U.S. patent application number 11/079418 was filed with the patent office on 2005-08-18 for lymphocyte surface receptor that binds caml and methods of use thereof.
This patent application is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Bram, Richard J., Von Bulow, Gotz.
Application Number | 20050183148 11/079418 |
Document ID | / |
Family ID | 25204137 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050183148 |
Kind Code |
A1 |
Bram, Richard J. ; et
al. |
August 18, 2005 |
Lymphocyte surface receptor that binds caml and methods of use
thereof
Abstract
A novel lymphocyte receptor protein, its DNA sequence, and its
role in the calcium activation pathway is described. The protein,
or genetically engineered constructs encoding it, is shown to
increase lymphocyte response, and to identify ligands of the
protein receptor. Antibodies to the proteins of the invention are
generated for diagnostic therapeutics. The protein and DNA can also
be used for diagnostic purposes and for identifying agents for
modulating the calcium induced activation pathway. A particular
advantage of the present invention is that it provides lymphocyte
activation of receptor found on all B cells, but only on a subset
of T cells. The receptor can thus be targeted to specifically
regulate B cell responses without affecting mature T cell activity.
Such targeting specificity is always advantageous, particularly
where an increase or decrease of antibody production is desired,
e.g., during an infection (increase) or to avoid immune complex
deposition complications (rheumatoid arthritis, glomerulonephritis,
and other auto immune conditions).
Inventors: |
Bram, Richard J.;
(Rochester, MN) ; Von Bulow, Gotz; (Carmel,
IN) |
Correspondence
Address: |
ALSTON AND BIRD LLP
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
St. Jude Children's Research
Hospital
Memphis
TN
|
Family ID: |
25204137 |
Appl. No.: |
11/079418 |
Filed: |
March 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11079418 |
Mar 14, 2005 |
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10293816 |
Nov 12, 2002 |
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10293816 |
Nov 12, 2002 |
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09782857 |
Feb 14, 2001 |
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6500428 |
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09782857 |
Feb 14, 2001 |
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09290333 |
Apr 12, 1999 |
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6316222 |
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09290333 |
Apr 12, 1999 |
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08810572 |
Mar 3, 1997 |
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5969102 |
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Current U.S.
Class: |
800/18 ;
435/354 |
Current CPC
Class: |
Y02A 50/411 20180101;
A61P 35/00 20180101; A61K 38/00 20130101; C07K 14/70578 20130101;
A01K 2217/075 20130101; Y10S 530/82 20130101; Y02A 50/30 20180101;
C07K 2317/75 20130101; C07K 16/2878 20130101 |
Class at
Publication: |
800/018 ;
435/354 |
International
Class: |
A01K 067/027; C12N
005/06 |
Goverment Interests
[0002] The research leading to the present invention was supported
in part by the Cancer Center CORE Grant CA-21765 from the National
Institutes of Health. The government may have certain rights in the
present invention. Support for this invention was also provided by
the AMERICAN LEBANESE SYRIAN ASSOCIATED CHARITIES, the American
Cancer Society Grant Bt-234, and the James McDonnell Foundation
Grant JSMF 93-40-03.
Claims
That which is claimed:
1. A transgenic mouse comprising a transgene encoding a fusion
protein comprising an extracellular fragment of the TACI protein
joined to the Fc domain of an immunoglobulin.
2. The transgenic mouse of claim 1, wherein said transgenic mouse
exhibits a suppressed immune system in comparison to a wild-type
mouse.
3. The transgenic mouse of claim 1, wherein said transgenic mouse
exhibits a decreased B-cell response in comparison to a wild-type
mouse.
4. The transgenic mouse of claim 1, wherein said extracellular
fragment of the TACI protein is an N-terminal fragment of the TACI
amino acid sequence shown in SEQ ID NO:2.
5. The transgenic mouse of claim 4, wherein the extracellular
fragment of the TACI protein is an N-terminal fragment the TACI
amino acid sequence shown in SEQ ID NO:6.
6. The transgenic mouse of claim 5, wherein the extracellular
fragment comprises the TNFR_NGFR pattern shown in SEQ ID NO:11.
7. The transgenic mouse of claim 4, wherein the extracellular
fragment of the TACI protein comprises amino acids 1-151 of SEQ ID
NO:2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/293,816, filed Nov. 12, 2002; which is a divisional of U.S.
application Ser. No. 09/782,857, filed Feb. 14, 2001, now U.S. Pat.
No. 6,500,428, issued Dec. 31, 2002; which is a divisional of U.S.
application Ser. No. 09/290,333, filed Apr. 12, 1999, now Pat. No.
6,316,222, issued Nov. 13, 2001; which is a divisional of U.S.
application Ser. No. 08/810,572, filed Mar. 3, 1997, now U.S. Pat.
No. 5,969,102, issued Oct. 19, 1999, each of which are hereby
incorporated in their entirety by reference herein.
FIELD OF THE INVENTION
[0003] The invention relates generally to the regulation of
transcription in lymphocytes, proteins involved therein, antibodies
thereof, nucleic acids that encode the proteins and uses of the
nucleic acids, antibodies and proteins.
BACKGROUND OF THE INVENTION
[0004] Investigators are only beginning to unravel the mechanisms
that control the cellular response to extrinsic factors. One basic
feature of many of such mechanisms is the initial binding of an
extrinsic factor, e.g., a ligand, to a cell surface membrane
protein, i.e., a receptor. The binding of a ligand to its receptor
usually effects a cellular change through a cascade of events.
These events commonly involve other proteins, such as protein
kinases, protein phosphatases, JAK proteins, Stat proteins, and/or
G-proteins. In addition, there is generally a requirement for a
transcription factor to bind to a specific DNA regulatory sequence
in the nucleus of the cell, and thereby initiate the transcription
of one or more particular genes.
[0005] Other factors are often involved. In antigen-stimulated
lymphocyte activation, for example, calcium (Ca.sup.2+) influx is
also necessary for the ultimate initiation of DNA transcription.
The increased cytoplasmic calcium concentration may originate as an
external influx or a release of internal stores. Increased calcium
concentration which activates the calcium-dependent protein
phosphatase calcineurin acts in conjunction with other agents to
signal the initiation of transcription. It is clear that the
pathway involving calcium influx is essential to a number of
processes involved with activation and proliferation of cells.
[0006] Intracellular calcium levels play a major function in a
number of different cell types involving a number of different
activities. In addition to the induction of gene transcription by
calcium influx, many other calcium-dependent events, such as those
which occur during muscle contraction (both cardiac and skeletal),
vesicle degranulation (such as in the response of neutrophils and
macrophages to infection, or basophil response to antigen
stimulation, or release of acetylcholine by neurons), and closure
of intracellular gap junctions offer opportunities for cellular
regulation. The cell cycle can also involve fluxes of calcium.
Intracellular chelators which block changes in intracellular
calcium concentration can block the cell cycle from progressing,
thereby arresting cell division. [Rabinovich et al. (1986) J. of
Immunol. 137:952-961]. Therefore, regulation of calcium can be
effective in modulating cell division in normal and diseased
cells.
[0007] Lymphocytes are a primary component of the cellular arm of
the immune system. Activation of one particular type of lymphocyte,
a T-cell, can result through the stimulation of a T-cell receptor
by e.g., the binding of a T-cell receptor (TCR) to an antigen
presented by an antigen-presenting cell. This stimulation results
in the activation a Ca.sup.2+-dependent phosphatase, calcineurin.
Activated calcineurin, in turn, activates NF-AT, a lymphocyte
specific transcription factor that together with a companion
transcription factor, AP-1, effects the expression of the inducible
T-cell growth factor, interleukin-2 (IL-2). Activation of AP-1 is a
calcium-independent process that involves protein kinase C, and can
be experimentally achieved with the addition of phorbol myristate
acetate (PMA). The immunosuppressant drug cyclosporin A (CsA) binds
to and inhibits the prolyl isomerase activity of cyclophilin and
the resulting drug-isomerase complex inactivates calcineurin, by a
direct interaction near the active site of the enzyme. [Liu et al.
(1991) Cell 66:807-15]; [Clipstone and Crabtree (1992) Nature
357:695-7]; [O'Keefe et al. (1992) Nature 357:692-4]. NF-.kappa.B
is a third key transcription factor which is important in the
activation of lymphocytes and which is activated following the
stimulation of the T-cell or B-cell antigen receptor.
[0008] Another protein associated with the calcium signaling
pathway in lymphocytes is the recently identified calcium-signal
modulating cyclophilin ligand (CAML) [Bram, R. J. and Crabtree, G.
R., DNA Encoding Calcium-Signal Modulating Cyclophilin Ligand, U.S.
Pat. No. 5,523,227, issued Jun. 4, 1996, hereby incorporated by
reference in its entirety]. CAML binds cyclophilin B with
reasonable specificity, i.e., CAML does not bind cyclophilin A or
C. Unlike the cyclosporin A-cyclophilin complex, however, the
CAML-cyclophilin B complex does not directly bind to calcineurin.
Thus CAML appears to affect calcineurin through its regulation of
Ca.sup.2+ influx. As expected, CsA can indirectly block the
activating effect of CAML on transcription, by inhibiting
calcineurin. In addition, CAML appears to have no effect on the
activation of AP-1, and so the CAML-dependent activation of NF-AT
experimentally requires the addition of PMA.
[0009] CAML acts downstream from an extrinsic signal but upstream
from calcineurin. The location of CAML in cytoplasmic vesicles
suggests that it can regulate Ca.sup.2+ influx by modulating
intracellular Ca.sup.2+ release. However, there remains a need to
determine the natural factor (or factors) that communicate the
external signal to the cellular CAML. Further, there is a need to
understand how CAML interacts with this factor in order to learn
how to better control the important cellular processes that CAML
helps to regulate. A different class of signaling molecule is the
TNFR family of cell surface receptors [Smith et al. (1994) Cell
76:959-62]. These receptors initiate intracellular signals leading
to the onset of cell growth, death, or gain of effector
function.
SUMMARY OF THE INVENTION
[0010] A novel lymphocyte receptor, its DNA sequence, and its role
in the calcium activation pathway is described. The protein, or
genetically engineered constructs encoding it, can be used to
enhance lymphocyte response, or to identify ligands of the protein
receptor. The soluble, extracellular domain can be used to inhibit
cellular activation. Antibodies to the protein can be generated for
diagnostic or therapeutic uses. The protein and DNA may also be
used for diagnostic purposes and for identifying agents for
modulating the calcium induced activation pathway. Knowledge of the
coding sequence allows for manipulation of cells to elucidate the
mechanism of which CAML is a part.
[0011] A particular advantage of the present invention is that it
provides lymphocyte activation of a receptor found on all B cells,
but only on a subset of T cells. The receptor can thus be targeted
to specifically regulate B cell responses without affecting mature
T cell activity. Such targeting specificity is always advantageous,
particularly where an increase or decrease of antibody production
independent of cellular immune responses is desired, e.g., during
an infection (increase) or to avoid immune complex deposition
complications (rheumatoid arthritis, glomerulonephritis, and other
autoimmune conditions).
[0012] Crosslinking the novel cell surface receptor of the present
invention activates B cells and some populations of T cells.
Activation of these cells increases the immune system activity. On
the other hand, blocking or inhibiting the novel cell surface
receptor of the present invention can result in immunosuppression.
Depending on the endogenous level of activation of the receptor,
which can be evaluated using the antibodies or nucleic acids of the
invention, receptor activity can be enhanced or suppressed to
achieve a desired outcome. Either activating or inhibiting the
function of the novel cell surface receptor of the present
invention can be used to treat cancers of T and B cells.
[0013] The present invention includes an isolated Transmembrane
Activator and CAML-Interactor (TACI) protein that functions as a
cell surface signaling protein and comprises an extracellular
domain, a membrane spanning segment, and a cytoplasmic domain. In
one embodiment, the TACI protein is a plasma membrane receptor in
which the extracellular domain resides at the N-terminal portion of
the protein and the cytoplasmic domain resides at the C-terminal
portion of the protein. The N-terminal portion of the TACI protein
functions as a binding site for a ligand that stimulates the
activation of the cell by inducing the binding of the C-terminal
portion of the TACI protein to the N-terminal domain of CAML. Since
CAML is an integral membrane protein that is localized to
cytoplasmic vesicles, the TACI protein is a plasma membrane
receptor that directly interacts with an intracellular organelle in
lymphocytes.
[0014] In one embodiment, the monomeric form of the isolated TACI
protein consists of about 295 amino acids. In a preferred
embodiment the monomeric form of a Transmembrane Activator and CAML
Interactor (TACI) protein contains 280 to 310 amino acids. In more
preferred embodiments the monomeric form of a TACI protein contains
290 to 296 amino acids. In a specific embodiment exemplified infra,
the monomeric form of a TACI protein contains 293 amino acids.
[0015] One embodiment of the isolated TACI protein contains two
TNFR superfamily cysteine-rich repeats [Bairoch (1993) Nucl. Acids
Res. 21:3097-3103]. In a preferred embodiment, a TACI protein that
is appropriately stimulated, in situ, such as by a ligand or an
anti-TACI antibody, initiates the activation of a transcription
factor through the combination of a Ca.sup.2+-dependent pathway and
a Ca.sup.2+-independent pathway.
[0016] The present invention includes an isolated nucleic acid that
consists of at least 18 nucleotides of a nucleotide sequence that
has at least 60% similarity with SEQ ID NO:1, or alternatively at
least 60% similarity with the coding sequence of SEQ ID NO:1. The
nucleotide sequence encodes a TACI protein which has a binding
affinity for CAML. In one such embodiment the isolated nucleic acid
encodes a TACI protein.
[0017] In a preferred embodiment of the present invention the
nucleotide sequence has at least 75% similarity with SEQ ID NO:1,
or has at least 75% similarity with the coding sequence of SEQ ID
NO:1. In a more preferred embodiment, the nucleotide sequence has
at least 90% similarity with SEQ ID NO:1, or has at least 90%
similarity with the coding sequence of SEQ ID NO:1. In an even more
preferred embodiment, the nucleotide sequence has between 95-98%
similarity with SEQ ID NO:1, or has between 95-98% similarity with
the coding sequence of SEQ ID NO:1. In a particular embodiment the
nucleotide sequence is SEQ ID NO:1. In a related embodiment, the
nucleotide sequence consists of the coding sequence of SEQ ID NO:1.
In a specific embodiment, exemplified infra, the isolated nucleic
acid has the nucleotide sequence of SEQ ID NO:1. In a related
embodiment, the isolated nucleic acid consists of the coding
sequence of SEQ ID NO:1.
[0018] In another related embodiment the present invention includes
an isolated nucleic acid which contains at least 18 nucleotides and
hybridizes to SEQ ID NO:1, or more particularly hybridizes to the
coding sequence of SEQ ID NO:1. In one such embodiment, the
hybridization is performed under moderate stringency. In another
embodiment, the hybridization is performed under standard
hybridization conditions. In yet a third embodiment, the
hybridization is performed under stringent hybridization
conditions.
[0019] In still another related embodiment the present invention
includes an isolated nucleic acid which contains at least 18
nucleotides of a nucleotide sequence that encodes a TACI protein
having an amino acid sequence of either SEQ ID NO:2, or SEQ ID NO:2
with one or more conservative substitutions. In one such
embodiments of this type, the isolated nucleic acid encodes an
N-terminal fragment of the TACI protein corresponding to the
extracellular domain. In another embodiment, the isolated nucleic
acid encodes a C-terminal fragment of the TACI protein that is
sufficient to bind to the N-terminal 146 amino acids of CAML. In
yet another embodiment, the isolated nucleic acid encodes the
transmembrane portion of the TACI protein. In still another
embodiment, the isolated nucleic acid encodes the full-length TACI
protein.
[0020] In a preferred embodiment of the present invention, the
isolated nucleic acid consists of at least 24 nucleotides. In a
more preferred embodiment, the isolated nucleic acid consists of at
least 30 nucleotides. In an even more preferred embodiment, the
isolated nucleic acid consists of at least 36 nucleotides.
Oligonucleotides of the invention can be used as nucleic acid
probes, PCR primers, antisense nucleic acids, and the like, for
diagnostic and therapeutic purposes.
[0021] In one embodiment of the present invention, an isolated
nucleic acid (SEQ ID NO:3) encodes a C-terminal fragment of the
TACI protein that is sufficient to bind to the N-terminal 146 amino
acids of CAML. In one particular embodiment of this type, the
C-terminal fragment contains about 126 amino acids. In another
embodiment of this type the C-terminal fragment has an amino acid
sequence of either SEQ ID NO:4, or SEQ ID NO:4 with one or more
conservative substitutions.
[0022] In another embodiment, an isolated nucleic acid of the
invention encodes an N-terminal fragment (SEQ ID NO:5) of the
CAML-binding protein corresponding to the extracellular domain. In
a particular embodiment of this type the N-terminal fragment has an
amino acid sequence of either SEQ ID NO:6, or SEQ ID NO:6 with one
or more conservative substitutions.
[0023] In a preferred embodiment, the isolated nucleic acid encodes
a TACI protein that has a binding affinity for CAML. When such a
TACI protein is appropriately stimulated, in situ, it initiates
activation of a transcription factor through the combination of a
Ca.sup.2+-dependent pathway and a Ca.sup.2+-independent pathway. In
a more preferred embodiment, the isolated nucleic acid encodes a
TACI protein having the amino acid sequence of SEQ ID NO:2.
[0024] The present invention also includes a DNA construct
comprising an isolated nucleic acid of the present invention that
is a recombinant DNA operatively linked to an expression control
sequence. The expression control sequence can be selected from the
group consisting of the early or late promoters of SV40 or
adenovirus, the lac system, the trp system, the TAC system, the TRC
system, the major operator and promoter regions of phage .lambda.,
the control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase, the promoters of acid phosphatase and
the promoters of the yeast .alpha.-mating factors.
[0025] In a preferred embodiment, the expression control sequence
is either a standard tet inducible promoter, a metallothionein
promoter, or an ecdysone promoter. In a more preferred embodiment,
the expression control sequence is the SR.sub..alpha. promoter.
[0026] The present invention also includes a unicellular host
transformed with a recombinant DNA construct of the present
invention. In one embodiment the unicellular host is a prokaryote.
In another embodiment the unicellular host is a eukaryote.
Preferably the eukaryotic host is a mammalian cell, for example, a
COS, CHO or Jurkat T cell, which could be useful for evaluating
activity of the TACI protein or to identify modulatory agents.
[0027] The present invention includes the isolated polypeptides
encoded by the nucleic acids of the present invention, fragments
thereof, and fusion proteins thereof. In one embodiment, the
polypeptide fragment consists of an N-terminal fragment of the TACI
protein corresponding to the regulatory extracellular domain. In a
particular embodiment the N-terminal fragment has an amino acid
sequence of SEQ ID NO:6 or SEQ ID NO:6 with one or more
conservative substitutions.
[0028] In another embodiment, the polypeptide fragment consists of
a C-terminal fragment of the TACI protein that is sufficient to
bind to the N-terminal 146 amino acids of CAML. In one such
embodiment, the C-terminal fragment contains 95 to 130 amino acids.
In a specific embodiment, the C-terminal fragment contains the
C-terminal 126 amino acids of SEQ ID NO:2. In an alternative
embodiment the C-terminal fragment of the TACI protein contains
about 110 amino acids. In a preferred embodiment of this type, the
C-terminal fragment contains 107 amino acids and has an amino acid
sequence of SEQ ID NO:4.
[0029] The present invention also includes the preparation of a
recombinant form of the extracellular portion of a TACI protein,
thereby creating a dominant-negative or blocking reagent. This
component intercepts the normal endogenous ligands which serve to
crosslink and activate the TACI protein. Administration of such a
polypeptide acts to suppress the immune system. Such administration
is useful in the treatment or prevention of autoimmune disease or
graft-rejection or graft-vs-host disease following
transplantation.
[0030] A chimeric TACI protein of the invention may be a protein
that is generated by joining the extracellular domain of another
receptor molecule with a transmembrane domain and the intracellular
domain of a TACI protein. In another embodiment, the extracellular
domain of a TACI protein can be joined with a transmembrane domain
and an intracellular domain of another receptor molecule. The
transmembrane domain can be the transmembrane domain of a TACI
protein, the transmembrane domain of the other receptor, or a
different transmembrane domain. Preferably, the transmembrane
domain is from the same protein component of the chimera as the
extracellular domain.
[0031] In a preferred embodiment the polypeptide is a TACI protein
encoded by a nucleic acid of the present invention that has a
binding affinity for CAML and when appropriately stimulated, in
situ, initiates activation of a transcription factor through the
combination of a Ca.sup.2+-dependent pathway and a
Ca.sup.2+-independent pathway.
[0032] The present invention also includes antisense nucleic acids
that hybridize under physiological conditions to the mRNAs that
encode the TACI proteins of the present invention. Such antisense
nucleic acids may be RNA transcribed from an antisense gene, or RNA
or DNA produced exogenously (whether by expression or chemical
synthesis). Preferably, a synthetic antisense nucleic acid is
prepared with non-naturally occurring bonds to prevent its rapid
hydrolysis and thus increase its effective half-life.
[0033] A knockout animal is also part of the present invention. The
knockout animal comprises a first and second allele which each
naturally encode and express functional TACI protein but in which
at least one of the two alleles is defective and thereby prevents
the animal from expressing an adequate amount of the TACI protein.
In one embodiment of this type, the first allele contains a defect
that prevents the animal from expressing any functional TACI
protein. In a preferred embodiment, a knockout animal contains both
a defective first allele and a defective second allele. These
defective alleles prevent the animal from expressing functional
TACI protein. In a preferred embodiment, the knockout animal is a
knockout mouse.
[0034] The present invention also includes antibodies to all of the
nucleic acids and polypeptides of the present invention. In a
specific embodiment, the antibody is prepared against the TACI
protein having an amino acid sequence of SEQ ID NO:2, or an
antigenic fragment thereof. The antibodies of the present invention
can be either monoclonal antibodies or polyclonal antibodies. In
one embodiment, the antibody is a monoclonal antibody that is a
chimeric antibody.
[0035] An immortal cell line that produces a monoclonal antibody of
the present invention is also part of the present invention. In a
specific embodiment of this immortal cell line, the monoclonal
antibody is prepared against the TACI protein having an amino acid
sequence of SEQ ID NO:2 or an antigenic fragment thereof.
[0036] The present invention also includes an N-terminal fragment
of CAML that is sufficient to bind to the C-terminal 126 amino acid
fragment of TACI-1. In one such embodiment, the N-terminal fragment
of CAML contains 146 amino acids. This N-terminal fragment of CAML
can serve as an inhibitor of TACI-CAML binding.
[0037] The present invention includes methods of making TACI
proteins, fragments thereof and fusion proteins thereof. In one
embodiment the method comprises introducing an expression vector
comprising a nucleic acid encoding a polypeptide that is a TACI
protein, or a fragment thereof, or a fusion protein thereof, into a
host cell and expressing the encoded polypeptide. In a preferred
embodiment the expressed polypeptide has a binding affinity for
CAML. In a more preferred embodiment the polypeptide, when
appropriately stimulated, in situ, initiates activation of a
transcription factor through the combination of a
Ca.sup.2+-dependent and a Ca.sup.2+-independent pathway. In the
most preferred embodiment of this type the expressed polypeptide is
a TACI protein having an amino acid sequence of SEQ ID NO:2.
[0038] Methods of purifying the expressed polypeptides encoding
TACI proteins, fragments thereof and fusion proteins thereof, are
also part of the present invention, as are the purified expressed
polypeptides themselves.
[0039] The present invention also includes methods for identifying
a ligand for a TACI protein. One embodiment of such a method
comprises contacting the N-terminal extracellular polypeptide of a
TACI protein with a candidate molecule and detecting the binding of
the N-terminal extracellular polypeptide with the candidate
molecule. The binding of the N-terminal extracellular polypeptide
with the candidate molecule indicates that the candidate molecule
is ligand.
[0040] In an alternative method for identifying a ligand for a TACI
protein, a functional TACI protein is used. In preferred
embodiments of this type the functional TACI protein is TACI-1. The
binding of the functional TACI protein with the candidate molecule
indicates that the candidate molecule is a ligand. In one such
embodiment, binding of the candidate molecule to the functional
TACI protein is determined by detecting cellular activation as a
function of the level of activation of the AP-1 pathway. In another
embodiment, binding of the candidate molecule to the functional
TACI protein is determined by detecting cellular activation as a
function of the level of activation of the CAML pathway.
[0041] In another embodiment, binding of the candidate molecule to
the functional TACI protein is determined by detecting cellular
activation as a function of the level of the concentration of the
NF-AT transcription factor. In still another embodiment, binding of
the candidate molecule to the functional TACI protein is determined
by detecting cellular activation as a function of the level of
activation of the NF-.kappa.B pathway. In yet another embodiment,
binding of the candidate molecule to the functional TACI protein is
determined by detecting cellular activation as a function of the
level of the activation of NF-AT. In this case, the level of
activation of NF-AT can be determined by methods including
demonstrating cytoplasm to nuclear translocation of NF-AT; the
relative dephosphorylation of NF-AT; and/or by NF-AT-dependent
transcription.
[0042] In preferred embodiments, more than one of the above
determinations of cellular activation is made, and the candidate
molecule is identified as a ligand when all the determinations made
indicate the binding of the candidate molecule to the TACI protein.
In the most preferred embodiment, all of the above determinations
of cellular activation are made and the candidate molecule is
identified as a ligand when all of these determinations indicate
that the candidate molecule binds to the TACI protein.
[0043] Methods for identifying a ligand for a TACI protein may be
performed in a large number of expression systems in the TACI
protein can be expressed. One embodiment employs the use of a yeast
two-hybrid expression system using the TACI protein as "bait." In
another embodiment, interaction cloning from E. coli
expression-libraries may be employed. In yet another embodiment,
functional expression cloning in mammalian cells of the TACI
protein can be utilized. In a preferred embodiment, the mammalian
cells are B-cell derived lines such as Burkitt's Lymphoma,
EBV-immortalized cell lines, or multiple myeloma cell lines. In a
more preferred embodiment of this type, the TACI protein is
expressed in Jurkat T cells containing a reporter gene under
control of an NF-AT promoter. In one such embodiment, the reporter
gene encodes secreted alkaline phosphatase (SEAP) as the
marker.
[0044] The present invention also includes methods of screening for
an immunosuppressant drug that inhibits the activation of B cells
to a greater extent than it inhibits the activation of mature T
cells. In preferred embodiments of this type, the immunosuppressant
drug inhibits the activation of B cells, but does not inhibit the
activation of mature T cells. Such methods may be performed in
transformed T cells, such as a Jurkat T cell, which can be
genetically manipulated to express the TACI protein; or in B cells
that naturally express the TACI protein. The present invention also
includes the immunosuppressant drugs identified which inhibit the
activation of B cells, but not the activation of mature T
cells.
[0045] The present invention includes methods of identifying an
immunosuppressant drug that selectively blocks the action of B
lymphocytes without effecting mature T lymphocytes. One such
embodiment comprises contacting a first lymphocyte with a potential
drug, wherein the first lymphocyte contains a TACI protein and a
first marker protein. The first marker protein is transcribed when
the TACI protein is stimulated in the absence of a candidate drug.
The TACI protein is stimulated, and the first marker protein is
detected under conditions in which if it is transcribed, it is
detectable. A potential drug is selected as a candidate drug when
the first marker protein cannot be detected. Next, a second
lymphocyte is contacted with the candidate drug, wherein the second
lymphocyte contains a T cell receptor, and a second marker protein
that is transcribed when the T cell receptor is stimulated either
in the absence or the presence of the immunosuppressant drug. The T
cell receptor is stimulated and the second marker protein is
detected under conditions in which if it is transcribed, it is
detectable. A candidate drug is identified as an immunosuppressant
drug when the second marker protein is detected, since the
immunosuppressant drug interferes with the pathway (or aspect
thereof) involving the TACI protein but not the pathway (or aspect
thereof) involving the T cell receptor.
[0046] In one embodiment, the first and second lymphocytes are
Jurkat T cells that have been modified to express a TACI protein.
In one such particular embodiment the method comprises contacting a
first Jurkat T cell with a potential drug, wherein the first Jurkat
T cell has been genetically engineered to express a TACI protein
and a first reporter gene. The first reporter gene is controlled by
an NF-AT promoter, and encodes a first marker protein. The TACI
protein is activated, and the amount of expression of the first
marker protein is quantified. A potential drug is selected as a
candidate drug when the amount of the first marker protein
expressed in the presence of the candidate drug is decreased
relative to the amount expressed in the absence of the candidate
drug. The candidate drug is then contacted with a second Jurkat T
cell that contains a T cell receptor and a second reporter gene.
The second reporter gene is controlled by an NF-AT promoter, and
encodes a second marker protein. The T cell receptor is activated
and the amount of expression of the second marker protein is
quantified and then compared to the amount of second marker protein
expressed in the absence of the candidate drug. A candidate drug is
identified as an immunosuppressant drug if either there is no
decrease in the amount of expression of the second marker protein
in the presence of the candidate drug, or the decrease in the
expression of the second marker protein is measurably less than the
corresponding decrease in expression of the first marker protein in
the presence of the candidate drug.
[0047] Any of the marker proteins described herein may be used for
this aspect of the invention including SEAP, LacZ or luciferase.
The first and second marker protein can be the same protein or two
different proteins. The TACI protein may be activated with an
antibody raised against a TACI protein, or an active fragment
thereof, or a fusion protein thereof. In a preferred embodiment,
the TACI protein is TACI-1. Several promoters can be used to
control the reporter gene including the NF-AT promoter mentioned
above and the AP-1 promoter. Potential drugs can be obtained from
any of the drug libraries currently available, and from the
chemical and phage libraries described herein.
[0048] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1. The tissue distribution, protein sequence and other
salient features of TACI-1. FIG. 1 depicts a Northern blot of the
tissues indicated probed with TACI-1 cDNA. Other tissues probed
include the heart, brain, placenta, lung, liver, skeletal muscle,
kidney and the pancreas, none of which showed any TACI-1 mRNA
expression.
[0050] FIG. 2A (SEQ ID NO:2) depicts the amino acid sequence of
TACI-1. The proposed transmembrane domain is shown in boxed print
and the Prosite TNFR_NGFR motifs are underlined. FIG. 2B depicts a
Kyte-Doolittle hydrophobicity plot and schematic diagram of TACI-1
showing the positions of the putative transmembrane domain (solid
black) and the TNFR_NGFR motifs (stippled). FIG. 2C depicts the
cysteine residues of the TACI protein and other TNFR family
members.
[0051] FIG. 3. TACI-1 is a cell surface protein. FIG. 3A depicts
the flow cytometry of TAg Jurkat T cells transiently transfected
with a TACI-1 expression plasmid and the transfection marker, pHook
(Invitrogen), or the transfection marker pHook alone. Cell surface
expression of TACI-1 and the transfection marker protein, HA-Hook,
were detected by indirect immunofluorescence using the
immunoaffinity-purified anti-TACI-1 polyclonal antibody and the
12CA5 monoclonal antibody. Depicted data represent TACI-1 staining
of cells gated for the transfection marker. FIG. 3B depicts a
photomicrograph showing surface the staining of Cos-7 cells
transiently expressing N-terminal FLAG-tagged TACI-1, stained with
M2 (anti-FLAG) antibodies and fluorescent anti-mouse IgG antibodies
[Bram & Crabtree (1994) Nature 371:355-358]. Surface staining
was present whether or not cells were permeabilised with
detergent.
[0052] FIG. 4. TACI-1 is a signaling protein that functions in the
activation of NF-AT-specific transcription. FIG. 4A depicts the
activation of an NF-AT-driven secreted alkaline phosphatase
reporter. TAg Jurkat T cells, co-transfected with the SXNFAT
reporter [Bram et al. (1993) Mol. Cell. Biol. 13:4760-4769] and the
expression plasmid pBJ5 [Takebe, Y., et al. (1988) Mol. Cell. Biol.
8:466-472] containing either TACI-1-encoding cDNA (solid triangles)
or nothing (circles), were treated with 50 ng/ml PMA and the
indicated amounts of ionomycin in the presence (closed symbols) and
absence (open symbols) of magnetic beads coated with
immunoaffinity-purified anti-TACI-1 polyclonal antibodies. FIG. 4B
depicts the activation of NF-AT by antibody-cross linked TACI-1
that is blocked by Cyclosporin A (CsA) or FK506. NF-AT activation
was determined in TAg Jurkat T cells co-transfected with SXNFAT and
pBJ5 (-TACI-1, left) or pBJ5-TACI-1 (+TACI-1, right), and treated
with the indicated combinations of PMA (50 ng/ml), ionomycin (2
.mu.M), CsA (100 ng/ml) and FK506 (500 pg/ml) in the presence of
anti-TACI-1 antibody-coated beads (`NS`, not stimulated). FIG. 4C
shows that NF-AT activation by antibody-cross linked TACI-1
requires extracellular calcium. NF-AT activation was measured in
the presence of EGTA in Jurkat T cells over expressing TACI-1. The
treatments included cross linked anti-TACI-1 (circles),
transfection with a C-terminally truncated, calcium-independent
calcineurin A subunit (triangles), or activation with the TCR
stimulating antibody OKT3 (squares). All cells were co-stimulated
by the addition of PMA to 50 ng/ml. FIG. 4D depicts the activation
of an AP-1-driven secreted alkaline phosphatase reporter. AP-1
activation was measured in TAg Jurkat T cells, co-transfected with
a mouse metallothionein AP-1-SEAP reporter [Bram et al., 1991,
supra] and pBJ5 containing no insert (-TACI-1, left) or TACI-1 cDNA
(+TACI-1, right). Cells were incubated in the absence, and with
serially increasing amounts, of cross linked anti-TACI-1
antibodies. To control for transfection efficiency, a plasmid
containing a constitutive promoter driving the expression of
luciferase (EF-Luc) was included.
[0053] FIG. 5. TACI-1 interaction with CAML is critical for
Ca.sup.2+ signaling. FIG. 5A shows the yeast 2-hybrid interaction.
Full-length cDNAs and indicated deletion mutants of TACI-1 and CAML
were cloned into the yeast expression plasmids pACT and/or pAS 1,
and the indicated combinations were tested for interaction with the
yeast 2-hybrid system (`+`, positive interaction; `-`, no
interaction; `ND`, not done). FIG. 5B depicts the
co-immunoprecipitation of CAML with TACI-1. 293T cells were
transfected with the indicated combinations of the expression
plasmid pBJ5 containing cDNAs for CAML, TACI-1 with an N-terminal
FLAG tag, the N-terminal 146 amino acids of CAML (CLX91), or no
insert. After incubation for 48 hours, the cells were lysed (1%
dodecyl maltoside, 20 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 2
mM MgSO.sub.4, 1 mM CaCl.sub.2, 1 mM PMSF) and the lysate was
clarified by centrifugation. FLAG-tagged TACI-1 and associated
proteins were immunoprecipitated with anti-FLAG monoclonal
antibody-conjugated agarose beads and subjected to Western transfer
using standard protocols. The Western blot was probed with
immunoaffinity-purified anti-CAML polyclonal antibodies followed by
chemiluminescent detection (Amersham). Parallel Western blots,
performed for each sample, confirmed the expected expression of
TACI-1, CAML or the truncated CAML mutant in all transfections.
FIG. 5C shows that the over expression of the N-terminal half of
CAML has a dominant negative effect on TACI-1-induced NF-AT
activation. NF-AT activation was determined in TAg Jurkat cells
transfected with pBJ5 alone, pBJ5-TACI-1 plus the control plasmid,
or pBJ5-TACI-1 with an equivalent amount of CLX91, following
treatment with TACI-1-specific antibodies (top). TACI-1 expression
in these transfections was determined by Western blot using
anti-TACI-1 polyclonal antibodies (bottom). FIG. 5D depicts a
schematic diagram showing the TACI-1/CAML signal transduction model
(`SOC`, stores-operated calcium channel).
DETAILED DESCRIPTION OF THE INVENTION
[0054] The present invention, in its broadest aspect, provides a
novel cell surface receptor that is normally present in
B-lymphocytes, and to a much lesser extent in immature
T-lymphocytes. The role of the cell surface receptor, the
Transmembrane Activator and CAML-Interactor (TACI) protein, is to
participate in alternate or co-stimulatory pathways to activate or
control lymphocyte function. These functions can include response
of lymphocytes to foreign antigens in infection, or to cancer, in
the graft-rejection, and graft-vs-host reaction. Additionally,
activation of lymphocyte signaling plays a key role during
lymphocyte development, thus allowing the positive selection of
functional lymphocytes and negative selection against self-reactive
clones. When activated, the TACI protein stimulates the influx of
calcium in lymphocytes. Such calcium influx can, under specific
circumstances, lead to the onset of programmed cell death
(apoptosis).
[0055] The terms "Transmembrane Activator and CAML-Interactor"
protein or "TACI" or "TACI protein" are used herein interchangeably
with "transmembrane CAML-binding protein" or "TCB" or "TCB protein"
and refer to proteinaceous material including single or multiple
proteins that act as a novel cell surface receptor that is normally
present in B-lymphocytes. This cell surface receptor has the
profile of activities set forth herein. Accordingly, proteins
displaying substantially equivalent or altered activity are
likewise contemplated. These modifications may be deliberate, for
example, such as modifications obtained through site-directed
mutagenesis, or may be accidental, such as those obtained through
mutations in hosts that are producers of the protein. Included
within the scope of these terms are proteins specifically recited
herein, as well as all substantially homologous analogs and allelic
variations.
[0056] In one embodiment the Transmembrane Activator and
CAML-Interactor is TACI-1, the human homologue. As shown in the
Example, infra, TACI-1 initiates a previously undetected signal
transduction mechanism that directly links cell surface stimuli to
the intracellular signaling molecule, CAML. TACI-1 therefore is a
member of a new class of lymphocyte-specific cell surface receptors
that modulate the immune response and thus may be used as tool to
regulate the immune system in either a positive or a negative
direction. In a specific embodiment, TACI-1 has the amino acid
sequence set forth in SEQ ID NO:2.
[0057] As used herein when a particular nucleic acid is said to
encode a protein or polypeptide of the present invention, it is
meant that the portion of the particular nucleic acid that consists
of the coding sequence for that protein or polypeptide is being
identified. For example, the coding region of SEQ ID NO:1 is from
nucleotide 14 to nucleotide 895, including the 3 nucleotide
translation stop codon at the 3/terminus.
[0058] For many purposes, there is a substantial interest in being
able to selectively prevent activation of lymphocytes or in the
alternative to selectively enhance their activation. For example,
for lymphocyte mediated autoimmune diseases, transplant rejection
syndrome, and graft-versus host disease, inhibiting the activation
of the lymphocyte involved is a viable or necessary treatment for
the disease. Furthermore, in the case of myelomas, lymphomas, and
leukemias, especially of B cells or immature T cells, there is an
interest in slowing the proliferation of cancer cells, which may
allow for therapies which are not as destructive to the host as
present day therapies. On the other hand, for infections or
anti-tumor immune responses, there would be interest in being able
to activate lymphocytes to more rapidly respond to the pathogen.
Thus, the present invention enables selection of useful agents,
e.g., synthetic organic compounds which can activate and/or
deactivate lymphocytes by providing a previously unknown key
component in the lymphocyte activation pathway.
[0059] In addition, as one understands the activation pathway more
completely, one is able to modulate the pathway more effectively,
such as providing for agents which are selective for a particular
set or subset of a cellular population. Since in many cases
activation requires co-stimulation, being able to manipulate agents
available to the cell may allow for such cellular activity. In this
context, the identification of a target protein that can be used to
develop drugs that modulate a particular pathway, such as the
CAML-mediated activation pathway, would allow physicians to
particularly treat distinct immune conditions without
over-stimulating or over-suppressing other delicate aspects of the
immune system that are otherwise functioning well. Such targeted
stimulation can be used to specifically amplify the effects of
immune stimulators, such as IL-2, thus allowing for the use of
lower doses of the immune stimulator and reducing side effects.
[0060] Furthermore, the invention is an important advance in
understanding the CAML-mediated activation pathway, by permitting
selective evaluation and control over the presence or the absence
of a particular intermediate in that pathway. This can be achieved
with a knock-out animal using homologous recombination, integration
of genes providing for antisense sequences, introduction of
expression constructs involving inducible promoters, and the like.
There is also an interest in being able to determine when a
particular gene is being expressed or is silent, the nature of the
cells in which the protein is expressed, and the like. Therefore,
there is substantial interest in identifying specific components of
cellular pathways to allow for understanding an activation pathway,
selectively modulating that pathway, and developing drugs which may
be active in binding to the target protein. In this way, drugs can
be screened to inhibit such specific pathways.
[0061] One particular aspect of the present invention includes a
drug screen that uses TACI as a tool for developing
immunosuppressant drugs specific for B-lymphocytes. Such
immunosuppressant drugs would selectively block the action of
B-lymphocytes, while leaving T-cells intact to protect patients
from viral pathogens. These drugs would be useful in treating
diseases such as Systemic Lupus Erythematosus, a disease due to an
over-activation of the B-lymphocyte response, or multiple myeloma
(e.g., Bence-Jones Myeloma).
[0062] Cross-linking the TACI protein activates calcium influx and
potentially other secondary messengers. Its mode of action can be
mediated by CAML. Unlike known cell surface signaling molecules
that are specific for B cells or T cells, such as CD4, CD8, TCR,
and CD3, the novel cell surface receptor of the present invention
physically interacts with CAML. In addition, there is no sequence
homology between the novel cell surface receptor of the present
invention and any of these known lymphocyte cell surface signaling
molecules, or any other cell surface signaling molecules.
[0063] TACI Proteins and Polypeptides
[0064] In a broad embodiment, the present invention provides TACI
proteins. Such proteins include an extracellular domain, a
transmembrane domain, and a cytoplasmic domain. The extracellular
domain binds ligand. Upon ligand binding, the cytoplasmic domain
binds CAML, thus initiating a Ca.sup.2+-dependent activation
pathway. Receptor oligomerization, e.g., by binding ligand or with
an anti-receptor antibody, also initiates a non-Ca.sup.2+-dependent
activation pathway.
[0065] The monomeric form of a TACI protein contains about 295
amino acids. As used herein "about 295 amino acids" means between
265 to 325 amino acids, i.e., roughly plus or minus 10%.
[0066] The invention further relates to functionally active
polypeptide components of TACI. In one aspect, a functionally
active component of TACI is an antigenic fragment, e.g., a peptide
reactive with anti-TACI antibodies, or which, when conjugated to a
carrier, can be used to generate anti-TACI antibodies. Another
functionally active fragment includes the extracellular domain,
which binds ligand. The extracellular domain corresponds to the
N-terminal fragment of TACI, e.g., from the first amino acid
residue of mature TACI to the transmembrane domain. In a specific
embodiment, the extracellular domain has the amino acid sequence
corresponding to about residue 1 to about residue 166 of SEQ ID
NO:6. The ligand-binding region of TACI is a sub-fragment of the
N-terminal fragment corresponding to the extracellular domain.
[0067] Still another functionally active fragment is the
cytoplasmic domain, e.g., from the C-terminal end of the
transmembrane domain to the C-terminus of TACI. The cytoplasmic
domain of TACI mediates signal transduction via Ca.sup.2+-dependent
and Ca.sup.2+-independent mechanisms. The cytoplasmic domain
includes the CAML-binding region of TACI. In particular, this
domain binds a polypeptide corresponding to the N-terminal 146
amino acid residues of CAML. In a specific embodiment, the
cytoplasmic domain corresponds to from about amino acid residue 187
to about amino acid residue 293 of SEQ ID NO:2.
[0068] In yet another embodiment, the present invention provides
proteolytic fragments of a TACI protein. Such fragments can be
prepared by enzymatic digestion, e.g., with Saureus Polypeptides V8
in papain trypsin, chymotrypsin, cathepsin, collagenase,
enteropeptidase, thrombin, or fibrinolytic or clotting enzymes; by
chemical cleavage, e.g., with cyanogen bromide, or sodium
borohydride; etc.
[0069] In a specific embodiment, the TACI protein is a receptor
protein having the amino acid sequence as shown in SEQ ID NO:2,
from residue 1 to residue 293. The present invention contemplates
allelic variants of TACI, homologous TACI proteins from other
species, and TACI analogs, e.g., prepared by making conservative
amino acid substitutions, whether by genetic engineering or by
chemical synthesis. A TACI analogue of the invention also includes
TACI antigenic fragments that contain, e.g., a terminal cysteine
residue to facilitate cross-linking to a carrier protein.
[0070] There are no reported DNA sequences that are closely related
to that for TACI-1. A search for Prosite motifs in TACI-1 reveals
one TNFR_NGFR pattern, which consists of
C-x(4,6)-[FYH]-x(5,10)-C-x(0,2)-C-x(-
2,3)-C-x(7,11)-C-x(4,6)-[DNEQSKP]-x(2)-C (SEQ ID NO:11) in the
N-terminal half of the protein (where e.g., C-x(4,6)-[FYH] is
indicative of an amino acid sequence starting with cysteine
followed by either 4, 5, or 6 unspecified amino acids, further
followed by either a phenylalanine, a tyrosine, or a histidine).
This motif is found in a number of proteins, most of which are
receptors for growth factors. Some of these proteins have one copy
of this motif. A comparison of the TACI-1 protein sequence with
itself reveals a significant repeat between the TNFR_NGFR motif at
residues 33-66 and residues 70-104. This analysis drew attention to
the presence of two TNFR-type cysteine-rich domains encompassing
these regions that indicate that TACI-1 is a member of the
superfamily of TNFR receptors.
[0071] Synthetic TACI Polypeptides.
[0072] The term "polypeptide" is used in its broadest sense to
refer to a compound of two or more subunit amino acids, amino acid
analogs, or peptidomimetics. The subunits may be linked by peptide
bonds. In another embodiment, the subunit may be linked by other
bonds, e.g., ester, ether, etc. As used herein the term "amino
acid" refers to either natural and or unnatural or synthetic amino
acids, including glycine and both the D or L optical isomers, and
amino acid analogs and peptidomimetics. A peptide of three or more
amino acids is commonly called an oligopeptide if the peptide chain
is short. If the peptide chain is long, the peptide is commonly
called a polypeptide or a protein. According to the invention, TACI
fragments, such as antigenic fragments, or potentially even
full-length TACI, can be prepared synthetically.
[0073] Synthetic polypeptides, prepared using the well known
techniques of solid phase, liquid phase, or peptide condensation
techniques, or any combination thereof, can include natural and
unnatural amino acids. Amino acids used for peptide synthesis may
be standard Boc (N.sup..alpha.-amino protected
N.alpha.-t-butyloxycarbonyl) amino acid resin with the standard
de-protecting, neutralization, coupling and wash protocols of the
original solid phase procedure of Merrifield [(1963) J. Am. Chem.
Soc. 85:2149-2154], or the base-labile N.sup..alpha.-amino
protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first
described by Carpino and Han [(1972) J. Org. Chem. 37:3403-3409].
Both Fmoc and Boc N.sup..alpha.-amino protected amino acids can be
obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge
Research Biochemical, Bachem, or Peninsula Labs or other chemical
companies familiar to those who practice this art. In addition, the
method of the invention can be used with other
N.sup..alpha.-protecting groups that are familiar to those skilled
in this art. Solid phase peptide synthesis may be accomplished by
techniques familiar to those in the art and provided, for example,
in Stewart and Young (1984) Solid Phase Synthesis (2d Ed., Pierce
Chemical Co., Rockford, Ill.); Fields and Noble (1990) Int. J.
Pept. Protein Res. 35:161-214, or using automated synthesizers,
such as sold by ABS. Thus, polypeptides of the invention may
comprise D-amino acids, a combination of D- and L-amino acids, and
various "designer" amino acids (e.g., .beta.-methyl amino acids,
C.alpha.-methyl amino acids, and N.alpha.-methyl amino acids, etc.)
to convey special properties. Synthetic amino acids include
ornithine for lysine, fluorophenylalanine for phenylalanine, and
norleucine for leucine or isoleucine. Additionally, by assigning
specific amino acids at specific coupling steps, .alpha.-helices,
.beta. turns, .beta. sheets, .gamma.-turns, and cyclic peptides can
be generated.
[0074] In a further embodiment, subunits of peptides that confer
useful chemical and structural properties will be chosen. For
example, peptides comprising D-amino acids will be resistant to
L-amino acid-specific proteases in vivo. In addition, the present
invention envisions preparing peptides that have more well defined
structural properties, and the use of peptidomimetics, and
peptidomimetic bonds, such as ester bonds, to prepare peptides with
novel properties. In another embodiment, a peptide may be generated
that incorporates a reduced peptide bond, i.e.,
R.sub.1--CH.sub.2--NH--R.sub.2, where R.sub.1 and R.sub.2 are amino
acid residues or sequences. A reduced peptide bond may be
introduced as a dipeptide subunit. Such a molecule would be
resistant to peptide bond hydrolysis, e.g., protease activity. Such
peptides would provide ligands with unique function and activity,
such as extended half-lives in vivo due to resistance to metabolic
breakdown, or protease activity. Furthermore, it is well known that
in certain systems constrained peptides show enhanced functional
activity [Hruby (1982) Life Sciences 31:189-199]; [Hruby et al.
(1990) Biochem J. 268:249-262]; the present invention provides a
method to produce a constrained peptide that incorporates random
sequences at all other positions.
[0075] Non-Classical Amino Acids that Induce Conformational
Constraints:
[0076] The following non-classical amino acids may be incorporated
in the peptide in order to introduce particular conformational
motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Kazmierski et
al. (1991) J. Am. Chem. Soc. 113:2275-2283];
(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,
(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine
[Kazmierski and Hruby (1991) Tetrahedron Lett.];
2-aminotetrahydronaphthalene-2-carboxylic acid [Landis, Ph.D.
Thesis, University of Arizona, (1989)];
hydroxy-1,2,3,4-tetrahydroisoquin- oline-3-carboxylate [Miyake et
al. (1989) J. Takeda Res. Labs. 43:53-76]; .beta.-carboline (D and
L) [Kazmierski, Ph.D. Thesis, University of Arizona, (1988)]; HIC
(histidine isoquinoline carboxylic acid) [Zechel et al. (1991) Int.
J. Pep. Protein Res. 43]; and HIC (histidine cyclic urea)
(Dharanipragada).
[0077] The following amino acid analogs and peptidomimetics may be
incorporated into a peptide to induce or favor specific secondary
structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a
.beta.-turn inducing dipeptide analog (Kemp et al. (1985) J. Org.
Chem. 50:5834-5838]; .beta.-sheet inducing analogs [Kemp et al.
(1988) Tetrahedron Lett. 29:5081-5082]; .beta.-turn inducing
analogs [Kemp et al. (1988) Tetrahedron Lett. 29:5057-5060];
.alpha.-helix inducing analogs [Kemp et al. (1988) Tetrahedron
Lett. 29:4935-4938]; .gamma.-turn inducing analogs [Kemp et al.
(1989) J. Org. Chem. 54:109:115]; and analogs provided by the
following references: Nagai and Sato (1985) Tetrahedron Lett.
26:647-650; DiMaio et al. (1989) J. Chem. Soc. Perkin Trans., p.
1687; also a Gly-Ala turn analog [Kahn et al. (1989) Tetrahedron
Lett. 30:2317]; amide bond isostere [Jones et al. (1988)
Tetrahedron Lett. 29:3853-3856]; tretrazol [Zabrocki et al. (1988)
J. Am. Chem. Soc. 110:5875-5880]; DTC [Samanen et al. (1990) Int.
J. Protein Pep. Res. 35:501:509]; and analogs taught in Olson et
al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990)
J. Org. Chem. 56:436. Conformationally restricted mimetics of beta
turns and beta bulges, and peptides containing them, are described
in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.
[0078] Protein Derivatives.
[0079] There are two major classes of peptide-carbohydrate linkages
to proteins. First, ether bonds join the serine or threonine
hydroxyl to a hydroxyl of the sugar. Second, amide bonds join
glutamate or aspartate carboxyl groups to an amino group on the
sugar. Acetal and ketal bonds may also bind carbohydrate to
peptide.
[0080] Generally, the TACI protein, or a fragment thereof, such as
the N-terminal extracellular domain fragment or the C-terminal
cytoplasmic CAML-binding domain fragment, may be derivatized by the
attachment of one or more chemical moieties to the protein moiety.
Chemical modification of biologically active component or
components may provide additional advantages under certain
circumstances, such as increasing the stability and circulation
time of the component or components and decreasing immunogenicity.
See U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979.
For a review, see Abuchowski et al., in Enzymes as Drugs [J. S.
Holcerberg and J. Roberts, eds. (1981) pp. 367-383]. A review
article describing protein modification and fusion proteins is
Francis (1992) Focus on Growth Factors 3:4-10, Mediscript:
Mountview Court, Friern Barnet Lane, London N20, OLD, UK.
[0081] The chemical moieties suitable for derivatization may be
selected from among water soluble and water insoluble polymers,
with water soluble polymers preferred. The polymer selected should
preferably be water soluble so that the component to which it is
attached does not precipitate in an aqueous environment, such as a
physiological environment. Preferably, for therapeutic use of the
end-product preparation, the polymer will be pharmaceutically
acceptable. One skilled in the art will be able to select the
desired polymer based on such considerations as whether the
polymer/component conjugate will be used therapeutically, and if
so, the desired dosage, circulation time, resistance to
proteolysis, and other considerations. For the present component or
components, these may be ascertained using the assays provided
herein.
[0082] Labeled TACI Proteins.
[0083] The TACI protein of the present invention, and fragments
thereof, may be labeled. Suitable labels include enzymes,
fluorophores (e.g., fluorescence isothiocyanate (FITC),
phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated
lanthanide series salts, especially Eu.sup.3+, to name a few
fluorophores), chromophores, radioisotopes, chelating agents, dyes,
colloidal gold, latex particles, ligands (e.g., biotin), and
chemiluminescent agents. When a control marker is employed, the
same or different labels may be used for the test sample and
control marker.
[0084] In the instance where a radioactive label, such as the
isotopes .sup.3H, .sup.14C, .sup.32p, .sup.35S, .sup.36Cl,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I,
.sup.131I, and .sup.186Re are used, known currently available
counting procedures may be utilized. In the instance where the
label is an enzyme, detection may be accomplished by any of the
presently utilized calorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques
known in the art.
[0085] Direct labels are one example of labels which can be used
according to the present invention. A direct label has been defined
as an entity, which in its natural state, is readily visible,
either to the naked eye, or with the aid of an optical filter
and/or applied stimulation, e.g. U.V. light to promote
fluorescence. Among examples of colored labels, which can be used
according to the present invention, include metallic sol particles,
for example, gold sol particles such as those described by
Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such as
described by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et
al. (WO 88/08534); dyed latex such as described by May, supra,
Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in
liposomes as described by Campbell et al. (U.S. Pat. No.
4,703,017). Other direct labels include a radio nucleotide, a
fluorescent moiety or a luminescent moiety. In addition to these
direct labeling devices, indirect labels comprising enzymes can
also be used according to the present invention. Various types of
enzyme linked immunoassays are well known in the art, for example,
alkaline phosphatase and horseradish peroxidase, lysozyme,
glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease,
these and others have been discussed in detail by Eva Engvall in
Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology (1980)
70:419-439 and in U.S. Pat. No. 4,857,453.
[0086] Suitable enzymes include, but are not limited to, alkaline
phosphatase and horseradish peroxidase.
[0087] Other labels for use in the invention include magnetic beads
or magnetic resonance imaging labels.
[0088] In another embodiment, a phosphorylation site can be created
on an antibody of the invention for labeling with .sup.32P, e.g.,
as described in European Patent No. 0372707 (application No.
89311108.8) by Pestka, or U.S. Pat. No. 5,459,240, issued Oct. 17,
1995 to Foxwell et al.
[0089] As exemplified herein, proteins, including antibodies, can
be labeled by metabolic labeling. Metabolic labeling occurs during
in vitro incubation of the cells that express the protein in the
presence of culture medium supplemented with a metabolic label,
such as [.sup.35S]-methionine or [.sup.32P]-orthophosphate. In
addition to metabolic (or biosynthetic) labeling with
[.sup.35S]-methionine, the invention further contemplates labeling
with [.sup.14C]-amino acids and [.sup.3H]-amino acids (with the
tritium substituted at non-labile positions).
[0090] Chimeric TACI Proteins
[0091] A chimeric TACI protein of the invention may be a protein
that is generated by joining a functional domain of a TACI protein,
such as the ligand binding domain or the CAML-binding domain, with
the complementary domain of another protein, e.g., an alternative
receptor. Chimeric constructs can also be prepared with a
functionally active fragment of a TACI protein and another
functionally active molecule. For example, the extracellular domain
of a TACI protein may be joined to the Fc domain of an
immunoglobulin. Alternatively, the cytoplasmic domain of a TACI
protein could be joined to a receptor ligand, such as transferrin
or a hormone, for intracellular targeting. In yet another
embodiment, a TACI domain could be joined to another targeting
molecule, such as an anti-immunoglobulin heavy chain or light chain
molecule (e.g., an Fv portion of an antibody) to specifically
target B cells. In still another embodiment, the functionally
active fragment of a TACI protein, preferably the N-terminal
extracellular domain, can be joined with a glycosylphospholipid,
such as glycosylphosphoinositol, anchor signal sequence, preferably
located at the C-terminus of the TACI fragment, so that a
glycolipid anchored protein is generated [Cross (1990) Annu. Rev.
Cell Biol. 6:1-39; Low (1987) Biochem. J. 244:1-13].
[0092] A chimeric TACI receptor can be prepared by joining the
extracellular domain of another receptor molecule with a
transmembrane domain and the intracellular domain of a TACI
protein. In another embodiment, the extracellular domain of TACI
can be joined with a transmembrane domain and an intracellular
domain of another receptor molecule. The transmembrane domain can
be the transmembrane domain of a TACI protein, the transmembrane
domain of the other receptor, or a different transmembrane domain.
Preferably, the transmembrane domain is from the same protein
component of the chimera as the extracellular domain. Chimeric
receptors have been described [International Patent Publications
WO96/23814; WO96/23881; and WO96/24671]. Chimeric antigen receptors
have been described [Capon et al., U.S. Pat. No. 5,359,046, issued
Oct. 25, 1994], including functional antigen-specific receptors
generated in B cells [Sanchez et al. (1993) J. Exp. Med.
178:1049-1055] and T cells [Burkhardt et al. (1994) Mol. Cell.
Biol. 14:1095-1103] by fusing the Ig.alpha. and Igf.beta. signal
transduction chains to IgM. Various type I and type II cytokine
receptors, including interferon-.alpha., interferon-.beta.,
interferon-.gamma., interleukin-1, interleukin-2, interleukin-3,
interleukin-4, interleukin-6, interleukin-8, interleukin-10,
interleukin-12, erythropoietin, granulocyte-macrophage colony
stimulating factor (CSF), granulocyte-CSF, macrophage-CSF,
.alpha.-chemokine receptors, and .beta.-chemokine receptors, can
provide complementary components in a chimeric receptor comprising
a functionally active fragment of a TACI protein.
[0093] As those of ordinary skill in the art can appreciate,
transmembrane domains are generally functionally equivalent for
anchoring a protein in a membrane. However, the presence of
specific amino acid residues in a transmembrane domain can affect
receptor interaction with, e.g., dimerization, or association with
other integral membrane proteins, such as in a multi-protein
receptor complex. Thus, selection of a transmembrane domain depends
on whether regulatory functions performed by the transmembrane
domain are desired or necessary.
[0094] Nucleic Acids Encoding TACI Proteins
[0095] The present invention contemplates isolation of a gene
encoding a TACI protein including a full length, or naturally
occurring form of TACI protein, and any antigenic fragments thereof
from any animal, particularly mammalian and more particularly
human, source. As used herein, the term "gene" refers to an
assembly of nucleotides that encode a polypeptide, and includes
cDNA and genomic DNA nucleic acids.
[0096] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis (1989) Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989");
DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover
ed. (1985)); Oligonucleotide Synthesis (M. J. Gait ed. (1984));
Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.
(1985)]; Transcription And Translation [B. D. Hames & S. J.
Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed.
(1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B.
Perbal (1984) A Practical Guide To Molecular Cloning; F. M. Ausubel
et al. (eds.) (1994) Current Protocols in Molecular Biology, John
Wiley & Sons, Inc.
[0097] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0098] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions
as an autonomous unit of DNA replication in vivo, i.e., capable of
replication under its own control.
[0099] A "cassette" refers to a segment of DNA that can be inserted
into a vector at specific restriction sites. The segment of DNA
encodes a polypeptide of interest, and the cassette and restriction
sites are designed to ensure insertion of the cassette in the
proper reading frame for transcription and translation.
[0100] A cell has been "transfected" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. A cell has
been "transformed" by exogenous or heterologous DNA when the
transfected DNA effects a phenotypic change. Preferably, the
transforming DNA should be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell.
[0101] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0102] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0103] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m of 55.degree., can be used,
e.g., 5.times.SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%
formamide, 5.times.SSC, 0.5% SDS. Moderate stringency hybridization
conditions correspond to a higher T.sub.m, e.g., 40% formamide,
with 5.times. or 6.times.SCC. High stringency hybridization
conditions correspond to the highest T.sub.m, e.g., 50% formamide,
5.times. or 6.times.SCC. Hybridization requires that the two
nucleic acids contain complementary sequences, although depending
on the stringency of the hybridization, mismatches between bases
are possible. The appropriate stringency for hybridizing nucleic
acids depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-0.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al, supra, 11.7-11.8). A minimum length for a hybridizable nucleic
acid is at least 10 nucleotides; preferably at least 18
nucleotides; more preferably the length is at least 24 nucleotides
and most preferably at least 30 nucleotides in length. In a
specific embodiment, a hybridizable nucleic acid of the invention
has a sequence corresponding to at least 12 nucleotides, preferably
at least 18 nucleotides, more preferably at least 24 nucleotides,
and most preferably at least 30 nucleotides in length of SEQ ID
NO:1, or more specifically the coding sequence of SEQ ID NO:1.
[0104] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C.
[0105] As used herein, the term "oligonucleotide" refers to a
nucleic acid, generally of at least 18 nucleotides, that is
hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA
molecule encoding a TACI protein. Oligonucleotides can be labeled,
e.g., with .sup.32P-nucleotides or nucleotides to which a label,
such as biotin, has been covalently conjugated (see the discussion,
supra, with respect to labeling TACI polypeptides). In one
embodiment, a labeled oligonucleotide can be used as a probe to
detect the presence of a nucleic acid encoding a TACI protein. In
another embodiment, oligonucleotides (one or both of which may be
labeled) can be used as PCR primers, either for cloning full length
or a fragment of a TACI protein, or to detect the presence of
nucleic acids encoding a TACI protein. In a further embodiment, an
oligonucleotide of the invention can form a triple helix with a
TACI DNA molecule. Generally, oligonucleotides are prepared
synthetically, preferably on a nucleic acid synthesizer.
Accordingly, oligonucleotides can be prepared with non-naturally
occurring phosphoester analog bonds, such as thioester bonds,
etc.
[0106] "Homologous recombination" refers to the insertion of a
foreign DNA sequence of a vector in a chromosome. Preferably, the
vector targets a specific chromosomal site for homologous
recombination. For specific homologous recombination, the vector
will contain sufficiently long regions of homology to sequences of
the chromosome to allow complementary binding and incorporation of
the vector into the chromosome. Longer regions of homology, and
greater degrees of sequence similarity, may increase the efficiency
of homologous recombination.
[0107] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is not limited to, prokaryotic sequences,
cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic
(e.g., mammalian) DNA, and even synthetic DNA sequences. If the
coding sequence is intended for expression in a eukaryotic cell, a
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence. In a specific
embodiment, a TACI coding sequence of the invention has the
nucleotide sequence depicted in SEQ ID NO:1.
[0108] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, terminators,
and the like, that provide for the expression of a coding sequence
in a host cell. In eukaryotic cells, polyadenylation signals are
control sequences.
[0109] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0110] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced and translated into the protein encoded by the coding
sequence.
[0111] A "signal sequence" may be included at the beginning of the
coding sequence of a protein to be expressed on the surface of a
cell. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide. The term "translocation signal sequence" is used
herein to refer to this sort of signal sequence. Translocation
signal sequences can be found associated with a variety of proteins
native to eukaryotes and prokaryotes, and are often functional in
both types of organisms. Interestingly, the TACI of the invention
is transported so that the N-terminus is extracellular in the
absence of a cleaved signal sequence. Thus, this transmembrane
protein is a type III transmembrane protein [see Wilson-Rawls et
al. (1994) Virology 201:66-76]. Thus, in a construct of the present
invention (including a chimeric construct as discussed above), if
the N-terminal portion of the construct encodes the N-terminus of
TACI, a signal peptide may not be required to obtain expression of
the transmembrane TACI protein.
[0112] As used herein, the term "sequence homology" in all its
grammatical forms refers to the relationship between proteins that
possess a "common evolutionary origin," including proteins from
superfamilies (e.g., the immunoglobulin superfamily) and homologous
proteins from different species (e.g., myosin light chain, etc.)
[Reeck et al. (1987) Cell 50:667]. The present invention naturally
contemplates homologues of the human TACI protein as falling within
the scope of the invention.
[0113] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences whether
or not they share a common evolutionary origin (see Reeck et al.,
supra). In common usage, the term "homologous," when modified with
an adverb such as "highly," may refer to sequence similarity and
not a common evolutionary origin.
[0114] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 50% (preferably at least about 75%, and most preferably at
least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I &
II, supra; Nucleic Acid Hybridization, supra. The present invention
contemplates nucleotides that are 50% similar to SEQ ID NO:1 (or
its complementary sequence), preferably 60% similar, and more
preferably 75% similar. Preferably such substantially similar
nucleic acids are homologous.
[0115] Similarly, in a particular embodiment, two amino acid
sequences are "substantially homologous" or "substantially similar"
when greater than 30% of the amino acids are identical, or greater
than about 60% are similar (functionally identical). Preferably,
the similar or homologous sequences are identified by alignment
using, for example, the GCG (Genetics Computer Group, Program
Manual for the GCG Package, Version 7, Madison, Wis.) pileup
program.
[0116] The term "corresponding to" is used herein to refer similar
or homologous sequences, whether the exact position is identical or
different from the molecule to which the similarity or homology is
measured. Thus, the term "corresponding to" refers to the sequence
similarity, and not the numbering of the amino acid residues or
nucleotide bases.
[0117] A gene encoding a TACI protein, whether genomic DNA or cDNA,
can be isolated from any source, particularly from a human cDNA or
genomic library. Methods for obtaining TACI protein gene are well
known in the art, as described above (see, e.g., Sambrook et al.,
1989, supra).
[0118] Accordingly, any animal cell potentially can serve as the
nucleic acid source for the molecular cloning of a TACI protein
gene. The DNA may be obtained by standard procedures known in the
art from cloned DNA (e.g., a DNA "library"), and preferably is
obtained from a cDNA library prepared from tissues with high level
expression of the protein (e.g., a thymic cDNA library, since
peripheral blood cells and in particular lymphocyte cells, appear
to have the highest levels of expression of TACI protein), by
chemical synthesis, by CDNA cloning, or by the cloning of genomic
DNA, or fragments thereof, purified from the desired cell (see, for
example, Sambrook et al., 1989, supra; Glover, D. M. (ed.) (1985)
DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K.
Vol. I, II). Clones derived from genomic DNA may contain regulatory
and intron DNA regions in addition to coding regions; clones
derived from cDNA will not contain intron sequences. Whatever the
source, the gene should be molecularly cloned into a suitable
vector for propagation of the gene.
[0119] In the molecular cloning of the gene from genomic DNA, DNA
fragments are generated, some of which will encode the desired
gene. The DNA may be cleaved at specific sites using various
restriction enzymes. Alternatively, one may use DNAse in the
presence of manganese to fragment the DNA, or the DNA can be
physically sheared, as for example, by sonication. The linear DNA
fragments can then be separated according to size by standard
techniques, including but not limited to, agarose and
polyacrylamide gel electrophoresis and column chromatography.
[0120] Once the DNA fragments are generated, identification of the
specific DNA fragment containing the desired TACI protein gene may
be accomplished in a number of ways. For example, a portion of a
TACI protein gene or its specific RNA, or a fragment thereof, can
be purified and labeled, the generated DNA fragments may then be
screened by nucleic acid hybridization to the labeled probe [Benton
and Davis (1977) Science 196:180]; [Grunstein and Hogness (1975)
Proc. Natl. Acad. Sci. U.S.A., 72:3961]. For example, a set of
oligonucleotides corresponding to the partial amino acid sequence
information obtained for the TACI protein can be prepared and used
as probes for DNA encoding a TACI protein, or as primers for cDNA
or mRNA (e.g., in combination with a poly-T primer for RT-PCR).
Preferably, a fragment is selected that is highly unique to the
TACI protein of the invention. Those DNA fragments with substantial
homology to the probe will hybridize. As noted above, the greater
the degree of homology, the more stringent hybridization conditions
can be used. In a specific embodiment, stringency hybridization
conditions are used to identify a homologous TACI protein gene.
[0121] Further selection can be carried out on the basis of the
properties of the gene, e.g., if the gene encodes a protein product
having the isoelectric, electrophoretic, amino acid composition, or
partial amino acid sequence of the TACI protein as disclosed
herein. Thus, the presence of the gene may be detected by assays
based on the physical, chemical, or immunological properties of its
expressed product. For example, cDNA clones, or DNA clones which
hybrid-select the proper mRNAs, can be selected which produce a
protein that, e.g., has similar or identical electrophoretic
migration, isoelectric focusing or non-equilibrium pH gel
electrophoresis behavior, proteolytic digestion maps, or antigenic
properties as known for TACI protein.
[0122] A TACI protein gene of the invention can also be identified
by mRNA selection, i.e., by nucleic acid hybridization followed by
in vitro translation. In this procedure, nucleotide fragments are
used to isolate complementary mRNAs by hybridization. Such DNA
fragments may represent available, purified TACI protein DNA, or
may be synthetic oligonucleotides designed from the partial amino
acid sequence information. Immunoprecipitation analysis or
functional assays (e.g., CAML binding activity) of the in vitro
translation products of the products of the isolated mRNAs
identifies the mRNA and, therefore, the complementary DNA
fragments, that contain the desired sequences. In addition,
specific mRNAs may be selected by adsorption of polysomes isolated
from cells to immobilized antibodies specifically directed against
TACI protein, such as the rabbit polyclonal anti-human TACI protein
antibody described herein.
[0123] A radiolabeled TACI protein cDNA can be synthesized using
the selected mRNA (from the adsorbed polysomes) as a template. The
radiolabeled mRNA or cDNA may then be used as a probe to identify
homologous TACI protein DNA fragments from among other genomic DNA
fragments.
[0124] The present invention also relates to cloning vectors
containing genes encoding analogs and derivatives of the TACI
protein of the invention, that have the same or homologous
functional activity as TACI protein, and homologs thereof from
other species. The production and use of derivatives and analogs
related to TACI protein are within the scope of the present
invention. In a specific embodiment, the derivative or analog is
functionally active, i.e., capable of exhibiting one or more
functional activities associated with a full-length, wild-type TACI
protein of the invention. In another embodiment, TACI protein
containing a different cytoplasmic domain, e.g., one unable to bind
CAML but still able to modulate the activation of AP-1. In another
aspect, a TACI protein of the invention can be prepared with a
lectin domain or domains from another protein, such as the mannose
receptor of macrophages or the phospholipase receptor on
muscle.
[0125] TACI protein derivatives can be made by altering encoding
nucleic acid sequences by substitutions, additions or deletions
that provide for functionally equivalent molecules. Preferably,
derivatives are made that have enhanced or increased functional
activity relative to native TACI protein. Alternatively, such
derivatives may encode soluble fragments of TACI protein
extracellular domain that have the same or greater affinity for the
natural ligand of TACI protein of the invention. Such soluble
derivatives may be potent inhibitors of ligand binding to TACI
protein.
[0126] Due to the degeneracy of nucleotide coding sequences, other
DNA sequences which encode substantially the same amino acid
sequence as a TACI protein gene may be used in the practice of the
present invention. These include but are not limited to allelic
genes, homologous genes from other species, and nucleotide
sequences comprising all or portions of TACI protein genes which
are altered by the substitution of different codons that encode the
same amino acid residue within the sequence, thus producing a
silent change. Likewise, the TACI protein derivatives of the
invention include, but are not limited to, those containing, as a
primary amino acid sequence, all or part of the amino acid sequence
of a TACI protein including altered sequences in which functionally
equivalent amino acid residues are substituted for residues within
the sequence resulting in a conservative amino acid substitution.
And thus, such a substitution is defined as a conservative
substitution.
[0127] For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity, which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the non-polar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Such alterations will not be
expected to affect apparent molecular weight as determined by
polyacrylamide gel electrophoresis, or isoelectric point.
Particularly preferred substitutions are:
[0128] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0129] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0130] Ser for Thr such that a free --OH can be maintained; and
[0131] Gln for Asn such that a free NH.sub.2 can be maintained.
[0132] Amino acid substitutions may also be introduced to
substitute an amino acid with a particularly preferable property.
For example, a Cys may be introduced at a potential site for
disulfide bridges with another Cys. A His may be introduced as a
particularly "catalytic" site (i.e., His can act as an acid or base
and is the most common amino acid in biochemical catalysis). Pro
may be introduced because of its particularly planar structure,
which induces .beta.-turns in the protein's structure.
[0133] The genes encoding TACI protein derivatives and analogs of
the invention can be produced by various methods known in the art.
The manipulations which result in their production can occur at the
gene or protein level. For example, the cloned TACI protein gene
sequence can be modified by any of numerous strategies known in the
art (Sambrook et al., 1989, supra). The sequence can be cleaved at
appropriate sites with restriction endonuclease(s), followed by
further enzymatic modification if desired, isolated, and ligated in
vitro. In the production of the gene encoding a derivative or
analog of TACI protein, care should be taken to ensure that the
modified gene remains within the same translational reading frame
as the TACI protein gene, uninterrupted by translational stop
signals, in the gene region where the desired activity is
encoded.
[0134] Additionally, the TACI protein-encoding nucleic acid
sequence can be mutated in vitro or in vivo, to create and/or
destroy translation, initiation, and/or termination sequences, or
to create variations in coding regions and/or form new restriction
endonuclease sites or destroy preexisting ones, to facilitate
further in vitro modification. Preferably, such mutations enhance
the functional activity of the mutated TACI protein gene product.
Any technique for mutagenesis known in the art can be used,
including but not limited to, in vitro site-directed mutagenesis
[Hutchinson et al. (1978) J. Biol. Chem. 253:6551]; [Zoller and
Smith (1984) DNA 3:479-488]; [Oliphant et al. (1986) Gene 44:177];
[Hutchinson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:710],
use of TAB.RTM. linkers (Pharmacia), etc. PCR techniques are
preferred for site directed mutagenesis (see Higuchi, "Using PCR to
Engineer DNA", in PCR Technology: Principles and Applications for
DNA Amplification, H. Erlich, ed. (1989) Stockton Press, Chapter 6,
pp. 61-70).
[0135] The identified and isolated gene can then be inserted into
an appropriate cloning vector. A large number of vector-host
systems known in the art may be used. Possible vectors include, but
are not limited to, plasmids or modified viruses, but the vector
system must be compatible with the host cell used. Examples of
vectors include, but are not limited to, E. coli, bacteriophages
such as lambda derivatives, or plasmids such as pBR322 derivatives
or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
The insertion into a cloning vector can, for example, be
accomplished by ligating the DNA fragment into a cloning vector
which has complementary cohesive termini. However, if the
complementary restriction sites used to fragment the DNA are not
present in the cloning vector, the ends of the DNA molecules may be
enzymatically modified. Alternatively, any site desired may be
produced by ligating nucleotide sequences (linkers) onto the DNA
termini; these ligated linkers may comprise specific chemically
synthesized oligonucleotides encoding restriction endonuclease
recognition sequences. Recombinant molecules can be introduced into
host cells via transformation, transfection, infection,
electroporation, etc., so that many copies of the gene sequence are
generated. Preferably, the cloned gene is contained on a shuttle
vector plasmid, which provides for expansion in a cloning cell,
e.g., E. coli, and facile purification for subsequent insertion
into an appropriate expression cell line, if such is desired. For
example, a shuttle vector, which is a vector that can replicate in
more than one type of organism, can be prepared for replication in
both E. coli and Saccharomyces cerevisiae by linking sequences from
an E. coli plasmid with sequences from the yeast 2.mu. plasmid.
[0136] In an alternative method, the desired gene may be identified
and isolated after insertion into a suitable cloning vector in a
"shot gun" approach. Enrichment for the desired gene, for example,
by size fractionation, can be done before insertion into the
cloning vector.
[0137] Expression of Transmembrane Activator and CAML Interactor
Polypeptides
[0138] The nucleotide sequence coding for the TACI protein, or
antigenic fragment, derivative or analog thereof, or a functionally
active derivative, including a chimeric protein, thereof, can be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted protein-coding sequence. Such elements
are termed herein a "promoter." Thus, the nucleic acid encoding the
TACI protein of the present invention is operationally associated
with a promoter in an expression vector of the invention. Both cDNA
and genomic sequences can be cloned and expressed under control of
such regulatory sequences. An expression vector also preferably
includes a replication origin.
[0139] The necessary transcriptional and translational signals can
be provided on a recombinant expression vector, or they may be
supplied by the native gene encoding a TACI protein and/or its
flanking regions.
[0140] As pointed out above, potential chimeric partners for TACI
protein include those having lectin domains, either from naturally
occurring multivalent lectin receptors, such as mannose receptor of
macrophages, natural lectins, or other sources, or a substitute
cytoplasmic domain, capable of mediating signal transduction or
modifying the endocytic processing.
[0141] Potential host-vector systems include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast containing yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities. Depending on the host-vector
system utilized, any one of a number of suitable transcription and
translation elements may be used.
[0142] A recombinant TACI protein of the invention, or functional
fragment, derivative, chimeric construct, or analog thereof, may be
expressed chromosomally, after integration of the coding sequence
by recombination. In this regard, any of a number of amplification
systems may be used to achieve high levels of stable gene
expression (see Sambrook et al., 1989, supra).
[0143] The cell containing the recombinant vector comprising the
nucleic acid encoding a TACI protein is cultured in an appropriate
cell culture medium under conditions that provide for expression of
TACI protein by the cell.
[0144] Any of the methods previously described for the insertion of
DNA fragments into a cloning vector may be used to construct
expression vectors containing a gene consisting of appropriate
transcriptional/translational control signals and the protein
coding sequences. These methods may include in vitro recombinant
DNA and synthetic techniques and in vivo recombination (genetic
recombination).
[0145] Expression of TACI protein may be controlled by any
promoter/enhancer element known in the art, but these regulatory
elements must be functional in the host selected for expression.
Promoters which may be used to control TACI protein gene expression
include, but are not limited to, the SV40 early promoter region
[Benoist and Chambon (1981) Nature 290:304-310], the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
[Yamamoto, et al. (1980) Cell 22:787-797], the herpes thymidine
kinase promoter [Wagner et al. (1981) Proc. Natl. Acad. Sci.
U.S.A., 78:1441-1445], the regulatory sequences of the
metallothionein gene [Brinster et al. (1982) Nature 296:39-42];
prokaryotic expression vectors such as the .beta.-lactamase
promoter [Villa-Kamaroff, et al. (1978) Proc. Natl. Acad. Sci.
U.S.A., 75:3727-3731], or the tac promoter [DeBoer, et al. (1983)
Proc. Natl. Acad. Sci. U.S.A. 80:21-25]; see also "Useful proteins
from recombinant bacteria" in Scientific American (1980) 242:74-94;
promoter elements from yeast or other fungi such as the Gal4
promoter, the ADC (alcohol dehydrogenase) promoter, PGK
(phosphoglycerol kinase) promoter, alkaline phosphatase promoter;
and the animal transcriptional control regions, which exhibit
tissue specificity and have been utilized in transgenic animals:
elastase I gene control region which is active in pancreatic acinar
cells [Swift et al. (1984) Cell 38:639-646]; [Omitz et al. (1986)
Cold Spring Harbor Symp. Quant. Biol. 50:399-409]; [MacDonald
(1987) Hepatology 7:425-515]; insulin gene control region which is
active in pancreatic beta cells [Hanahan (1985) Nature
315:115-122], immunoglobulin gene control region which is active in
lymphoid cells [Grosschedl et al. (1984) Cell 38:647-658]; [Adames
et al. (1985) Nature 318:533-538]; [Alexander et al. (1987) Mol.
Cell. Biol. 7:1436-1444], mouse mammary tumor virus control region
which is active in testicular, breast, lymphoid and mast cells
[Leder et al. (1986) Cell 45:485-495], albumin gene control region
which is active in liver [Pinkert et al. (1987) Genes and Devel.
1:268-276], alpha-fetoprotein gene control region which is active
in liver [Krumlauf et al. (1985) Mol. Cell. Biol. 5:1639-1648];
[Hammer et al. (1987) Science 235:53-58], alpha 1-antitrypsin gene
control region which is active in the liver [Kelsey et al. (1987)
Genes and Devel. 1:161-171], beta-globin gene control region which
is active in myeloid cells [Mogram et al. (1985) Nature
315:338-340]; [Kollias et al. (1986) Cell 46:89-94], myelin basic
protein gene control region which is active in oligodendrocyte
cells in the brain [Readhead et al. (1987) Cell 48:703-712], myosin
light chain-2 gene control region which is active in skeletal
muscle [Sani (1985) Nature 314:283-286], and gonadotropic releasing
hormone gene control region which is active in the hypothalamus
[Mason et al. (1986) Science 234:1372-1378].
[0146] Expression vectors containing a nucleic acid encoding a TACI
protein of the invention can be identified by four general
approaches: (a) PCR amplification of the desired plasmid DNA or
specific mRNA, (b) nucleic acid hybridization, (c) presence or
absence of selection marker gene functions, and (d) expression of
inserted sequences. In the first approach, the nucleic acids can be
amplified by PCR to provide for detection of the amplified product.
In the second approach, the presence of a foreign gene inserted in
an expression vector can be detected by nucleic acid hybridization
using probes comprising sequences that are homologous to an
inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the
presence or absence of certain "selection marker" gene functions
(e.g., .beta.-galactosidase activity, thymidine kinase activity,
resistance to antibiotics, transformation phenotype, occlusion body
formation in baculovirus, etc.) caused by the insertion of foreign
genes in the vector. In another example, if the nucleic acid
encoding a TACI protein is inserted within the "selection marker"
gene sequence of the vector, recombinants containing the TACI
protein insert can be identified by the absence of the TACI protein
gene function. In the fourth approach, recombinant expression
vectors can be identified by assaying for the activity,
biochemical, or immunological characteristics of the gene product
expressed by the recombinant, provided that the expressed protein
assumes a functionally active conformation.
[0147] A wide variety of host/expression vector combinations may be
employed in expressing the DNA sequences of this invention. Useful
expression vectors, for example, may consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable
vectors include derivatives of SV40 and known bacterial plasmids,
e.g., E. coli plasmids co1 E1, pCR1, pBR322, pMal-C2, pET, pGEX
[Smith et al. (1988) Gene 67:31-40], pMB9 and their derivatives,
plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of
phage .lambda., e.g., NM989, and other phage DNA, e.g., M13 and
filamentous single stranded phage DNA; yeast plasmids such as the
2.mu. plasmid or derivatives thereof; vectors useful in eukaryotic
cells, such as vectors useful in insect or mammalian cells; vectors
derived from combinations of plasmids and phage DNAs, such as
plasmids that have been modified to employ phage DNA or other
expression control sequences; and the like.
[0148] For example, in a baculovirus expression systems, both
non-fusion transfer vectors, such as but not limited to pVL941
(BamH1 cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1,
NotI, XmaIII, BglII, and PstI cloning site; Invitrogen), pVL1392
(BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning
site; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI,
NcoI, and HindIII cloning site, with blue/white recombinant
screening possible; Invitrogen), and fusion transfer vectors, such
as but not limited to pAc700 (BamH1 and KpnI cloning site, in which
the BamH1 recognition site begins with the initiation codon;
Summers), pAc701 and pAc702 (same as pAc700, with different reading
frames), pAc360 (BamH1 cloning site 36 base pairs downstream of a
polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B,
C (three different reading frames, with BamH1, BglII, PstI, NcoI,
and HindIII cloning site, an N-terminal peptide for ProBond
purification, and blue/white recombinant screening of plaques;
Invitrogen (220)) can be used.
[0149] Mammalian expression vectors contemplated for use in the
invention include vectors with inducible promoters, such as the
dihydrofolate reductase (DHFR) promoter, e.g., any expression
vector with a DHFR expression vector, or a DHFR/methotrexate
co-amplification vector, such as pED (PstI, SalI, SbaI, SmaI, and
EcoRI cloning site, with the vector expressing both the cloned gene
and DHFR; see Kaufman (1991) Current Protocols in Molecular Biology
16:12. Alternatively, a glutamine synthetase/methionine sulfoximine
co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI,
EcoRI, and BclI cloning site, in which the vector expresses
glutamine synthase and the cloned gene; Celltech). In another
embodiment, a vector that directs episomal expression under control
of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,
SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning
site, constitutive RSV-LTR promoter, hygromycin selectable marker;
Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI,
PvuII, and KpnI cloning site, constitutive hCMV immediate early
gene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI,
NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible
metallothionein IIa gene promoter, hygromycin selectable marker:
Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI
cloning site, RSV-LTR promoter, histidinol selectable marker;
Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and
BamHI cloning site, RSV-LTR promoter, G418 selectable marker;
Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable
marker, N-terminal peptide purifiable via ProBond resin and cleaved
by enterokinase; Invitrogen). Selectable mammalian expression
vectors for use in the invention include pRc/CMV (HindIII, BstXI,
NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),
pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418
selection; Invitrogen), and others. Vaccinia virus mammalian
expression vectors (see, Kaufinan, 1991, supra) for use according
to the invention include but are not limited to pSC11 (SmaI cloning
site, TO- and .beta.-gal selection), pMJ601 (SalI, SmaI, AflI,
NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI, and HindIII cloning
site; TK- and , .beta.-gal selection), and pTKgptF1S (EcoRI, PstI,
SalI, AccI, HindII, SbaI, BamHI, and Hpa cloning site, TK or XPRT
selection). Yeast expression systems can also be used according to
the invention to express the TACI protein. For example, the
non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI,
BstXI, BamH1, SacI, Kpn1, and HindIII cloning sit; Invitrogen) or
the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI,
BamH1, SacI, KpnI, and HindIII cloning site, N-terminal peptide
purified with ProBond resin and cleaved with enterokinase;
Invitrogen), to mention just two, can be employed according to the
invention.
[0150] Once a particular recombinant DNA molecule is identified and
isolated, several methods known in the art may be used to propagate
it. Once a suitable host system and growth conditions are
established, recombinant expression vectors can be propagated and
prepared in quantity. As previously explained, the expression
vectors which can be used include, but are not limited to, the
following vectors or their derivatives: human or animal viruses
such as vaccinia virus or adenovirus; insect viruses such as
baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda),
and plasmid and cosmid DNA vectors, to name but a few.
[0151] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Different host cells have characteristic and specific mechanisms
for the translational and post-translational processing and
modification (e.g., glycosylation, cleavage e.g., of signal
sequence) of proteins. Appropriate cell lines or host systems can
be chosen to ensure the desired modification and processing of the
foreign protein expressed. For example, expression in a bacterial
system can be used to produce an non-glycosylated core protein
product.
[0152] Vectors are introduced into the desired host cells by
methods known in the art, e.g., transfection, electroporation,
micro injection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, lipofection (lysosome fusion), use of a
gene gun, or a DNA vector transporter (see, e.g., Wu et al. (1992)
J. Biol. Chem. 267:963-967; Wu and Wu (1988) J. Biol. Chem.
263:14621-14624; Hartmut et al., Canadian Patent Application No.
2,012,311, filed Mar. 15, 1990).
[0153] Gene Therapy and Transgenic Vectors
[0154] A genetic deficiency of a TACI protein can be one of the
many factors involved in inherited immunodeficiency. The present
invention includes gene therapy with the TACI protein cDNA that
restores normal lymphocyte function in patients having a genetic
defect in the inlet Transmembrane Activator and CAML Interactor
protein gene. The present invention also includes modified forms of
TACI to be used as gene-therapeutic tools through inserting them
into blood stem cells in preparation for re-infusion into patients
as part of bone marrow transplant regimes. In one embodiment an
epitope tagged-TACI derivative is used as a way to selectively
activate only those lymphocytes derived from the marrow transplant
by injection of the specific antibody into the patient. In an
alternative embodiment transduction of a TACI gene lacking its
intracellular portion is used to create a relatively innocuous
lymphocyte with diminished responsiveness. This embodiment is
useful in autoimmune diseases involving activation of B cells.
[0155] In one embodiment, a gene encoding a TACI protein or
polypeptide domain fragment thereof is introduced in vivo in a
viral vector. Such vectors include an attenuated or defective DNA
virus, such as but not limited to herpes simplex virus (HSV),
papilloma virus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective viruses,
which entirely or almost entirely lack viral genes, are preferred.
Defective virus is not infective after introduction into a cell.
Use of defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the vector can
infect other cells. Thus, lymphocytes can be specifically targeted.
Examples of particular vectors include, but are not limited to, a
defective herpes virus 1 (HSV 1) vector [Kaplitt et al. (1991)
Molec. Cell. Neurosci. 2:320-330], an attenuated adenovirus vector,
such as the vector described by Stratford-PerricaudetI [(1992) J.
Clin. Invest. 90:626-630], and a defective adeno-associated virus
vector [Samulski et al. (1987) J. Virol. 61:3096-3101]; [Samulski
et al. (1989) J. Virol. 63:3822-3828].
[0156] Preferably, for in vivo administration, an appropriate
immunosuppressive treatment is employed in conjunction with the
viral vector, e.g., adenovirus vector, to avoid immuno-deactivation
of the viral vector and transfected cells. For example,
immunosuppressive cytokines, such as interleukin-12 (IL-12),
interferon-.gamma. (IFN-.gamma.), or anti-CD4 antibody, can be
administered to block humoral or cellular immune responses to the
viral vectors [see, e.g., Wilson (1995) Nature Medicine]. In
addition, it is advantageous to employ a viral vector that is
engineered to express a minimal number of antigens.
[0157] In another embodiment the gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. (1983) Cell 33:153; Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al. (1988) J. Virol. 62:1120; Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995, by Dougherty et al.; and Kuo et al. (1993)
Blood 82:845.
[0158] Targeted gene delivery is described in International Patent
Publication WO 95/28494, published October 1995.
[0159] Alternatively, the vector can be introduced in vivo by
lipofection. For the past decade, there has been increasing use of
liposomes for encapsulation and transfection of nucleic acids in
vitro. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker [Felgner et. al. (1987) Proc. Natl. Acad. Sci.
U.S.A. 84:7413-7417; see Mackey, et al. (1988) Proc. Natl. Acad.
Sci. U.S.A. 85:8027-8031]. The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes [Felgner and Ringold
(1989) Science 337:387-388]. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as
pancreas, liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting [see
Mackey, et al., supra]. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins such as antibodies, or non-peptide
molecules could be coupled to liposomes chemically.
[0160] It is also possible to introduce the vector in vivo as a
naked DNA plasmid. Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in the art,
e.g., transfection, electroporation, micro injection, transduction,
cell fusion, DEAE dextran, calcium phosphate precipitation, use of
a gene gun, or use of a DNA vector transporter [see, e.g., Wu et
al. (1992) J. Biol. Chem. 267:963-967; Wu and Wu (1988) J. Biol.
Chem., 263:14621-14624; Hartmut et al., Canadian Patent Application
No. 2,012,311, filed Mar. 15, 1990].
[0161] In a preferred embodiment of the present invention, a gene
therapy vector as described above employs a transcription control
sequence operably associated with the sequence for the TACI protein
inserted in the vector. That is, a specific expression vector of
the present invention can be used in gene therapy.
[0162] Gene Targeting
[0163] As used herein "Gene targeting" is a type of homologous
recombination that occurs when a fragment of genomic DNA is
introduced into a mammalian cell and that fragment locates and
recombines with endogenous homologous sequences.
[0164] As used herein a "knockout mouse" is a mouse that contains
within its genome a specific gene that has been inactivated by the
method of gene targeting. A knockout mouse includes both the
heterozygote mouse (i.e., one defective allele and one wild-type
allele) and the homozygous mutant (i.e., two defective
alleles).
[0165] As used herein a "marker gene" is a selection marker that
facilitates the isolation of rare transfected cells from the
majority of treated cells in the population. A non-comprehensive
list of such markers includes neomycin phosphotransferase,
hygromycin B phosphotransferase, Xanthine/guanine phosphoribosyl
transferase, herpes simplex thymidine kinase, and diphtheria
toxin.
[0166] The functional activity of Transmembrane Activator and CAML
Interactor protein can be evaluated transgenically. In this
respect, a transgenic mouse model can be used. The Transmembrane
Activator and CAML Interactor protein gene can be used in
complementation studies employing a transgenic mouse. Transgenic
vectors, including viral vectors, or cosmid clones (or phage
clones) corresponding to the wild type locus of a candidate gene,
can be constructed using the isolated TACI protein gene. Cosmids
may be introduced into transgenic mice using published procedures
[Jaenisch (1988) Science 240:1468-1474]. In a genetic sense, the
transgene acts as a suppressor mutation.
[0167] Alternatively, a transgenic animal model can be prepared in
which expression of the TACI protein gene is disrupted. Gene
expression is disrupted, according to the invention, when no
functional protein is expressed. One standard method to evaluate
the phenotypic effect of a gene product is to employ knock-out
technology to delete the gene. Alternatively, recombinant
techniques can be used to introduce mutations, such as nonsense and
amber mutations, or mutations that lead to expression of an
inactive protein. In another embodiment, TACI protein genes can be
tested by examining their phenotypic effects when expressed in
antisense orientation in wild-type animals. In this approach,
expression of the wild-type allele is suppressed, which leads to a
mutant phenotype. RNA.times.RNA duplex formation (antisense-sense)
prevents normal handling of mRNA, resulting in partial or complete
elimination of wild-type gene effect. This technique has been used
to inhibit TK synthesis in tissue culture and to produce phenotypes
of the Kruppel mutation in Drosophila, and the Shiverer mutation in
mice [Izant et al. Cell (1984) 36:1007-1015; Green et al. (1986)
Annu. Rev. Biochem. 55:569-597; Katsuki et al. (1988) Science
241:593-595]. An important advantage of this approach is that only
a small portion of the gene need be expressed for effective
inhibition of expression of the entire cognate mRNA. The antisense
transgene will be placed under control of its own promoter or
another promoter expressed in the correct cell type, and placed
upstream of the SV40 polyA site. This transgene will be used to
make transgenic mice, or by using gene knockout technology.
[0168] Thus the present invention extends to the preparation of
antisense nucleotides and ribozymes that may be used to interfere
with the expression of the TACI protein at the translational level.
This approach utilizes antisense nucleic acid and ribozymes to
block translation of a specific mRNA, either by masking that mRNA
with an antisense nucleic acid or cleaving it with a ribozyme.
Genes encoding TACI mRNA-specific antisense or ribozyme nucleic
acids can be introduced, e.g., using techniques as described above
for "Gene Therapy." Alternatively, synthetic antisense or ribozyme
oligonucleotides can be prepared.
[0169] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
[see Marcus-Sekura (1988) Anal. Biochem. 172:298]. In the cell,
they hybridize to that mRNA, forming a double stranded molecule.
The cell does not translate an mRNA in this double-stranded form.
Therefore, antisense nucleic acids interfere with the expression of
mRNA into protein. Oligomers of about fifteen nucleotides and
molecules that hybridize to the AUG initiation codon will be
particularly efficient, since they are easy to synthesize and are
likely to pose fewer problems than larger molecules when
introducing them into organ cells. Antisense methods have been used
to inhibit the expression of many genes in vitro [Marcus-Sekura
(1988), supra; Hambor et al. (1988) J. Exp. Med. 168:1237].
Preferably synthetic antisense nucleotides contain phosphoester
analogs, such as phosphorothiolates, or thioesters, rather than
natural phophoester bonds. Such phosphoester bond analogs are more
resistant to degradation, increasing the stability, and therefore
the efficacy, of the antisense nucleic acids.
[0170] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single stranded RNA molecules in a manner
somewhat analogous to DNA restriction endonucleases. Investigators
have identified two types of ribozymes, Tetrahymena-type and
"hammerhead"-type. Hammerhead-type ribozymes are preferable to
Tetrahymena-type ribozymes for inactivating a specific mRNA
species, and eighteen base recognition sequences are preferable to
shorter recognition sequences.
[0171] The DNA sequences encoding the TACI protein described and
enabled herein may thus be used to prepare antisense molecules
against and ribozymes that cleave mRNAs for the TACI protein, thus
inhibiting expression of the gene encoding the TACI protein, which
may reduce the level of immune stimulation by dendritic cells, or
the level of clonal detection by mediated by thymic epithelial
cells.
[0172] Gene targeting in embryonic stem cells is a relatively new
technique that allows the precise manipulation of genes in vivo.
This technique allows the creation of mice with defined mutations
in the structure of any given gene. This ability to generate
predetermined mutations gives investigators the ability to apply
the power of genetics to the complex human immune system, as it has
successfully been applied in neuronal systems for such organisms as
D. melanogaster and C. elegans.
[0173] A key to finding treatments for many disorders has been the
development of appropriate animal models. The present invention
includes a knockout mouse containing a non-functional allele for
the gene that naturally encodes and expresses functional TACI
protein. Included within this aspect of the invention is a knockout
mouse containing two non-functional alleles for the gene that
naturally encodes and expresses functional TACI protein, and
therefore is unable to express functional TACI protein.
[0174] Non-functional alleles can be generated in any number of
ways that are well known in the art, all of which may be used in
the present invention. In some embodiments, a non-functional allele
is made defective by an insertion of extraneous DNA into the coding
region of TACI protein allele. In a preferred embodiment, the
insertion is placed in the first exon of the coding region of the
TACI protein gene. In more preferred embodiments, the insertion
contains a signal to terminate transcription prior to the
transcription of a region of the allele that encodes TACI protein.
In these preferred embodiments it is still more preferred to remove
a section of DNA at the beginning of the coding region for the TACI
protein and replacing it with the above insertion.
[0175] The present invention also includes a method for producing
the knockout mouse of the instant invention that includes:
obtaining genomic DNA encoding a TACI protein, constructing a
vector containing said genomic DNA and a marker gene wherein said
marker gene is placed within the exon of said genomic DNA. The
vector is then electroporated into an embryonic stem cell and an
embryonic stem cell is selected that has integrated the vector into
the genome, wherein the selected cell has integrated the marker
gene into the endogenous site of the gene for the TACI protein in
the mouse genome. The cell is then injected into a mouse blastocyst
which is then re-implanted into a pseudopregnant female mouse,
which gives birth to a chimeric mouse containing a defective allele
for the TACI protein in its germ line. The chimeric mouse is then
mated to a mouse of a standard in-bred line to generate a
heterozygous knockout mouse. Two heterozygous mice are then bred
generating a homozygous knockout mouse offspring. Detailed
protocols for successful gene targeting are well known in the art,
and for example, as described by Joyner, A. L. (1993) Gene
Targeting: A Practical Approach. The Practical Approach Series
(Rickwood, D. and Hames, B. D., Eds.), IRL Press, Oxford which is
hereby incorporated by reference in its entirety.
[0176] Another aspect of the invention is a method for selecting a
therapeutic agent for possible use as an immunosuppressant which
comprises administering a suspected therapeutic agent to the
knockout mouse of the present invention and measuring and/or
determining the putative therapeutic agent's effect on any of the
phenotypic characteristics which may be believed to be related to
the immunodeficiency.
[0177] A preferred embodiment of this aspect of the invention
includes administering a suspected therapeutic agent to the
knockout mouse of the present invention and measuring a test
response in the knockout mouse, wherein the normal response of the
knockout mouse in the absence of a therapeutic agent is
characteristically different from that of wild-type mice. The
potential therapeutic agents are selected on the basis of whether
there is a statistical significance between test response and the
normal response. Potential therapeutic agents are selected that
show a statistically significant change in the characteristic
measured/determined. In a preferred embodiment, the normal response
of the knockout mouse in the absence of a therapeutic agent is
characteristically different by being characteristically lower than
that of wild-type mice and the selected therapeutic agents act to
raise the sensitivity of that characteristic.
[0178] The suspected therapeutical agents may be obtained from any
number of drug or peptide libraries including those commercially
available from drug chemical companies.
[0179] Purification of TACI Proteins and Homologues thereof:
[0180] The TACI protein of the present invention and homologues
thereof can be purified by any number of procedures that encompass
a wide variety of known purification steps. Those with skill in the
art would know to refer to references, such as the Methods of
Enzymology series, for greater detail and breadth. Initial steps
for purifying the proteins of the present invention include salting
in or salting out, such as in ammonium sulfate fractionations;
solvent exclusion fractionations, e.g., an ethanol precipitation;
detergent extractions to free membrane bound proteins using such
detergents as Triton X-100, Tween-20 etc.; or high salt
extractions. Solubilization of proteins may also be achieved using
aprotic solvents such as dimethyl sulfoxide and
hexamethylphosphoramide. In addition, high speed
ultracentrifugation may be used either alone or in conjunction with
other extraction techniques.
[0181] Generally good secondary isolation or purification steps
include solid phase absorption using calcium phosphate gel or
hydroxyapatite; or solid phase binding. Solid phase binding may be
performed through ionic bonding, with either an anion exchanger,
such as diethylaminoethyl (DEAFE), or diethyl [2-hydroxy propyl]
amino ethyl (QAE) Sephadex or cellulose; or with a cation exchanger
such as carboxymethyl (CM) or sulfo propyl (SP) Sephadex or
cellulose. Alternative means of solid phase binding includes the
exploitation of hydrophobic interactions e.g., the using of a solid
support such as phenylSepharose and a high salt buffer;
affinity-binding, using, e.g., CAML (or a TACI-binding fragment
thereof) bound to an activated support; immuno-binding, using e.g.,
an antibody to the TACI protein bound to an activated support; as
well as other solid phase supports including those that contain
specific dyes or lectins etc. A further solid phase support
technique that is often used at the end of the purification
procedure relies on size exclusion, such as Sephadex and Sepharose
gels, or pressurized or centrifugal membrane techniques, using size
exclusion membrane filters.
[0182] Solid phase support separations are generally performed
batch-wise with low-speed centrifugations or by column
chromatography. High performance liquid chromatography (HPLC),
including such related techniques as FPLC, is presently the most
common means of performing liquid chromatography. Size exclusion
techniques may also be accomplished with the aid of low speed
centrifugation.
[0183] In addition size permeation techniques such as gel
electrophoretic techniques may be employed. These techniques are
generally performed in tubes, slabs or by capillary
electrophoresis.
[0184] Almost all steps involving protein purification employ a
biological buffer at a pH close to the pKa of that buffer. Typical
buffers can be purchased from most biochemical catalogues and
include the classical buffers such as Tris, pyrophosphate,
monophosphate, and diphosphate, or the Good buffers [Good et al.
(1966) Biochemistry 5:467]; [Good, N. E. and Izawa, S. (1972) Meth.
Enzymol. 24, Part B, 53]; and [Fergunson, W. J. and Good, N. E.
(1980) Anal. Biochem. 104:300] such as Mes, Hepes, Mops, tricine
and Ches.
[0185] Materials to perform all of these techniques are available
from a variety of sources such as Sigma Chemical Company in St.
Louis, Mo.
[0186] Antibodies to the TACI Protein
[0187] The present invention discloses the protein sequence and
properties of a specific Transmembrane Activator and CAML
Interactor protein, TACI, thereby enabling the development of
antibody reagents specific for the extracellular portion of the
receptor. Polyvalent antibody reagents can act to cross-link and
activate TACI signaling in lymphocytes, a useful process in
situations where enhanced lymphocyte responsiveness is beneficial.
Such situations include for example, in the treatment of
immunodeficiencies that are either congenital or acquired e.g.,
AIDS. In addition, these antibody reagents may be used as an
adjuvant treatment in cancer in instances that the immune system
can aid in the eradication of neoplastic cells. Similarly
monovalent antibody reagents can act to block access to TACI in
lymphocytes, a process that is useful in situations where depressed
lymphocyte responsiveness is beneficial such as during organ
transplants.
[0188] According to the present invention, the TACI protein
produced naturally, recombinantly or by chemical synthesis, and
fragments or other derivatives or analogs thereof, including fusion
proteins, may be used as an immunogen to generate antibodies that
recognize the TACI protein. Such antibodies include but are not
limited to polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and an Fab expression library. The anti-TACI protein
antibodies of the invention may be cross reactive, e.g., they may
recognize the TACI protein from different species. Polyclonal
antibodies have greater likelihood of cross reactivity.
Alternatively, an antibody of the invention may be specific for a
single form of the TACI protein. Preferably, such an antibody is
specific for human TACI protein. Antibodies of the invention can be
labeled, as described above for TACI proteins and polypeptides.
[0189] Various procedures known in the art may be used for the
production of polyclonal antibodies to the TACI protein or
derivative or analog thereof. For the production of antibody,
various host animals can be immunized by injection with the TACI
protein, or a derivative (e.g., fragment or fusion protein)
thereof, including but not limited to rabbits, mice, rats, sheep,
goats, etc. In one embodiment, the TACI protein or fragment thereof
can be conjugated to an immunogenic carrier, e.g., bovine serum
albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants
may be used to increase the immunological response, depending on
the host species, including but not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0190] For preparation of monoclonal antibodies directed toward the
TACI protein, or fragment, analog, or derivative thereof, any
technique that provides for the production of antibody molecules by
continuous cell lines in culture may be used. These include but are
not limited to the hybridoma technique originally developed by
Kohler and Milstein [(1975) Nature 256:495-497], as well as the
trioma technique, the human B-cell hybridoma technique [Kozbor et
al. (1983) Immunology Today 4:72]; [Cote et al. (1983) Proc. Natl.
Acad. Sci. U.S.A. 80:2026-2030], and the EBV-hybridoma technique to
produce human monoclonal antibodies [Cole et al. (1985) Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96]. In
an additional embodiment of the invention, monoclonal antibodies
can be produced in germ-free animals utilizing recent technology
[PCT/US90/02545]. In fact, according to the invention, techniques
developed for the production of "chimeric antibodies" [Morrison et
al. (1984) J. Bacteriol. 159:870; Neuberger et al. (1984) Nature
312:604-608; Takeda et al. (1985) Nature 314:452-454] by splicing
the genes from a mouse antibody molecule specific for a TACI
protein together with genes from a human antibody molecule of
appropriate biological activity can be used; such antibodies are
within the scope of this invention. Such human or humanized
chimeric antibodies are preferred for use in therapy of human
diseases or disorders (described infra), since the human or
humanized antibodies are much less likely than xenogeneic
antibodies to induce an immune response, in particular an allergic
response, themselves.
[0191] According to the invention, techniques described for the
production of single chain antibodies [U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to
produce the TACI protein-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries [Huse et
al. (1989) Science 246:1275-1281] to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for a TACI protein, or its derivatives, or analogs.
[0192] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0193] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of a TACI protein, one may assay generated hybridomas for a
product which binds to a TACI protein fragment containing such
epitope. For selection of an antibody specific to a TACI protein
from a particular species of animal, one can select on the basis of
positive binding with the TACI protein expressed by or isolated
from cells of that species of animal.
[0194] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of the TACI protein,
e.g., for Western blotting, imaging the TACI protein in situ,
measuring levels thereof in appropriate physiological samples, etc.
using any of the detection techniques mentioned above or known in
the art.
[0195] In a specific embodiment, antibodies that agonize or
antagonize the activity of the TACI protein can be generated. Such
antibodies can be tested using the assays described infra for
identifying ligands.
[0196] Ligands to the TACI Protein
[0197] The identity of the endogenous ligand of the TACI protein is
unknown. The TACI protein can be used, to screen clones in order to
identify the endogenous ligand(s). This ligand is likely to be
involved in the regulation of the immune system as well, and thus
should have similar or complementary uses to those described
herein. Methods for screening for TACI-1 ligands include: (i)
through the use of a yeast two-hybrid system with TACI-1 as "bait",
as described, e.g., in Chien et al. (1991) Proc. Natl. Science,
USA, 88:9578-9582 and Durfee et al. (1993) Genes Dev. 7:555-69;
(ii) interaction cloning from E. coli expression-libraries as
described above; and (iii) functional expression cloning in
mammalian cells as described above.
[0198] Identification and isolation of a gene encoding a TACI
protein of the invention provides for expression of the TACI
protein or fragments thereof in quantities greater than can be
isolated from natural sources, or in cells that are specially
engineered to be regulated by the TACI protein expressed after
transfection or transformation of the cells. Accordingly, in
addition to rational design of agonists and antagonists based on
the structure of the TACI protein, the present invention
contemplates an alternative method for identifying specific ligands
of the TACI protein using various screening assays known in the
art.
[0199] Any screening technique known in the art can be used to
screen for TACI protein agonists or antagonists. The present
invention contemplates screens for small molecule ligands or ligand
analogs and mimics, as well as screens for the natural ligand(s)
that bind to and agonize or antagonize the TACI protein in vivo.
For example, natural products libraries can be screened using
assays of the invention for molecules that agonize or antagonize
the TACI protein activity, or that bind to the extracellular domain
or cytoplasmic domain of TACI.
[0200] Knowledge of the primary sequence of the TACI protein, and
the similarity of that sequence with proteins of known function,
can provide an initial clue as the inhibitors or antagonists of the
protein. Identification and screening of antagonists is further
facilitated by determining structural features of the protein,
e.g., using X-ray crystallography, neutron diffraction, nuclear
magnetic resonance spectrometry, and other techniques for structure
determination. These techniques provide for the rational design or
identification of agonists and antagonists.
[0201] Another approach uses recombinant bacteriophage to produce
large libraries. Using the "phage method" [Scott and Smith (1990)
Science 249:386-390; Cwirla, et al. (1990) Proc. Natl. Acad. Sci.
87:6378-6382; Devlin et al. (1990) Science 249:404-406], very large
libraries can be constructed (10.sup.6-10.sup.8 chemical entities).
A second approach uses primarily chemical methods, of which the
Geysen method [Geysen et al. (1986) Molecular Immunology
23:709-715; Geysen et al. (1987) J. Immunologic Method 102:259-274]
and the method of Fodor et al. [(1991) Science 251:767-773] are
examples. Furka et al. [14th International Congress of
Biochemistry, Volume 5, Abstract FR:013 (1988); Furka (1991) Int.
J. Peptide Protein Res. 37:487-493], Houghton [U.S. Pat. No.
4,631,211, issued December 1986] and Rutter et al. [U.S. Pat. No.
5,010,175, issued Apr. 23, 1991] describe methods to produce a
mixture of peptides that can be tested as agonists or
antagonists.
[0202] In another aspect, synthetic libraries [Needels et al.
(1993) Proc. Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al.
(1993) Proc. Natl. Acad. Sci. USA 90:10922-10926; Lam et al.,
International Patent Publication No. WO 92/00252; Kocis et al.,
International Patent Publication No. WO 9428028, each of which is
incorporated herein by reference in its entirety], and the like can
be used to screen for the TACI protein ligands according to the
present invention.
[0203] Alternatively, assays for binding of soluble ligand to cells
that express recombinant forms of the TACI N-Terminal extracellular
domain can be performed. The soluble ligands can be provided
readily as recombinant or synthetic polypeptides.
[0204] The screening can be performed with recombinant cells that
express TACI, or a fragment thereof, or alternatively, using
purified protein, e.g., produced recombinantly, as described above.
For example, the ability of labeled, soluble or solubilized TACI
fragment to bind ligand can be used to screen libraries, as
described in the foregoing references.
[0205] Screening For Novel Immunosuppressant Drugs
[0206] Certain diseases result from over activation of the
B-lymphocyte response. One example is Systemic Lupus Erythematosus
(SLE), in which antibodies are created that react with antigens
(proteins, DNA, etc.) naturally present in the patient. The
antibodies form complexes with the antigens, and circulate as
reactive protein agglomerates. These complexes deposit in different
organs and lead to the many symptoms of SLE, including kidney
failure, neurologic symptoms, and death. Current treatments include
the use of relatively non-specific immunosuppressants such as
cyclosporin A or steroids which suppress responses in both T and B
cells. Although they can often effectively treat SLE and similar
diseases, there is a significant risk of over-immunosuppression, in
which the patient develops serious infections because of lack of
functioning T-cells. A new immunosuppressant drug that selectively
blocks the action of B-lymphocytes, while leaving T-cells intact to
protect patients from viral pathogens would be extremely useful to
treat these diseases. Therefore, TACI-1 can be used as a novel tool
for developing immunosuppressant drugs specific for
B-lymphocytes.
[0207] The TACI protein is not naturally present in mature
T-lymphocytes or Jurkat T cells, a cell line that has phenotypic
characteristics of mature T-cells. Indeed, the highest level of
expression of the TACI protein is on peripheral B lymphocytes,
whereas peripheral T-cells do not express the TACI protein.
However, Jurkat T cells can be transfected with a TACI expression
plasmid and the TACI expressed can be readily stimulated by cross
linking to a TACI specific antibody. This stimulation leads to the
activation of a pair of second messenger pathways that are both
necessary and sufficient to stimulate the early T-cell
transcription factor NF-AT.
[0208] In a particular embodiment Jurkat T cells are transfected
with TACI-1 expression plasmid and a NF-AT-reporter plasmid. Jurkat
T cells naturally express the T-cell receptor (TCR). The cells are
stimulated by the addition of antibodies to TACI-1, antibodies to
TCR, or antibodies to both. Candidate drugs are mixed with the
Jurkat T cells and the effect of these drugs is determined. Phage
and chemical libraries described above may be used as sources for
drug candidates.
[0209] These candidate drugs are then applied in a parallel
experiment in which antibodies to TCR are used in place of the
antibodies to TACI-1. If the candidate drug has no effect on the
SEAP signal stimulation due to the antibody-dependent activation of
TCR, the candidate drug is identified as having selective
inhibition of the TACI-1 activated response. Such selected drugs
include that class of drugs which can selectively inhibit the
B-cell (antibody producing) response, while allowing T-cell
mediated (cellular) immunity to proceed. Such selected drugs may be
used to treat the autoimmune diseases described above. Because
TACI-1 activates lymphocytes by a novel mechanism (i.e. through
direct contact with CAML) it is likely that a significant number of
drugs are discoverable that can interfere with the CAML-dependent
pathway, yet leave normal signaling through the T-cell receptor
intact.
[0210] In a preferred embodiment of this type, the NF-AT-reporter
plasmid contains the SEAP reporter. This signal is used to measure
the degree of inhibition of activation. The SEAP reporter assay can
be scaled up so as to be performed by a robot screening apparatus.
Drugs that block the activation of TACI-1 but not TCR as measured
by the SEAP reporter assay are identified as having selective
inhibition of the TACI-1 activated response.
[0211] Alternatively, a phage library can be employed. Phage
libraries have been constructed which when infected into host E.
coli produce random peptide sequences of approximately 10 to 15
amino acids [Parmley and Smith (1988) Gene 73:305-318, Scott and
Smith (1990) Science 249:386-249]. Specifically, the phage library
can be mixed in low dilutions with permissive E. coli in low
melting point LB agar which is then poured on top of LB agar
plates. After incubating the plates at 37.degree. C. for a period
of time, small clear plaques in a lawn of E. coli will form which
represents active phage growth and lysis of the E. coli. A
representative of these phages can be absorbed to nylon filters by
placing dry filters onto the agar plates. The filters can be marked
for orientation, removed, and placed in washing solutions to block
any remaining absorbent sites. The filters can then be placed in a
solution containing, for example, a radioactive fragment of a TACI
protein containing the N-terminal extracellular domain e.g., for
human TACI-1 it is a peptide having the amino acid sequence of SEQ
ID NO:6. After a specified incubation period, the filters can be
thoroughly washed and developed for autoradiography. Plaques
containing the phage that bind to the radioactive N-terminal
extracellular domain can then be identified. These phages can be
further cloned and then retested for their ability to inhibit the
stimulation of TACI by an anti-TACI antibody while not inhibiting
the corresponding stimulation of TCR as described above.
[0212] Once the phages have been purified, the binding sequence
contained within the phage can be determined by standard DNA
sequencing techniques. Once the DNA sequence is known, synthetic
peptides can be generated which represents these sequences.
[0213] The effective peptide(s) can be synthesized in large
quantities for use in in vivo models and eventually in humans as
immunosuppressant drugs, for example. It should be emphasized that
synthetic peptide production is relatively non-labor intensive,
easily manufactured, quality controlled and thus, large quantities
of the desired product can be produced quite cheaply. Similar
combinations of mass produced synthetic peptides have recently been
used with great success [Patarroyo, (1990) Vaccine 10:175-178].
[0214] Therapeutic Methods and Compositions
[0215] The Transmembrane Activator CAML Interactor protein of the
present invention can activate two signals normally used to
initiate cell growth and division. This receptor is likely to be
involved in the neoplastic transformation of T or B lymphocytes in
lymphoma or leukemia. Therefore, dominant negative forms of TACI-1
are useful to suppress growth of such cancer cells. Alternatively,
TACI-1 over-stimulation can lead to programmed cell death. Taking
advantage of this property, leukemia or lymphomas with TACI-1 cell
surface expression may be induced to die by such over-stimulation.
Activation of the TACI protein with antibody or crosslinking may
activate an endogenous pathway leading to apoptosis. Binding with a
monomeric form of an antibody or analogous ligand can block the
TACI protein-associated endogenous pathway and interfere with
growth simulation.
[0216] Therapeutic Stimulation of TACI Activity.
[0217] As discussed above, the present invention advantageously
provides for selective stimulation of the immune system by
agonizing TACI activity. TACI agonists include the TACI ligand or
ligands discovered as described supra, and antibodies that
crosslink the receptor. Ligand agonists or antibody agonists can be
administered as described below for treatment of subjects in whom
immune stimulation, particularly of B cells, is desired.
[0218] B cell responses can be particularly important in fighting
infectious diseases, including, but not limited to, bacterial,
viral, protozoan, and parasitic infections. Antibodies against
infectious microorganisms can rapidly immobilize the organisms by
binding to antigen, followed by complement attack or cell mediated
attack. Thus, a TACI agonist of the invention can be provided to a
subject who is suffering from an infection to boost humoral immune
responses. TACI agonists may be particularly useful for boosting
immune responses after vaccination, during challenge with the
infectious organism. Thus, subjects particularly at risk for
infectious diseases, such as influenza, can supplement a
vaccination or memory immunity with a TACI agonist during the flu
season. Comparable immune boosting could be used with subjects who
are entering or reentering an area with an endemic infectious
disease, such as malaria.
[0219] In addition, B cell responses may be important in amplifying
immune responses to tumors that express tumor-specific antigens.
Thus, a TACI agonist may be provided where endogenous anti-tumor
antibodies are detected. Indeed, B cells or plasma cells expressing
anti-tumor cell surface immunoglobulin can be selected, such as by
panning, and transduced with TACI to allow for an amplification of
their antibody production.
[0220] In addition to amplifying beneficial immune responses, the
present TACI agonists, e.g., ligands and antibodies, can be used to
over-stimulate cells, such as B cells tumors (multiple myelomas,
lymphomas, and leukemias), immature T cell tumors (leukemias and
thymomas), and autoimmune or inflammatory cells and induce
apoptosis in such cells, thereby reducing or eliminating the cancer
or autoimmune/inflammatory condition.
[0221] Therapies for Boosting Cellular Immune Responses.
[0222] Although TACI is found in only a subset of immature T cells,
mature T cells can be transfected or transduced in vivo or ex vivo
to express a functional TACI receptor, to amplify cellular immune
responses. Preferably, tumor infiltrating cells are selected for
such boosting, and reintroduced into the subject to more vigorously
fight the tumor.
[0223] Therapeutic Methods by Antagonizing TACI Activity.
[0224] As discussed above, the present invention contemplates
inhibiting TACI activity by various means, including but not
limited to use of the free N-terminal extracellular domain;
expression of a non-functional extracellular domain lacking a
signal transduction domain, e.g., GPI-linked N-terminal TACI; use
of antisense or ribozyme technologies to suppress expression of
TACI; and use of TACI antagonist ligands or antibodies.
[0225] Suppression of TACI activity is useful for treating
undesirable immune responses, including autoimmune and inflammatory
diseases, transplantation rejection, and graft-versus host (GVH)
disease. Autoimmune and inflammatory diseases include immune
complex-induced vasculitis [Cochrane, (1984) Springer Seminar
Immunopathol. 7:263], glomerulonephritis [Couser et al. (19815)
Kidney Inst. 29:879], hemolytic anemia [Schreiber and Frank (1972)
J. Clin. Invest. 51:575], myasthenia gravis [Lennon, et al. (1978)
J. Exp. Med. 147:973; Biesecker and Gomez (1989) J. Immunol.
142:2654], type II collagen-induced arthritis [Watson and Townes
(1985) J. Exp. Med. 162:1878]; experimental allergic and hyperacute
xenograft rejection [Knechtle et al. (1985) J. Heart Transplant
4(5):541; Guttman (1974) Transplantation 17:383; Adachi et al.
(1987) Trans. Proc. 19(1):1145]; rheumatoid arthritis, and systemic
lupus erythematosus (SLE).
[0226] In another embodiment, where a lymphocyte cancer such as
myeloma, lymphoma, or leukemia expresses TACI, and TACI contributes
to cancer growth, use of a TACI antagonist of the invention can be
used to suppress the cancer cell growth.
[0227] Accordingly, a component of a therapeutic composition such
as a polyvalent or mono-valent antibody of the present invention
may be introduced parenterally, transmucosally, e.g., orally,
nasally, or rectally, or transdermally. Preferably, administration
is parenteral, e.g., via intravenous injection, and also including,
but is not limited to, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration.
[0228] In another embodiment, the therapeutic compound can be
delivered in a vesicle, in particular a liposome [see Langer (1990)
Science 249:1527-1533; Treat et al. (1989) Liposomes in the Therapy
of Infectious Disease and Cancer, Lopez-Berestein and Fidler
(eds.), Liss: New York, pp. 353-365; Lopez-Berestein, ibid., pp.
317-327; see generally ibid.]. To reduce its systemic side effects,
this may be a preferred method for introducing TACI.
[0229] In yet another embodiment, the therapeutic compound can be
delivered in a controlled release system. For example, an agonist
or antagonist to the transmembrane binding protein of the present
invention, such as an antibody, may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used [see Langer, supra; Sefton (1987)
CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery
88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574]. In another
embodiment, polymeric materials can be used [see (1974) Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Press: Boca Raton, Fla.; (1984) Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New York; Ranger and Peppas (1983) J. Macromol. Sci. Rev. Macromol.
Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et
al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg.
71:105]. In yet another embodiment, a controlled release system can
be placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose [see, e.g.,
Goodson (1984) Medical Applications of Controlled Release, supra,
vol. 2, pp. 115-138]. Preferably, a controlled release device is
introduced into a subject in proximity of the site of inappropriate
immune activation or a tumor.
[0230] Other controlled release systems are discussed in the review
by Langer (1990) Science 249:1527-1533.
[0231] In a further aspect, recombinant cells that have been
transformed with the TACI protein gene and that express high levels
of the polypeptide can be transplanted in a subject in need of the
TACI protein. Preferably autologous cells transformed with TACI
protein are transplanted to avoid rejection.
[0232] The therapeutic compounds of the present invention can be
delivered by intravenous, intraarterial, intraperitoneal,
intramuscular, or subcutaneous routes of administration.
Alternatively, these compounds, properly formulated, can be
administered by nasal or oral administration. A constant supply of
these therapeutic compounds can be ensured by providing a
therapeutically effective dose (i.e., a dose effective to induce
metabolic changes in a subject) at the necessary intervals, e.g.,
daily, every 12 hours, etc. These parameters will depend on the
severity of the disease condition being treated, other actions,
such as diet modification, that are implemented, the weight, age,
and sex of the subject, and other criteria, which can be readily
determined according to standard good medical practice by those of
skill in the art.
[0233] A subject in whom administration of these therapeutic agents
is an effective therapeutic regiment for an immunodeficiency
disease is preferably a human, but can be any animal. Thus, as can
be readily appreciated by one of ordinary skill in the art, the
methods and pharmaceutical compositions of the present invention
are particularly suited to administration to any animal,
particularly a mammal, and including, but by no means limited to,
domestic animals, such as feline or canine subjects, farm animals,
such as but not limited to bovine, equine, caprine, ovine, and
porcine subjects, wild animals (whether in the wild or in a
zoological garden), research animals, such as mice, rats, rabbits,
goats, sheep, pigs, dogs, cats, etc., avian species, such as
chickens, turkeys, songbirds, etc., i.e., for veterinary medical
use.
[0234] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broad scope of the
invention.
EXAMPLE
Calcium Signaling by a Lymphocyte Surface Receptor Mediated through
CAML
Introduction
[0235] Ca.sup.2+ influx is a key regulator of antigen-stimulated
lymphocyte activation [Imboden et al. (1985) Immunol. 134:663-665];
[Crabtree & Clipstone (1994) Annu. Rev. Biochem. 63:1045-1083];
[Weiss & Littman (1994) Cell 76:263-274]. The CAML protein has
been identified as a regulator of Ca.sup.2+ signaling that is
necessary but not sufficient for the activation of lymphocyte
transcription factor, NF-AT (Bram & Crabtree, 1994, supra). The
location of CAML in cytoplasmic vesicles is consistent with it
regulating Ca.sup.2+ influx by modulating intracellular Ca.sup.2+
release. Here a novel human CAML-interacting receptor expressed by
B lymphocytes that acts as a cell-surface signaling molecule is
disclosed. This receptor, TACI (Transmembrane Activator CAML
Interactor), initiates Ca.sup.2+-dependent activation of NF-AT when
cross linked with an antibody. The signal can be blocked by a
dominant negative mutant of CAML. As shown herein, the TACI protein
also can independently activate the AP-1 transcription factor, thus
providing both signals required for lymphocyte activation. The TACI
protein initiates a novel signal transduction mechanism directly
linking cell surface stimuli to the intracellular signaling
molecule, CAML, and thereby defines a new class of
lymphocyte-specific cell surface receptors that modulate the immune
response. In addition, the TACI protein is a novel tool that can be
used to regulate the immune system in either a positive or a
negative direction. TACI-1 is the human homologue of TACI
illustrated in the instant example.
Materials And Methods
[0236] Molecular Cloning and Screening.
[0237] A human B-lymphocyte cDNA library is screened by the
two-hybrid system [Fields & Song (1989) Nature 340:245-246];
[Durfee et al. (1993) Genes Dev. 7:555-569], with the full coding
region of CAML used as bait. The human CAML cDNA is inserted into
the yeast two-hybrid bait vector pAS1. This construct directs the
expression of a GAL4-DNA binding domain fused to the entire protein
sequence of CAML. A B-lymphocyte library in plasmid pACT is
transformed into yeast Y153 and potential interacting plasmids are
identified by growth of colonies on media lacking histidine and
containing 3-amino triazole.
[0238] One clone (TACI-1), out of eight primary positives was
identified. The TACI-1 cDNA is subcloned into a mammalian
expression plasmid which adds an epitope tag to the amino-terminal
end of the expressed protein. This construct is then transfected
into the Jurkat T-lymphocyte cell line, COS cells, or NIH3T3 cells.
In each case, cell surface expression of the TACI protein is
demonstrated. The orientation of the protein is with the N-terminus
outside the cell, as the epitope is available for reaction with a
specific antibody even without permeablizing the cell membrane.
This result facilitated the functional studies described
herein.
[0239] Secondary screening relied on enforced over expression of
positive clones in Jurkat T cells, and assaying for NF-AT
activation. (Bram et al. (1993), supra) Jurkat T cells transiently
transfected with the tagged-TACI-1 construct and an NFAT-reporter
plasmid are incubated in medium containing the monoclonal
epitope-specific antibody. To maximize cross-linking of TACI-1, the
antibodies are bound to beads before addition to the cell
suspensions. Following a 24 hour incubation, the activity of the
NFAT-reporter is determined. A dramatic induction of NFAT reporter
activity is found when cells are stimulated in this manner. Control
transfections without the TACI-1 construct do not show activation
following such treatment. Likewise, transfection of an unrelated
cell surface molecule (CD8) followed by anti-CD8 stimulation did
not activate NFAT in these cells. The degree of activation was
70-80% of the maximal stimulation that could achieved in these
Jurkat T cells by addition of phorbol ester plus ionomycin.
[0240] After screening a multiple tissue Northern blot (Clontech)
with TACI-1 cDNA (excised from the yeast two-hybrid vector), an
independent TACI-1 clone is obtained from a human fetal spleen cDNA
library (Stratagene). The 5'-terminal coding region is confirmed by
rapid amplification of cDNA ends (RACE) using a `Marathon-ready`
human spleen cDNA library (Clontech), nested TACI-1-specific
primers (5'-TCTGAATTGTTTTCAACTTCTC-3' (SEQ ID NO:9) and
5'-CAGCAGAGGATCCCAGTACTGC- TC-3' (SEQ ID NO:10)), and Pfu
polymerase (Stratagene) according to the manufacturers'
recommendations.
[0241] Antisera.
[0242] cDNAs encoding the N-terminal 146 amino acid residues of
CAML and the N-terminal 151 residues of TACI-1 are each cloned into
a GST-fusion bacterial expression vector (Pharmacia). Rabbit
polyclonal antisera are raised against purified GST-fusion proteins
[Smith & Johnson (1988) Gene 67:31-40], and specific antibodies
are purified by immunoaffinity chromatography over the purified
proteins coupled to agarose (Pierce), using standard techniques.
Cross-linked anti-TACI-1 is prepared by incubating
immunoaffinity-purified polygonal anti-TACI-1 antibodies with
anti-rabbit IgG antibody-coupled magnetic beads (PerSeptive
Diagnostics).
[0243] Screen for Identifying Novel Immunosuppressant Drugs:
[0244] Jurkat T cells are transfected with TACI-1 expression
plasmid and a NF-AT-reporter plasmid. Jurkat T cells naturally
express the T-cell receptor (TCR). The cells are stimulated by the
addition of antibodies to TACI-1, antibodies to TCR, or antibodies
to both. Candidate drugs are mixed with the Jurkat T cells and the
effect of these drugs is determined.
[0245] The NF-AT-reporter plasmid contains the SEAP reporter. This
signal is used to measure the degree of inhibition of activation.
The SEAP reporter assay is scaled up so as to be performed by a
robot screening apparatus. Drugs that block the activation of
TACI-1 but not TCR as measured by the SEAP reporter assay are
identified as having selective inhibition of the TACI-1 activated
response.
Results and Discussion
[0246] Proteins that can interact with CAML are identified by using
a two-hybrid screen (Fields & Song (1989), supra); (Durfee et
al. (1993), supra) with CAML as bait. To determine whether one of
these identified CAML-binding proteins can affect Ca.sup.2+
signaling in T-cells, their ability to modulate the activity of the
Ca.sup.2+-dependent transcription factor NF-AT is examined [Truneh
et al. (1985) Nature 313:318-321]; [Verweij et al. (1990) J. Biol.
Chem. 265:15788-15795]; [Karttunen & Shastri (1991) Proc. Natl.
Acad. Sci. USA 88:3972-3976]; [Emmel et al. (1989) Science
246:1617-1620]. Enforced over expression of the two-hybrid clones
in Jurkat T-cells reveals that one clone (encoding the TACI-1
protein), replaced the requirement for Ca.sup.2+ influx, implying
that TACI-1 lies in the same signal pathway as CAML. Northern blot
analysis for TACI-1 mRNA demonstrates a 1.4 kb mRNA expressed only
in spleen, small intestine, thymus and peripheral blood lymphocytes
suggesting a limited expression of TACI-1 (FIG. 1). The pattern
observed is consistent with the expression of TACI-1 being
predominantly in peripheral blood cells, since peripheral blood
cells, and in particular lymphocytes, can be present in all of
these organs (including the Peyer's patches lining the small
intestine.) Furthermore, there is no detection of expression in
colon, testis, ovary, or prostate. In addition, the TACI-1 protein
is detected in all normal peripheral B lymphocytes using
specific-antibody staining. There is no detectable protein
expressed in peripheral T-lymphocytes, monocytes or
neutrophils.
[0247] Determination of the DNA sequence from both strands of the
DNA isolated reveals a complete open reading frame of 1325 base
pairs, which is predicted to encode a protein of 293 amino acids.
The deduced amino acid sequence of TACI-1 (FIG. 2A) includes a
single hydrophobic region (residues 167 to 186) (SEQ ID NO:8, which
is encoded by the nucleic acid sequence provided in SEQ ID NO:7),
that has features of a membrane spanning segment (FIG. 2B).
Analysis of the protein sequence by the method of Sipos [Sipos
& von Heijne (1993) Eur. J. Biochem. 213:1333-1340]; [Claros
& von Heijne (1994) Comput. Appl. Biosci. 10:685-686], predicts
extracellular exposure for the N-terminus and cytoplasmic exposure
for the C-terminus. Although TACI-1 lacks an N-terminal signal
sequence, the presence of an upstream stop codon indicates that the
complete open reading frame is contained within the clone. TACI-1
is relatively rich in cysteine residues, but there is no
significant sequence similarity or homology to any other disclosed
protein. A search for Prosite motifs in TACI-1 reveals one
TNFR_NGFR motif [Bairoch (1993) Nucleic Acids Res. 21:3097-3103]
(residues 33-71) N-terminal to a putative transmembrane region,
which consists of
C-x(4,6)-[FYH]-x(5,10)-C-x(0,2)-C-x(2,3)-C-x(7,11)-C-x(4,6)-[DNEQSKP]-x(2-
)-C (SEQ ID NO:11) in the N-terminal half of the protein. This
motif is found in a number of proteins, some of which are receptors
for growth factors. Some of these proteins have one copy of this
motif. A comparison of the TACI-1 protein sequence with itself
reveals a significant repeat between the TNFR_NGFR motif at
residues 33-66 and residues 70-104. This analysis drew attention to
the presence of two TNFR-type cysteine-rich domains encompassing
these regions that indicate that TACI-1 is a member of the
superfamily of TNFR receptors. (FIG. 2C)
[0248] To confirm that TACI-1 is a transmembrane protein, its
expression on Jurkat T cells transfected with a TACI-1-encoding
plasmid using flow cytometry was examined. Cells transfected with
TACI-1 show surface staining with rabbit polyclonal antibodies
raised against a fusion protein that includes the N-terminal 12
kilodalton portion of TACI-1 (FIG. 3A). Additional evidence that
TACI-1 is localized at the cell surface is derived from
immunofluorescence microscopy, where surface staining of intact
cells transfected with an N-terminal FLAG-epitope-tagged TACI-1
expression plasmid is observed (FIG. 3B). Since the N-terminus of
TACI-1 is extracellular in the absence of a cleaved signal
sequence, it is a type III transmembrane protein [Wilson-Rawls et
al., (1994) Virology 201:66-76].
[0249] To assess the effect of TACI-1 on NF-AT activity in T-cells,
the protein is transiently expressed in TAg Jurkat T cells with a
secreted alkaline phosphatase reporter driven by the NF-AT-binding
sequences from the IL-2 promoter (Bram & Crabtree (1994),
supra); [Fiering et al. (1990) Genes Dev. 4:1823-1834]; (Bram et
al. (1993), supra). TACI-1 over expression can partially replace
the requirement for ionomycin in this assay. The addition of
anti-TACI-1 antibodies to the cells further increased NF-AT
activation (more than twofold, see FIG. 4A), demonstrating that
TACI-1 responds to cross-linking at the cell surface. The degree of
NF-AT activation varies in different experiments due to
transfection efficiency but is typically 40-50% of the maximal
response to the corresponding treatment of the cells with PMA plus
ionomycin. TACI-1-mediated NF-AT activation is dependent on
calcineurin, as is demonstrated by the loss of NF-AT activity in
the presence of an immunosuppressive drugs, such as Cyclosporin A
or FK506 [Friedman & Weissman (1991) Cell 66:799-806]; (Lui et
al. (1991), supra) (FIG. 4B).
[0250] To examine the requirement for Ca.sup.2+ influx in
TACI-1-mediated activation of NF-AT, extracellular calcium can be
removed by the addition of increasing concentrations of EGTA. This
results in the inhibition of TACI-1-mediated NF-AT activation, as
has been shown previously for T-cell receptor-mediated activation
(FIG. 4C). As a control, the effect of expressing in the cells, a
constitutively active, calcium-independent mutant of calcineurin A
[Hubbard & Klee (1989) Biochemistry 28:1868-74]; (O'Keefe et
al. (1992), supra); (Clipstone & Crabtree (1993), supra) is
examined. As expected, in these cells NF-AT activation is seen even
in the presence of EGTA (FIG. 4C). Thus, in T-cells, TACI-1
mediates the calcineurin-dependent aspect of the activation of
NF-AT by initiating the influx of extracellular Ca.sup.2+ (most
likely through the capacitative Ca.sup.2+ influx pathway following
the depletion of intracellular stores [Putney & Bird (1993)
Cell 75:199-201]; Hoth & Prenner (1993) Physiol. 465:359-386];
[Zweifach & Lewis (1993) Proc. Natl. Acad. Sci. USA
90:6295-6299]; [Premack et al. (1994) J. Immunol.
152:5226-5240).
[0251] Activation of NF-AT by CAML requires exogenous stimulation
of protein kinase C by the addition of phorbol ester (Bram &
Crabtree (1994), supra). Antibody-cross linked TACI-1, however, is
able to activate NF-AT in the absence of either PMA or ionomycin
(FIG. 4B, solid bars). Experiments examining the activation of an
AP-1 reporter by the over expression of TACI-1 shows that AP-1
activation is elevated (over four-fold) in TACI-1-transfected
Jurkat T cells. This effect can be further enhanced with the
addition of cross-linked anti-TACI-1 antibodies (FIG. 4D).
Therefore, TACI-1 initiates Ca.sup.2+ influx, which in turns
activates calcineurin, as well as activates the AP-1 pathway
following stimulation, thereby mediating the fulfillment of both
requirements for the activation of NF-AT.
[0252] Further confirmation that TACI-1 interacts with CAML can be
demonstrated by their specific interaction in a two-hybrid reverse
swap experiment (Durfee et al. (1993), supra) (FIG. 5A). To define
the critical amino acid residues involved in the interaction,
deletion mutants of both TACI-1 and CAML are tested for their
ability to physically associate (FIG. 5A). The C-terminal 126 amino
acids of TACI-1 are found to be sufficient for binding to the
N-terminal 146 amino acids of CAML. Additional evidence for the in
vivo association of TACI-1 with CAML is provided by experiments in
which full length CAML and a mutant comprising the 146 N-terminal
amino acid residues of CAML are co-immunoprecipitated with TACI-1
from cell lysates (FIG. 5B). Therefore, it may be concluded that
the cytoplasmic C-terminal tail of TACI-1 can physically associate
with the N-terminal half of CAML.
[0253] To examine whether TACI-1 signaling depends on the
association of TACI-1 with CAML, the interacting domain of CAML
(residues 1-146) is tested to determine if it can inhibit
TACI-1-induced NF-AT activation in a dominant negative fashion.
Co-transfection of the mutant CAML(1-146) expression plasmid
completely eliminates TACI-1-induced NF-AT activation in Jurkat T
cells. On the other hand, there is no inhibitory effect on PMA plus
ionomycin-induced NF-AT activity, thus ruling out a nonspecific
toxic effect. Co-expression of CAML(1-146) also does not influence
the accumulation of TACI-1 protein as detected by Western blot
analysis (FIG. 5C). CAML(1-146) lacks the hydrophobic transmembrane
domains that are required for Ca.sup.2+ influx activity [Holloway
& Bram (1996) Biol. Chem. 271:8549-8552]. Hence the elimination
of NF-AT-inducing activity in these cells can be attributed to
binding of the CAML(1-146) fragment to the intracellular C-terminal
portion of TACI-1, preventing association with endogenous
full-length CAML.
[0254] CAML is an integral membrane protein localized to
cytoplasmic vesicles (Bram & Crabtree (1994), supra). Analysis
of deletion mutants has shown that hydrophobic domains in the
C-terminal half of the protein are essential for activity (Holloway
& Bram (1996), supra), and that the hydrophilic N-terminal half
of the protein may have a regulatory role. Trypsin digestion
experiments further demonstrated that the N-terminal half of the
molecule is cytoplasmic. Here, the interaction between TACI-1 and
CAML is required for TACI-1-mediated NF-AT activation in Jurkat T
cells is demonstrated. Taken together, these data indicate that a
physical interaction between TACI-1 in the plasma membrane and
intracellular CAML-containing vesicles can initiate a calcium
influx signal (FIG. 5D). These findings provide the first evidence
for direct communication between cell surface receptors and
intracellular organelles in lymphocytes. This mechanism may be
somewhat analogous to the dihydropyridine-ryanodine receptor model
in muscle cells, in which stimulation of one molecule can directly
modulate the activity of the other [Marty et al. (1994) Proc. Natl.
Acad. Sci. USA 91:2270-2274]; [Sham et al. (1995) Proc. Natl. Acad.
Sci. USA 92:121-125]; [Nakai et al. (1996) Nature 380:72-75].
[0255] Other cell surface proteins have been shown to activate
lymphocyte function, including the CD3 T-cell receptor, CD2, CD20,
and Thy-1. These proteins have no sequence-homology with TACI-1,
and it is likely that they play different roles from each other,
either in terms of response to different extracellular signals,
and/or in terms of developmental stage of expression on
lymphocytes. TACI-1 must also play a role in the modulation of the
function of lymphocytes in alternate and/or co-stimulatory
pathways. Thus, in addition to defining a new signaling mechanism,
TACI-1 is a novel lymphocyte-specific receptor capable of
activating T-cells.
[0256] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0257] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
11 1 1377 DNA Homo sapiens 1 agcatcctga gtaatgagtg gcctgggccg
gagcaggcga ggtggccgga gccgtgtgga 60 ccaggaggag cgctttccac
agggcctgtg gacgggggtg gctatgagat cctgccccga 120 agagcagtac
tgggatcctc tgctgggtac ctgcatgtcc tgcaaaacca tttgcaacca 180
tcagagccag cgcacctgtg cagccttctg caggtcactc agctgccgca aggagcaagg
240 caagttctat gaccatctcc tgagggactg catcagctgt gcctccatct
gtggacagca 300 ccctaagcaa tgtgcatact tctgtgagaa caagctcagg
agcccagtga accttccacc 360 agagctcagg agacagcgga gtggagaagt
tgaaaacaat tcagacaact cgggaaggta 420 ccaaggattg gagcacagag
gctcagaagc aagtccagct ctcccggggc tgaagctgag 480 tgcagatcag
gtggccctgg tctacagcac gctggggctc tgcctgtgtg ccgtcctctg 540
ctgcttcctg gtggcggtgg cctgcttcct caagaagagg ggggatccct gctcctgcca
600 gccccgctca aggccccgtc aaagtccggc caagtcttcc caggatcacg
cgatggaagc 660 cggcagccct gtgagcacat cccccgagcc agtggagacc
tgcagcttct gcttccctga 720 gtgcagggcg cccacgcagg agagcgcagt
cacgcctggg acccccgacc ccacttgtgc 780 tggaaggtgg gggtgccaca
ccaggaccac agtcctgcag ccttgcccac acatcccaga 840 cagtggcctt
ggcattgtgt gtgtgcctgc ccaggagggg ggcccaggtg cataaatggg 900
ggtcagggag ggaaaggagg agggagagag atggagagga ggggagagag aaagagaggt
960 ggggagaggg gagagagata tgaggagaga gagacagagg aggcagaaag
ggagagaaac 1020 agaggagaca gagagggaga gagagacaga gggagagaga
gacagagggg aagagaggca 1080 gagagggaaa gaggcagaga aggaaagaga
caggcagaga aggagagagg cagagaggga 1140 gagaggcaga gagggagaga
ggcagagaga cagagaggga gagagggaca gagagagata 1200 gagcaggagg
tcggggcact ctgagtccca gttcccagtg cagctgtagg tcgtcatcac 1260
ctaaccacac gtgcaataaa gtcctcgtgc ctgctgctca cagcccccga gagcccctcc
1320 tcctggagaa taaaaccttt ggcagctgcc cttcctcaaa aaaaaaaaaa aaaaaaa
1377 2 293 PRT Homo sapiens 2 Met Ser Gly Leu Gly Arg Ser Arg Arg
Gly Gly Arg Ser Arg Val Asp 1 5 10 15 Gln Glu Glu Arg Phe Pro Gln
Gly Leu Trp Thr Gly Val Ala Met Arg 20 25 30 Ser Cys Pro Glu Glu
Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met 35 40 45 Ser Cys Lys
Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys Ala Ala 50 55 60 Phe
Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp 65 70
75 80 His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly Gln
His 85 90 95 Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu Arg
Ser Pro Val 100 105 110 Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg Ser
Gly Glu Val Glu Asn 115 120 125 Asn Ser Asp Asn Ser Gly Arg Tyr Gln
Gly Leu Glu His Arg Gly Ser 130 135 140 Glu Ala Ser Pro Ala Leu Pro
Gly Leu Lys Leu Ser Ala Asp Gln Val 145 150 155 160 Ala Leu Val Tyr
Ser Thr Leu Gly Leu Cys Leu Cys Ala Val Leu Cys 165 170 175 Cys Phe
Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg Gly Asp Pro 180 185 190
Cys Ser Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala Lys Ser 195
200 205 Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr Ser
Pro 210 215 220 Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu Cys
Arg Ala Pro 225 230 235 240 Thr Gln Glu Ser Ala Val Thr Pro Gly Thr
Pro Asp Pro Thr Cys Ala 245 250 255 Gly Arg Trp Gly Cys His Thr Arg
Thr Thr Val Leu Gln Pro Cys Pro 260 265 270 His Ile Pro Asp Ser Gly
Leu Gly Ile Val Cys Val Pro Ala Gln Glu 275 280 285 Gly Gly Pro Gly
Ala 290 3 321 DNA Homo sapiens 3 aagaagaggg gggatccctg ctcctgccag
ccccgctcaa ggccccgtca aagtccggcc 60 aagtcttccc aggatcacgc
gatggaagcc ggcagccctg tgagcacatc ccccgagcca 120 gtggagacct
gcagcttctg cttccctgag tgcagggcgc ccacgcagga gagcgcagtc 180
acgcctggga cccccgaccc cacttgtgct ggaaggtggg ggtgccacac caggaccaca
240 gtcctgcagc cttgcccaca catcccagac agtggccttg gcattgtgtg
tgtgcctgcc 300 caggaggggg gcccaggtgc a 321 4 107 PRT Homo sapiens 4
Lys Lys Arg Gly Asp Pro Cys Ser Cys Gln Pro Arg Ser Arg Pro Arg 1 5
10 15 Gln Ser Pro Ala Lys Ser Ser Gln Asp His Ala Met Glu Ala Gly
Ser 20 25 30 Pro Val Ser Thr Ser Pro Glu Pro Val Glu Thr Cys Ser
Phe Cys Phe 35 40 45 Pro Glu Cys Arg Ala Pro Thr Gln Glu Ser Ala
Val Thr Pro Gly Thr 50 55 60 Pro Asp Pro Thr Cys Ala Gly Arg Trp
Gly Cys His Thr Arg Thr Thr 65 70 75 80 Val Leu Gln Pro Cys Pro His
Ile Pro Asp Ser Gly Leu Gly Ile Val 85 90 95 Cys Val Pro Ala Gln
Glu Gly Gly Pro Gly Ala 100 105 5 498 DNA Homo sapiens 5 atgagtggcc
tgggccggag caggcgaggt ggccggagcc gtgtggacca ggaggagcgc 60
tttccacagg gcctgtggac gggggtggct atgagatcct gccccgaaga gcagtactgg
120 gatcctctgc tgggtacctg catgtcctgc aaaaccattt gcaaccatca
gagccagcgc 180 acctgtgcag ccttctgcag gtcactcagc tgccgcaagg
agcaaggcaa gttctatgac 240 catctcctga gggactgcat cagctgtgcc
tccatctgtg gacagcaccc taagcaatgt 300 gcatacttct gtgagaacaa
gctcaggagc ccagtgaacc ttccaccaga gctcaggaga 360 cagcggagtg
gagaagttga aaacaattca gacaactcgg gaaggtacca aggattggag 420
cacagaggct cagaagcaag tccagctctc ccggggctga agctgagtgc agatcaggtg
480 gccctggtct acagcacg 498 6 166 PRT Homo sapiens 6 Met Ser Gly
Leu Gly Arg Ser Arg Arg Gly Gly Arg Ser Arg Val Asp 1 5 10 15 Gln
Glu Glu Arg Phe Pro Gln Gly Leu Trp Thr Gly Val Ala Met Arg 20 25
30 Ser Cys Pro Glu Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met
35 40 45 Ser Cys Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys
Ala Ala 50 55 60 Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly
Lys Phe Tyr Asp 65 70 75 80 His Leu Leu Arg Asp Cys Ile Ser Cys Ala
Ser Ile Cys Gly Gln His 85 90 95 Pro Lys Gln Cys Ala Tyr Phe Cys
Glu Asn Lys Leu Arg Ser Pro Val 100 105 110 Asn Leu Pro Pro Glu Leu
Arg Arg Gln Arg Ser Gly Glu Val Glu Asn 115 120 125 Asn Ser Asp Asn
Ser Gly Arg Tyr Gln Gly Leu Glu His Arg Gly Ser 130 135 140 Glu Ala
Ser Pro Ala Leu Pro Gly Leu Lys Leu Ser Ala Asp Gln Val 145 150 155
160 Ala Leu Val Tyr Ser Thr 165 7 60 DNA Homo sapiens 7 ctggggctct
gcctgtgtgc cgtcctctgc tgcttcctgg tggcggtggc ctgcttcctc 60 8 20 PRT
Homo sapiens 8 Leu Gly Leu Cys Leu Cys Ala Val Leu Cys Cys Phe Leu
Val Ala Val 1 5 10 15 Ala Cys Phe Leu 20 9 22 DNA Artificial
Sequence Primer Sequence 9 tctgaattgt tttcaacttc tc 22 10 24 DNA
Artificial Sequence Primer Sequence 10 cagcagagga tcccagtact gctc
24 11 48 PRT Artificial Sequence TNFR_NGFR motif 11 Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35
40 45
* * * * *