds CAML, initiating a
Ca.sup.2+-dependent activation pathway, which includes the
activation of the transcription factors, NF-AT, AP-1 and NF.sub.kB,
a factor implicated in the actions of other members of the
TNF-receptor family. The regions for the interaction between TACI
and CAML were defined as the cytoplasmic COOH-terminal 126 amino
acids of TACI and the NH.sub.2-terminal 201 amino acids of CAML.
CAML's ability to act as a signaling intermediate was verified by
the inhibition of TACI-induced activation of the transcription
factor when blocked by a dominant-negative mutant. (Von Bulow, G.
et al., Science, Vol. 278, p.138-141 (1997).)
[0007] Although this interaction between the cytoplasmic domain of
TACI and CAML has been identified, little is known about the
extracellular ligand with which TACI interacts to initiate the
intracellular cascades. Given the important role TACI plays in
signal transduction and given the potential therapies that may
arise from the manipulation of the signaling cascades, there is a
need in the art for the identification and understanding of the
interaction of TACI with its signaling ligand. Further, there is a
need for the development of assays and therapeutic methods using
the interaction between TACI and its signaling ligand.
[0008] Another TNF protein that has been recently discovered is a
ligand that has been designated Neutrokine .alpha., which is
described in WO 98/18921. Identical nucleotide and polypeptide
sequences have also been disclosed as "TL5" in EP 0869180A1 and as
"63954" in WO 98/27114. As a member of the TNF family, Neutrokine
.alpha. polypeptides were described as useful in the treatment of
tumor and tumor metastasis, infections by bacteria, virus and other
parasites, immunodeficiencies, inflammatory disease,
lymphadenopathy, autoimmune diseases, and GVHD. Neutrokine .alpha.
was also described as useful to mediate cell activation and
proliferation. Further, Neutrokine .alpha. polypeptides were
described as primary mediators of immune regulation and
inflammatory response. (WO 98/18921 at 11; EP 0869180A1 at 3.)
[0009] As Neutrokine .alpha. polypeptides may inhibit immune cell
functions, the ligand was described as also having a variety of
anti-inflammatory activities. (WO 98/18921 at 49.) Specifically, it
was said that Neutrokine .alpha. polypeptides could be used as an
anti-neovascularizing agent to treat solid tumors and for other
non-cancer indications in which blood vessel proliferation is not
wanted. (Id.) The polypeptides could also be employed to enhance
host defenses against resistant chronic and acute infections and to
inhibit T-cell proliferation by the inhibition of IL-2
biosynthesis. Finally, Neutrokine .alpha. polypeptides could also
be used to stimulate wound healing and to treat other fibrotic
disorders. (Id.)
[0010] As such activities may be modulated by the Neutrokine
.alpha. polypeptides, knowledge of how the ligand functions would
be of significant interest to the scientific community. WO
98/18921, EP 0869180A1 and WO 98/27114, however, fail to identify
specific receptors with which Neutrokine .alpha. polypeptides bind.
Identification of the related TNF receptor would allow those
skilled in the art to identify antagonists which may then be used
in therapies to treat the disorders associated with the Neutrokine
.alpha. polypeptides. Thus, there is a need to greater understand
this TNF ligand, identify the receptors with which it interacts,
and determine how the interaction functions.
SUMMARY OF THE INVENTION
[0011] This invention aids in fulfilling these needs in the art by
identifying a novel interaction between the extracellular domain of
TACI and the Neutrokine .alpha. polypeptide (hereinafter referred
to as TACI ligand (TACI-L)), and uses thereof. Specifically, the
invention encompasses the identification of a novel interaction
between TACI (SEQ. ID. NO.: 2) and TACI-L (SEQ. ID. NO.: 4).
[0012] The present invention provides a screening method for
identifying molecules that enhance or inhibit the TACI/TACI-L
interaction, or that prevent or inhibit dissociation of a complex
formed by TACI and TACI-L. This screening method involves
contacting a mixture of cells which express TACI and cells which
express TACI-L with a candidate molecule, measuring cellular
responses, and detecting the ability of the candidate molecule to
inhibit or enhance the interaction between TACI and TACI-L or
inhibit the dissociation of the complex formed by TACI and TACI-L.
Successful inhibition indicates that the candidate molecule is an
antagonist. Increased activation of TACI or TACI-L indicates that
the candidate molecule is an agonist. The candidate molecules are
preferably small molecules, antibodies or peptides.
[0013] In a further aspect of the present invention, a solid phase
method may be used to identify small molecules which inhibit the
interaction between TACI and TACI-L. Using this method, TACI may be
bound and is placed in a mixture with labeled TACI-L. After
contact, the amount of signal is measured. Diminished levels of
signal indicate that the candidate molecule inhibited the
interaction between TACI and TACI-L.
[0014] In a still further aspect, the present invention provides a
screening method for identifying molecules which mimic the
biological activity of the TACI/TACI-L interaction. This screening
method involves adding a candidate molecule that binds to TACI or
TACI-L to a biological assay and comparing the biological effect of
the candidate molecule to the biological effect of TACI/TACI-L
complex.
[0015] In yet a further aspect, the invention provides for a
therapeutic use of agonists and antagonists of the TACI/TACI-L
complex in the treatment of diseases modulated by the complex.
[0016] In still a further aspect, the invention provides for the
antagonists and agonists of the TACI/TACI-L complex.
[0017] Finally, the invention relates to a kit to aid in the above
determinations and uses.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows the nucleotide (SEQ. ID. NO.:1) (FIG. 1a) and
deduced amino acid (SEQ. ID. NO.:2)(FIG. 1b) sequences of the TACI
protein.
[0019] FIG. 2 shows the nucleotide (SEQ. ID. NO.:3) (FIG. 2a) and
deduced amino acid (SEQ. ID. NO.:4)(FIG. 2b) sequences of the
TACI-L protein.
[0020] FIG. 3 shows the amino acid sequence of a polypeptide (SEQ.
ID. NO.:5), in which a CMV leader followed by a leucine zipper
motif is fused to the N-terminal region of the amino acid sequence
of TACI-L.
[0021] FIG. 4 shows the results of a plate binding assay capturing
TACI-L in which the ligand is diluted 1:2. FIG. 4a demonstrates the
results of the assay and shows the complete saturation of the
receptor binding sites. FIG. 4b, the Scatchard plot corresponding
to FIG. 4a, demonstrates the actual number of sites that were
bound. From these results, an affinity constant of
1.53.times.10.sup.-9 can be generated.
[0022] FIG. 5 shows the results of a plate binding assay capturing
TACI-L in which the ligand is diluted 1:5. FIG. 5a demonstrates the
results of the assay and shows the complete saturation of the
receptor binding sites. FIG. 5b, the Scatchard plot corresponding
to FIG. 5a, demonstrates the actual number of sites that were
bound. From these results, an affinity constant of
2.2.times.10.sup.-9 can be generated.
[0023] FIG. 6 shows the results of a plate binding assay capturing
HuTACI/Fc. FIG. 6a graphs the complete saturation of the receptor
binding sites. FIG. 6b, the Scatchard graph which corresponds to
FIG. 6a, demonstrates the actual number of sites that were bound.
The Scatchard plot of FIG. 6b demonstrates a curvilinear binding,
with a low affinity constant of 5.7.times.10.sup.-10 and a high
affinity constant of 1.0.times.10.sup.-10.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The terms "TACI" and "TACI protein" are used interchangeably
to define the TNF receptor disclosed by WO 98/39361. TACI comprises
an extracellular domain, a transmembrane domain, and a cytoplasmic
domain.
[0025] "Fragments" of TACI encompass truncated amino acid sequences
of the TACI protein that retain the biological ability to bind to
TACI-L. An example of such a fragment is the extracellular domain
of TACI. Such fragments are identified in WO 98/39361, which is
incorporated in this application in its entirety.
[0026] "Soluble TACI" includes truncated proteins that lack a
functional transmembrane domain of the protein but retain the
biological activity of binding to TACI-L. The soluble,
extracellular domain can be used to inhibit cellular
activation.
[0027] "Homologous analogs" of TACI include isolated nucleic acids
of the TACI protein that are at least about 75% identical to
SEQ.ID.NO.:1 and retain the biological activity of binding to
TACI-L. Also contemplated by the term are embodiments in which a
nucleic acid molecule comprises a sequence that is at least 80%
identical, at least 90% identical, at least 95% identical, at least
98% identical, at least 99% identical, or at least 99.9% identical
to SEQ.ID.NO.:1 and retain the biological activity of binding to
TACI-L. Further included are nucleic acids which are at least 85%
similar, at least 95% similar, or at least 99% similar to nucleic
acids that encode the amino acids of the TACI protein, as described
in SEQ. ID. NO.:2, and that maintain a binding affinity to TACI-L.
Still further included are all substantially homologous analogs and
allelic variations.
[0028] The percent identity and percent similar may be determined
by visual inspection and mathematical calculation. Alternatively,
the percent identity of two nucleic acid molecules can be
determined by comparing their sequences using the GAP computer
program, version 6.0 described by Devereux et al. (Nucl. Acids Res.
12:387, 1984) and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The preferred default parameters
for the GAP program include: (1) a unary comparison matrix
(containing a value of 1 for identities and 0 for non-identities),
and the weighted comparison matrix of Gribskov and Burgess, Nucl.
Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,
eds., Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0029] The terms "TACI-L" and "TACI ligand" are used
interchangeably to define the member of the TNF ligand family
disclosed by WO 98/18921. TACI-L is also disclosed as "TL5" in EP
0869180A1 and as "63954" in WO 98/27114. The full-length TACI-L
comprises an extracellular domain, a transmembrane domain, and a
cytoplasmic domain. Although the exact location of the
extracellular, transmembrane, and cytoplasmic domains may differ
slightly due to different analytical criteria for identifying the
functional domains, the range of amino acids 1 to 46 generally
represents the intracellular domain; amino acids 47 to 72 represent
the transmembrane domain, and amino acids 73-285, the extracellular
domain.
[0030] "Fragments" of TACI-L encompass truncated amino acids of the
TACI-L protein that retain the biological ability to bind to TACI.
An example of such a fragment is the extracellular domain of
TACI-L, which binds TACI. Another example of a TACI-L fragment is
amino acids 123-285 of the extracellular domain of the TACI
ligand.
[0031] "Soluble TACI-L" includes truncated proteins that lack a
functional transmembrane domain of the protein but retain the
biological activity of binding to TACI. The soluble, extracellular
domain can be used to inhibit cellular activation.
[0032] "Homologous analogs" of TACI-L include isolated nucleic
acids of the TACI-L protein that are at least about 75% identical
to SEQ.ID.NO.:3 and retain the biological ability to bind to TACI.
Also contemplated by the term are embodiments in which a nucleic
acid molecule comprises a sequence that is at least 80% identical,
at least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at least 99.9% identical to
SEQ.ID.NO.:3 and retain the biological ability to bind to TACI.
Further included are nucleic acids which are at least 85% similar,
at least 95% similar, or at least 99% similar to nucleic acids that
encode the amino acids of TACI-L, as described in SEQ. ID. NO.:4
and that maintain a binding affinity to TACI. Still further
included are all substantially homologous analogs and allelic
variations.
[0033] Sequences are substantially homologous when at least 50%
(preferably 60%, more preferably 65%, more preferably 75%, more
preferably 85%, and most preferably 99%) of the nucleotides match
over the defined length of the DNA sequences. Sequences which are
substantially homologous can be identified by comparing the
sequences using software known in the art or by the well-known
Southern hybridization experiment. Substantially homologous analogs
and allelic variations must maintain the same biological activity
as the protein they are homologous to (e.g. bind to the same
receptor or ligand).
[0034] The terms "TACI/TACI-L complex" or "TACI/TACI-L interaction"
are used interchangeably and refer to the protein unit formed by
the binding interaction of TACI to TACI-L.
[0035] The term "TACI/TACI-L fragment complex" includes the protein
units formed in which at least one binding partner is either a
fragment of TACI or TACI-L (e.g. the binding interaction of a TACI
fragment to TACI-L, TACI to a TACI-L fragment, or a TACI fragment
to a TACI-L fragment) or a homologous analog of TACI or TACI-L. The
TACI/TACI-L fragment complex has the same biological activity,
effects, and uses as the TACI/TACI-L complex, as described below.
The term "biological activity" includes the binding of TACI to
TACI-L or fragments thereof.
[0036] The term "biological effects" includes any cellular changes
or effects which result from a protein-protein interaction or the
interaction of a protein with an agonist or antagonist. Examples of
a biological effect of the TACI/TACI-L complex include an increase
or decrease in Ca.sup.2+ ions resulting from a protein-protein
interaction or the activation of the transcription factors, NF-AT,
AP-1 and NF.sub.KB.
[0037] The TACI/TACI-L interaction is a protein-protein
interaction. Protein-protein interactions can be observed and
measured in binding assays using a variety of detection
methodologies that include, but are not limited to, surface plasmon
resonance (Biacore), radioimmune based assays, and fluorescence
polarization binding assays. When performed in the presence of a
test compound, the ability of the test compound to modulate (e.g.
enhance or inhibit) the protein-protein binding affinity is
measured. In one embodiment of the instant invention, the binding
interaction between TACI and TACI-L occurs between the
extracellular domain of the TACI protein and amino acids 123-285 of
the extracellular domain of the TACI ligand.
[0038] The discovery of the interaction between TACI and TACI-L is
described in detail in Examples 1-3. Briefly, a ligand expression
construct was transfected into cells. The cells were incubated with
TACI:Fc, bound with an antibody of TACI:Fc, and followed by a
detecting agent. A soluble form of TACI-L was used in verifying the
interaction and was produced by fusing a CMV leader sequence
followed by a leucine zipper motif to the polypeptide. Other useful
leader sequences include IgKappa and Growth Hormone. PCR was used
to amplify the cDNA sequence which encodes the extracellular domain
(amino acids 123-285) of TACI-L by using the restriction sites of
specific oligonucleotides. CMV and leucine zipper sequences can be
obtained by methods well known in the art, such as by PCR or by
enzymatic digestion of previously cloned sequences. These fragments
are ligated and inserted into the appropriate expression vector.
(Smith et al., Cell, Vol. 73, 1349-1360.)
[0039] The interaction between TACI and TACI-L was further
characterized by plate binding assays, as described in Examples 4
and 5. Plate binding assays were conducted capturing either the
TACI protein or the TACI ligand. In each instance, a high affinity
constant was obtained, demonstrating the close binding interaction
between TACI and TACI-L.
[0040] The discovery and understanding of the interaction between
the extracellular region of TACI and TACI-L can be used to
determine potential agonists or antagonists and to further develop
understanding of which cell types TACI-L acts upon. Assays may
utilize the interaction between TACI-L and TACI to screen for
potential inhibitors (antagonists) or enhancers (agonists) of
activity associated with TACI-L molecules and identify candidate
molecules which may serve as therapeutically active agents that
enhance, inhibit or modulate the TACI/TACI-L complex. Potential
antagonists to the TACI/TACI-L interaction may include small
molecules, peptides, and antibodies that bind to and occupy the
binding site of either TACI or TACI-L, causing them to be
unavailable to bind to each other and therefore preventing normal
biological activity. Other potential antagonists are antisense
molecules which may hybridize to mRNA in vivo and block translation
of the mRNA into the TACI-L protein. Potential agonists include
small molecules, peptides and antibodies which bind to TACI or
TACI-L and elicit the same or enhanced biological effects as those
caused by the binding of TACI to TACI-L.
[0041] Small molecules are usually less than 10 K molecular weight
and possess a number of physiochemical and pharmacological
properties to enhance cell penetration, resist degradation and
prolong their physiological half-lives. (Gibbs, J., Pharmaceutical
Research in Molecular Oncology, Cell, Vol. 79 (1994).) Antibodies,
which include intact molecules as well as fragments such as Fab and
F(ab')2 fragments, may be used to bind to and inhibit the
TACI/TACI-L complex by blocking the commencement of the signaling
cascade. Such activity by the antibodies could be useful in the
treatment of Acute Respiratory Disease Syndrome (ARDS). (WO
98/18921 at 57.) It is preferable that the antibodies are
humanized, and more preferable that the antibodies are human. The
antibodies of the present invention may be prepared by any of a
variety of well-known methods.
[0042] Antagonists may be employed to inhibit (antagonize) the
interaction between TACI and TACI-L for therapeutic purposes to
treat tumor and tumor metastasis and to combat various autoimmune
diseases that may be modulated by the TACI/TACI-L complex, e.g.
multiple sclerosis and diabetes, as well as other disorders, such
as viral infection, rheumatoid arthritis, graft rejection, and
IgE-mediated allergic reactions. A further disorder that may be
treated by antagonists of the TACI/TACI-L interaction is
inflammation mediated by the interaction. In general, the
interaction may be used to study cellular processes associated with
TNF-receptors such as immune regulation, cell proliferation, cell
death, and inflammatory responses.
[0043] Specific screening methods are known in the art and many are
extensively incorporated in high throughput test systems so that
large numbers of test compounds can be screened within a short
amount of time. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays, cell based assays, etc. These assay
formats are well known in the art. The screening assays of the
present invention are amenable to screening of chemical libraries
and are suitable for the identification of small molecule drug
candidates, antibodies, peptides.
[0044] A particular example of an assay for the identification of
potential TACI antagonists is a competitive assay which combines
TACI-L and a candidate molecule with TACI under the appropriate
conditions for a competitive assay. Either TACI or TACI-L can be
labeled so that the binding may be measured and the effectiveness
of the antagonist judged. The label allows for detection by direct
or indirect means. Direct means include, but are not limited to
luminescence, radioactivity, optical or electron density. Indirect
means include but are not limited to an enzyme or epitope tag.
[0045] By observing the effect that candidate molecules have on
TACI/TACI-L complexes in various binding assays, on TACI/TACI-L
mediated activity in functional tests, and in cell based screens,
molecules that are potential therapeutics because they can modulate
the TACI/TACI-L-binding interaction are identified. Such molecules
either mimic the biological activity of the TACI/TACI-L complex,
prevent the formation of the TACI/TACI-L complex or inhibit
dissociation of the TACI/TACI-L complex already formed. Molecules
preventing the interaction of TACI and TACI-L may be useful when
enhancement of the immune system is desired. Antagonists of the
dissociation of the TACI/TACI-L complex may be useful as
immunosuppressants or antiinflammatory agents.
[0046] Molecules which inhibit or prevent the dissociation of the
TACI/TACI-L complex can be identified by forming the complex in the
absence of a candidate molecule, then adding the candidate molecule
to the mixture, and changing the conditions so that, but for the
presence of the candidate molecule, TACI would be released from the
complex. The concentration of the free or bound TACI could then be
measured and the dissociation constant of the complex could be
determined and compared to a control.
[0047] Another method by which molecules which inhibit the
interaction between TACI and TACI-L can be identified is the solid
phase method, in which TACI is bound and placed in a medium with
labeled TACI-L. After contact with a candidate molecule, the amount
of signal produced by the interaction between TACI and TACI-L is
measured. Diminished levels of signal, in comparison to a control,
indicate that the candidate molecule inhibited the interaction
between TACI and TACI-L. In a further embodiment of this method,
TACI-L could be bound and TACI labeled.
[0048] Screening assays can further be designed to find molecules
that mimic the biological activity of the TACI/TACI-L complex.
Molecules which mimic the biological activity of the TACI/TACI-L
complex may be useful for enhancing the interaction. To identify
compounds for therapeutically active agents that mimic the
biological activity of the TACI/TACI-L complex, it must first be
determined whether a candidate molecule binds to TACI or TACI-L. A
binding candidate molecule is added to a biological assay to
determine its biological effects. The biological effects of the
candidate molecule are then compared to the those of the
TACI/TACI-L complex.
[0049] Thus, the present invention encompasses methods of screening
candidate molecules for their ability to modulate TACI/TACI-L
complexes and their ability to modulate activities mediated by
TACI/TACI-L complexes. By observing the effect that the candidate
molecule has on the known binding characteristics of TACI, TACI-L
or fragments thereof, compounds that inhibit or enhance TACI/TACI-L
binding can be identified. Typical candidate molecules are small
molecules, antibodies, or peptides and may be part of extensive
small molecule libraries developed for use in screening methods. In
this context, the identification of small molecules which may
interact with the TACI protein or the TACI ligand can be used to
develop drugs that modulate the activation pathway and may allow
physicians to treat distinct immune conditions without the negative
side effects present in current therapies. For such therapeutic
uses, the agonists or antagonists of the TACI/TACI-L complex
identified can be administered through well-known means, including
parenterally (subcutaneous, intramuscular, intravenous,
intradermal, etc. injection) and with a suitable carrier.
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation instonic with the blood of the recipient; and aqueous
and non-aqueous sterile suspensions which may include suspending
agents or thickening agents. The dosage will depend on the specific
activity of the vaccine and can be readily determined by routine
experimentation.
[0050] Generally, the conditions for an assay are conditions under
which TACI and TACI-L would normally interact. In other words, for
an assay to identify the inhibitor of the TACI//TACI-L interaction,
the conditions would be such that, but for the candidate molecule,
TACI and TACI-L would bind.
[0051] The following examples are offered by way of illustration,
and not by way of limitation. Those skilled in the art will
recognize that variations of the invention embodied in the examples
can be made, especially in light of the teachings of the various
references cited herein, the disclosures of which are incorporated
by reference in their entirety.
EXAMPLE 1
Generation of TACI-FC
[0052] This Example describes a method of generating TACI-Fc. The
cDNA sequence encoding the extracellular domain of TACI (amino
acids 2-166) was amplified by PCR using a sense primer
(5'-ataaccggtagtggcctgggccggagc- aggcgag-3') (SEQ. ID. NO. 6) and
an antisense primer (5'-ataagatctgggctcgctgtagaccagggccacctgatc)
(SEQ. ID. NO. 7). The amplified PCR fragment was digested with the
appropriate restriction enzyme and then ligated into the mammalian
expression vector pDC409, in-frame with the Ig kappa leader
sequence at the 5' end and with the Fc portion of human IgG1 at the
3' end. The plasmid was transfected transiently in CV1/EBNA cells
and the soluble protein TACI-FC was purified on a protein
G-sepharose column. Protein concentration was determined by BCA
analysis. Purity was assessed by SDS-PAGE analysis which, under
reducing conditions, showed a single band at 42 kDa.
EXAMPLE 2
Ligand Screening by Slide Binding Assay
[0053] This Example describes the method of a slide binding assay
and demonstrates that the TACI-Fc protein interacted only with
TACI-L. The purified TACI-Fc was used to screen against a cDNA
panel containing known members of the ligand family (4-1BBL, CD40L,
OX40L, CD27L, CD30L, RANKL, LT-alpha, LT-beta, LIGHT, TWEAK, FasL,
TRAIL, proTNF and TACI-L). TACI-Fc was then bound to the slides by
adding 2 .mu.g of the DNA encoding the members of the ligand family
to a sterile tube and adding 75 .mu.M choloroquine in
transfection/growth medium to a final volume of 175 .mu.l. 25 .mu.l
of DEAE-dextran (4 mg/ml in PBS) was then added to the DNA solution
and mixed.
[0054] The growth medium was aspirated from the slides and replaced
with 3 ml of 75 .mu.M chloroquine in the transfection/growth
medium, followed by the addition of the DNA/DEAE-dextran mixture to
the cells. The slides were rocked side-to-side and back-and-forth
to distribute the precipitated DNA evenly. The slides were
incubated at 37.degree. C. for 4.5 hours.
[0055] The medium was aspirated and 3 ml 10% DMSO was added in the
transfection/growth medium. After a 5 minute incubation period at
room temperature, the medium was aspirated again and replaced with
3 ml fresh transfection/growth medium. The cells were then
incubated at 37.degree. C. for 2 days to allow for expression of
the transfected cDNAs.
[0056] To screen for positive pools expressing the cell-bound
protein, slides were incubated with TACI:Fc and then with a
radioiodinated protein probe (labeled goat antihuman Fc F(ab')2)
for 30 minutes at room temperature. The probe solution is then
removed by aspiration and washed to remove the non-specifically
bound probe. Finally, the slides were fixed by incubating each
slide with 1 ml 2.5% glutaraldehyde in PBS for 30 minutes at room
temperature to retain specifically bound label. The slides were
then washed twice with 1 ml PBS and air-dried.
[0057] The dried slides were dipped in liquid photographic emulsion
that has been warmed to 42.degree. C., dried at room temperature
and exposed for 2 days at room temperature before developing. The
slides were examined at 25.times. magnification under bright-field
illumination to detect cell types upon which the ligand is acting.
TACI-Fc protein was found to bind only to cells transfected with
the TACI-L. The ability of the TACI-FC to bind to CV1 expressing
the TACI-L was also demonstrated by the well-known methods of flow
cytometry.
EXAMPLE 3
Immunoprecipitation of Membrame-Associated TACI with the
TACI-Ligand
[0058] This Example demonstrates the interaction between the TACI
protein and TACI-L. CV1 cells were transfected with soluble TACI-L
plasmid and the two day supernatant was harvested. CV1 cells were
transfected with membrane associated TACI and metabolically labeled
with 35S-CYS-MET two days post-transfection (labeled cell lysate).
Supernatant containing TACI-L was used in immunoprecipitation
experiments with labeled cell lysate. A specific band at 45 kDa
which was consistent with the predicted size of TACI was obtained,
as shown in FIG. 1. Thus, the interaction between the TACI protein
and TACI-L was confirmed.
EXAMPLE 4
Plate Binding Assay Capturing TACI-L
[0059] This example further characterizes the interaction between
TACI and TACI-L by conducting a plate binding assay and
demonstrates the high affinity between the proteins. Equilibrium
binding isotherms were determined in 96-well microtiter plates that
had been coated with TACI-L COS expressed supernatants, captured
through Leucine Zipper M15 antibody. Plates were incubated with 5
.mu.g/ml LZ M15 in PBS for 4 hr at 4.degree. C. After being washed
3 times with PBS, the plates were incubated with a 1:2 or 1:5
dilution of the COS expressing TACI supernatant in PBS/0.05% Tween
20 for 12 hours at 4.degree. C. The plates were then washed for an
additional 3 times with PBS and nonspecific binding sites were
blocked with 300 .mu.l/well of a binding media (RPMI 1640, 2.5%
BSA, 20 MM HEPES, 0.02% sodium azide pH 7.2) and 2.5% non-fat dried
milk. The plates were incubated for 1 hour at room temperature and
washed 3 times with PBS.
[0060] TACI/Fc was diluted to 2 .mu.g/ml to the first well, and
serial dilutions were performed against the binding media.
Incubation occurred for 2 hours at 4.degree. C. Plates were then
washed 3 times with PBS. A final incubation occurred for 30 minutes
at room temperature with 125 ng/ml 125-I goat anti-human F(ab')2.
The goat anti-human F(ab')2 was labeled with 125-I using solid
phase chloramine T analog (Iodogen; Pierce Chemical, Rockford,
Ill.) to a specific radioactivity of 8.73e14 cpm/mmol. Nonspecific
binding was determined in the presence of 1000-fold excess of
unlabeled goat anti-human F(ab')2. Plates were washed 3 times in
PBS and the specifically bound ligand was released with 50 mM
citrate (pH 3.0) and then gamma counted. Data was processed as
described (Dower et al., 1984).
[0061] FIG. 4a demonstrates the results of the assay using 1:2
dilution and shows the complete saturation of the receptor binding
sites. FIG. 4b, the Scatchard graph corresponding to FIG. 4a,
demonstrates the actual number of sites that were actually bound.
From these results, the affinity constant of 1.53.times.10.sup.-9
can be generated.
[0062] FIG. 5a demonstrates the results of the assay using 1:5
dilution and shows the complete saturation of the receptor binding
sites. FIG. 5b, the Scatchard graph corresponding to FIG. 5a,
demonstrates the actual number of sites that were actually bound.
From these results, the affinity constant of 2.2.times.10.sup.-9 is
shown.
EXAMPLE 5
Plate Binding Assay Capturing HuTACI/FC
[0063] This example also characterizes the interaction between TACI
and TACI-L by use of a plate binding assay and further demonstrates
the high affinity between the proteins. Equilibrium binding
isotherms were determined in 96-well microtiter plates that had
been coated with HuTACI/Fc, captured through goat anti-human Fc
polyclonal antibody. Plates were incubated with 5 .mu.g/ml goat
anti-human FC in PBS for 4 hours at 4.degree. C. After being washed
3 times with PBS, the plates were incubated with 0.1 .mu.g/ml Fc
chimera in PBS/0.05% Tween 20 for 12 hours at 4.degree. C. and then
washed for an additional 3 times with PBS. Nonspecific binding
sites were blocked with 300 .mu.l/well of a binding media (RPMI
1640, 2.5% BSA, 20 MM HEPES, 0.02% sodium azide pH 7.2) and 2.5%
non-fat dried milk. The plates were incubated for 1 hour at room
temperature and then washed 3 times with PBS. TACI-L was expressed
in COS cells and concentrated 10-fold.
[0064] TACI-L supernatant was diluted 1:10 to the first well, and
serial dilutions were performed against the binding media.
Incubation occurred for 2 hours at 4.degree. C. Plates were then
washed 3 times with PBS. A final incubation occurred for 30 minutes
at room temperature with 125-I Leucine Zipper M15. Leucine Zipper
M15 (LZM15) was labeled with 125-I using solid phase chloramine T
analog (Iodogen; Pierce Chemical, Rockford, Ill.) to a specific
radioactivity of 8.73e14 cpm/mmol. Nonspecific binding was
determined in the presence of 1000-fold excess of unlabeled LZM15.
Plates were washed 3 times in PBS and specifically bound ligand was
released with 50 mM citrate (pH 3.0) and then gamma counted. Data
was processed as described (Dower et al., 1984).
[0065] FIG. 6a demonstrates the complete saturation of the receptor
binding sites. FIG. 6b, the Scatchard graph which corresponds to
FIG. 6a, demonstrates the actual number of sites that were actually
bound. The Scatchard graph of FIG. 6b demonstrates a curvilinear
binding, with a low affinity constant of 5.7.times.10.sup.-10 and a
high affinity constant of 1.0.times.10.sup.-10. FIG. 6b
demonstrates that the majority of the binding occurred at an
affinity constant between 2-3.times.10.sup.-9.
* * * * *