Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR REGULATING T CELL SUBSETS BY
MODULATING TRANSCRIPTION FACTOR ACTIVITY
Back~round of the Invention
CD4+ T helper cells are not a homogeneous population but can be divided on the
basis of cytokine secretion into at least two subsets terrned T helper typc 1 (Thl) and T
helper type 2 (Th2) (see e.g, Mosmann, T.R. et al. (1986) J. Immunol. 136:2348 2357;
Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251; Seder, R.A. and Paul, W.E. (1994)
Ann. Rev. Immunol. 12:635 673). Thl cells secrete interleukin-2 (IL-2) and interferon-~
(IFN-~) while Th2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin- 10
(IL-10) and interleukin-13 (IL-13). Both subsets produce cytokines such as tumornecrosis factor (TNF) and granulocyte/macrophage-colony stimulating factor (GM-
CSF). In addition to their different pattern of cytokine expression, Thl and Th2 cells are
thought to have differing functional activities. For example, Thl cells are involved in
inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in
providing efficient "help" to B Iymphocytes and stimulating production of IgGl and IgE
antibodies.
There is now abundant evidence that the ratio of Thl to Th2 cells is highly
relevant to the outcome of a wide array of immunologically-me~ te~l clinical diseases
including autoimmune, allergic and infectious ~li.ce~ec For example, in experimental
leishm~ni~ infections in mice, ~nim~1s that are resistant to infection mount
predominantly a Thl response, whereas ~3nim~l~ that are susceptible to progressive
infection mount predomin~ntly a Th2 response (Heinzel, F.P., et al. (1989) J: Exp. Med.
169:59 72; Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1 :A58-A61). In murine schistosomiasis, a Thl to Th2 switch is observed coincident with the
release of eggs into the tissues by female parasites and is associated with a worsening of
the disease condition (Pearce, E. J., et al. ( 1991) J. Exp. Med. 173: 159- 166; Grzych, J-
M., etal. (1991)~. Immunol. 141:1322 1327; Kullberg, M.C., etal. (1992)J. Immunol.
148:3264-3270). Many human ~ eaces, including chronic infections (such as with
human imrnunodei~lciency virus (HIV) and tuberculosis) and certain metastatic
carcinomas, also are characterized by a Thl to Th2 switch (see e.g., Shearer, G.M. and
Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G.M.
(1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J. Clin. Invest.91:1005-1010; Pisa, P., et al. (1992) Proc. Nall. Acad. Sci. USA 89:7708-7712; Fauci,
A.S. (1988) Science 239:617-623). Furthermore, certain autoimmune diseases have
been shown to be associated with a predominant Thl response. For example, patients
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with rheumatoid arthritis have predominantly Thl cells in synovial tissue (Simon, A.K.,
etal. (1994) Proc. Natl. Acad Sci. USA 91:8562-8566) and experimental autoimmuneencephalomyelitis (EAE) can be induced by autoreactive Thl cells (Kuchroo, V.K., et
al. (1993) J. Immunol 151:4371-4381).
S The ability to alter or manipulate ratios of Thl and Th2 subsets requires an
understanding of the mech~ni.cm.c by which the differentiation of CD4 T helper
precursor cells (Thp), which secrete only IL-2, choose to become Thl or Th2 effector
cells. It is clear that the cytokines themselves are potent Th cell inducers and form an
autoregulatory loop (see e.g, Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251; Seder,
R.A. and Paul, W.E. (1994) Ann. Rev. Immunol. 12:635 673). Thus, IL-4 promotes the
differentiation of Th2 cells while preventing the differentiation of precursors into Thl
cells, while IL- 12 and IFN-y have the opposite effect. One possible means therefore to
alter Thl :Th2 ratios is to increase or decrease the level of selected cytokines. Direct
~q(lmini~tration of cytokines or antibodies to cytokines has been shown to have an effect
on certain diseases mediated by either Thl or Th2 cells. For example, ~rlmini.stration of
recombinant IL-4 or antibodies to IL-12 ameliorate EAE, a Thl-driven autoimmune
disease (see Racke; M.K. et al. (1994) J. Exp. Med. 180:1961-1966; and Leonard, J.P. et
al. (1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies cure the Th2-mediated
parasitic disease, Leishmania major (Sadick, M.D. et al. (1990) J. Exp. Med. 171: 115-
127). However, as therapeutic options, systemic ~mini~tration of cytokines or
antibodies may have unwanted side effects and, accordingly, alternative approaches to
manipulating Thl/Th2 subsets are still needed.
The molecular basis for the tissue-specific expression of IL-4 in Th2 cells, or any
T cell cytokine, has remained elusive. One possibility is the presence of repressor
proteins that selectively silence cytokines. Transcriptional silencing has been well
documented for bacteria, yeast and m~mm~ n genes. Examples include E. coli
thermoregulation genes (Goransson, M. et al. ~1990) Nature 344:682-685), yeast a2
mating type genes (Keleher, C.A. et al. (1988) Cell 53:927-936) and m~mm~ n MHC
class I and TcRa genes (Weisman, J.D. and Singer, D.S. (1991) Mol. Cell. Biol.
11 :4228-4234; Winoto, A. and Baltimore, D. (1989) Cell 59:649-655). Indeed, early
experiments involving injection of IL-2 genomic DNA into Xenopus oocytes suggested
the existence of a repressor protein for IL-2 in resting versus activated T cell extracts
(Mouzaki, A. et al. (1991) EMBO J. 10:1399-1406). These studies suggested that the
absence of IL-2 production in resting T cells was due to proteins that silenced the
transcription of IL-2 by interacting with negative elements in the IL-2 promoter.
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A second possibility is the existence of Th selective transactivators. Study of the
transcriptional elements of cytokine genes has provided insight into the regulation of
these cytokine genes. Analysis of the IL-4 cytokine promoter, for example, has revealed
functionally critical sites for several transcription factors including members of the NF-
S AT and AP-1 families (Rooney, J.W. et al. (1995) lmmunity 2:473-483; Szabo, S.J. et al.
(1993) Mol. Cell. Biol. 13:4793 4805). NF-AT is a multisubunit transcription complex
that contains a cyclosporin A sensitive cytoplasmic phosphoprotein and an inducible
nuclear component composed of AP-l family member proteins (Fl~n~n, W.M. et al.
(1991) Nature 352:803-807; Jain, J. et al. (1992) Nature 356:801-804). Purification and
cloning of NF-ATp revealed a region of limited sequence identity to the Rel Homology
Domain (RHD) of the NFKB family of transcription factors (McCaffrey, P.G. et al.(1993) Science 262:750-754). Subsequent cloning and sequencing ofthree related
genes, NF-ATc, NF-AT4/x/c3, and NF-AT3/c4 revealed similar domains. NF-AT
family members share approximately 70% sequence similarity within this domain and
approximately 18% sequence similarity to the RHD of the Rel/NFKB family of
transcription factors. Consistent with their very limited sequence similarity in the RHD,
there are marked differences in the behavior of NFKB and NF-AT proteins, and much
less is known about the pathways that mediate transcriptional regulation of NF-AT target
genes. However, considering that NF-AT family members can bind to and transactivate
the promoters of multiple cytokine genes including I~-2 and IL-4 (Rooney, J. et al.
(1995) Immunity 2:545-553; Szabo, S.J. et al. (1993) Mol. Cell. Biol. 13:4793 4805;
Fl~n~n, W.M. et al. (1991) Nature 352:803-807; Northrop, J.P. et al. (1994) Nature
369:497), NF-AT proteins are not likely to be directly responsible for metli~tin~ Thl- or
Th2-specific cytokine transcription.
Most, if not all, NF-AT binding sites in cytokine promoter regulatory regions are
accompanied by nearby sites that bind auxiliary transcription factors, usually members
of the AP- 1 family. It has been shown that NF-AT and AP- I proteins bind coordinately
and cooperatively and are required for full activity of the IL-2 and IL-4 promoters.
Different AP- 1 proteins, specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have
been shown to bind to these sites (Rao, A. et al. (1994) Immunol. Today 15:274-281;
Jain, J. et al. (1993) Nature 365:352-355; Boise, L.H. et al. (1993) Mol. Cell. Biol.
13:1911-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995)
Mol. Cell. Biol. 15:6299 6310). However, none ofthese AP-1 proteins is expressed in a
Thl- or Th2-specific manner and there is no evidence for the differential recruitment of
,~
AP-1 family members to the IL-2 or IL-4 composite sites (Rooney, J. et al. ( 1995) Mol.
Cell. Biol. 15 :6299 6310). Thus, neither NF-AT proteins nor the AP- l family members
, . . . ,. , .. . ., ~ .
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c-Jun, c-Fos, Fra- 1, Fra-2, Jun B and Jun D can account for the tissue-specifictranscription of IL-4 in Th2 cells.
Sl~mlnary of the Invention
S This invention pertains to methods for regulating production of Th2-associated
cytokines and for regulating Th 1 or Th2 subsets by modulating the activity of one or
more transcription factors that regulate expression of Th2-specific cytokine genes, and
to compositions useful for such modulation As described further herein, it has now been
discovered that the tissue-specific expression of IL-4 in Th2 cells is not due to a
repressor protein but rather to a Th2-specific transactivator protein. The proto-oncogene
c-Maf has now been demonstrated to be responsible for the tissue-specific expression of
the Th2-associated cytokine interleukin-4. Moreover, ectopic expression of
c-Maf in cells other than Th2 cells (e.g., Thl cells, B cells and non-lymphoid cells) leads
to activation of the I~-4 promoter and, under ap~lopliate conditions, production of
endogenous IL-4. It further has been discovered that c-Maf and NF-AT synergize to
activate Th2-associated cytokine gene expression.
Furthermore, a 45 kDa protein that interacts with members of the NF-AT family
of proteins, termed NIP45 (for NF-AT lnteracting Protein 45), has now been isolated
and characterized. NIP45 was isolated based upon its ability to interact with the Rel
Homology Domain (RHD) of NF-AT. NIP45 has been shown to synergize with NF-AT
and c-Maf to stimulate cytokine gene expression. NIP45 potentiates gene expression
mediated by c-Maf and NF-AT such that when all three factors (c-Maf, NF-AT and
NIP45) are active in a cell, high levels of endogenous IL-4 production is stimulated. It
still further has been discovered that a small maf protein lacking a transactivation
domain, such as pl 8, can repress Th2-associated cytokine gene expression, e.g.,expression mediated by c-Maf.
Accordingly, this invention pertains to methods for modulating Th2-associated
cytokines expression by modnlztting the expression or activity of one or more
transcription factors that cooperate with an NF-AT family protein to regulate the
expression of Th2-associated cytokine genes. In one embodiment, the transcription
factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-
associated cytokine gene, and thus whose expression or activity is mod~ te-l is a Th2-
specific transcription factor (e.g, a Th2-specific maf family protein). In another
embodiment, the transcription factor that cooperates with an NF-AT family protein to
regulate the expression of a Th2-associated cytokine gene, and thus whose expression or
activity is modtll~tç~l~ is a maf family protein, such as c-Maf. In yet another
, . . . . . . . .
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embodiment, the transcription factor that cooperates with an NF-AT family protein to
regulate the expression of a Th2-associated cytokine gene, and thus whose expression or
activity is modulated, is a protein that interacts with an NF-AT family protein, such as
- NIP45. In yet another embodiment, the expression or activity of a small maf protein,
such as pl 8, is modulated. The methods of the invention may involve modulating the
expression or activity of one transcription factor (e.g., c-Maf or NIP45 or pl 8) or a
combination of transcription factors (e.g, c-Maf + NF-AT, or NF-AT + NIP45, or
c-Maf + NF-AT + NIP45).
The modulatory methods of the invention generally involve contacting a cell
with an agent that modulates the expression or activity of a transcription factor(s) such
that production of the Th2-associated cytokine by a cell is modulated. In particular,
preferred agents of the invention act intracellularly to modulate the activity of the
transcription factor. In one embodiment, the modulatory method of the invention
stimulates production of a Th2-associated cytokinc. For example, Th2-associated
cytokine production can be stimulated in Thl cells, B cells or non-lymphoid cells. In
another embodiment, the modulatory method of the invention inhibits production of a
Th2-associated cytokine. A Th2-associated cytokine modulated in the method
preferably is interleukin-4.
A variety of agents can be used to stimulate the expression or activity of a
transcription factor that regulates expression of a Th2-associated cytokine gene. For
example, a stimulatory agent of the invention can be a nucleic acid molecule encoding
the transcription factor that is introduced into and expressed in the cell. Alternatively,
chemical agents that enhance the expression or activity of the transcription facto can be
used as stimulatory agents.
A variety of agents can be used to inhibit the expression or activity of a
transcription factor that regulates expression of a Th2-associated cytokine gene.
Examples of suitable inhibitory agents include antisense nucleic acid molecules that are
complementary to a gene encoding the transcription factor, intracellular antibodies that
bind the transcription factor (e.g, in the cell nucleus), inhibitory forms of the
transcription factor (e.g, dominant negative forms) and chemical agents that inhibit the
expression or activity of the transcription factor.
Combination methods, involving modulation of the expression or activity of two,
three or more transcription factors that regulate Th2-associated cytokine gene
expression, are also encompassed by the invention. Accordingly, in other embodiments
of the invention, a cell is contacted with at least one additional agent that modulates the
activity of at least one additional transcription factor that contributes to the regulation of
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the Th2-associated cytokine gene. Preferably, the at least one additional transcription
factor whose expression or activity is modulated is selected from the group consisting of
NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-I
family proteins.
Cytokine production by a cell can be modulated in vitro or in vivo in accordancewith the methods of the invention. In one embodiment, a cell is contacted with amodulating agent(s) in vitro and then the cell is ~(lministered to a subject to thereby
regulate the development of Thl and/or Th2 subsets in the subject. Accordingly, in
another aspect, the invention provides methods for regulating the development of Thl or
Th2 subsets in a subject. In addition to the embodiment wherein ex vivo modified cells
are ~dmini~tered to the subject, in another embodiment, these methods involve direct
a-lmini~tration to the subject of an agent that modulates the activity of one or more
transcription factors that regulate expression of a Th2-associated cytokine gene such that
development of Thl or Th2 cells in the subject is mod~ te~l.
The modulatory methods of the invention can be used to manipulate Thl :Th2
ratios in a variety of clinical situations. For example, inhibition of Th2 formation may
be useful in allergic ~ice~ces, malignancies and infectious diseases whereas
enhancement of Th2 formation may be useful in autoimmune diseases and organ
transplantation .
Yet another aspect of the invention pertains to NIP45 compositions. This
invention provides isolated compositions of NIP45 protein and isolated nucleic acid
sequences encoding NIP45, other compositions related thereto and methods of use
thereof. The amino acid sequence of NIP45 protein has been determined (shown in SEQ
ID NO: 6~ and a cDNA encoding NIP45 protein has been isolated (the nucleotide
sequence of which is shown in SEQ ID NO: 5). In one aspect, the invention provides
isolated nucleic acid molecules encoding NIP45, or fragments thereof. In one
embodiment, the invention provides an isolated nucleic acid molecule comprising a
nucleotide sequence encoding NIP45 protein. In another embodiment, the inventionprovides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a
protein, wherein the protein comprises an amino acid sequence that is homologous to the
amino acid sequence of SEQ ID NO: 6 and interacts with the Rel Homology Domain of
an NF-AT family protein. In yet another embodiment, the invention provides an
isolated nucleic acid molecule which hybridizes under stringent conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO: 5. In yet anotherembodiment, the invention provides an isolated nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO: 5. In still other embodiments, the invention
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provides an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID
NO: 6. Isolated nucleic acid molecules encoding NIP45 fusion proteins and isolated
antisense nucleic acid molecules are also encompassed by the invention.
Another aspect of the invention pertains to vectors, such as recombinant
expression vectors, containing a NIP45 nucleic acid molecule of the invention and host
cells into which such vectors have been introduced. In one embodiment, such a host cell
is used to produce NIP45 protein by culturing the host cell in a suitable medium. If
desired, NIP45 protein can be then isolated from the host cell or the medium.
Still another aspect of the invention pertains to isolated NIP45 proteins, or
portions thereof. In one embodiment, the invention provides an isolated NIP45 protein,
or a portion thereof that interacts with an NF-AT family protein. In yet anotherembodiment, the invention provides an isolated protein which comprises an amino acid
sequence homologous to the amino acid sequence of SEQ ID NO: 6 and that interacts
with an NF-AT family protein. NIP45 fusion proteins are also encompassed by the
invention.
The NIP45 proteins of the invention, or fragments thereof, can be used to prepare
anti-NIP45 antibodies. Accordingly, the invention further provides an antibody that
specifically binds NIP45 protein. In one embodiment, the antibody is monoclonal. In
another embodiment, the antibody is labeled with a detectable substance.
The NIP45-encoding nucleic acid molecules of the invention can be used to
prepare nonhuman transgenic ~nim~l~ that contain cells carrying a transgene encoding
NIP45 protein or a portion of NIP45 protein. Accordingly, such transgenic ~nim~l~ are
also provided by the invention. In one embodiment, the NIP45 transgene carried by the
transgenic animal alters an endogenous gene encoding endogenous NIP45 protein (e.g.,
a homologous recombinant animal).
Another aspect of the invention pertains to methods for detecting the presence of
NIP45 protein or mRNA in a biological sample. To detect NIP45 protein or mRNA, the
biological sample is contacted with an agent capable of detecting NIP45 protein (such as
a labeled anti-NIP45 antibody) or NIP45 mRNA (such as a labeled nucleic acid probe
capable of hybridizing to NIP45 mRNA) such that the presence of NIP45 protein ormRNA is detected in the biological sample.
Still another aspect of the invention pertains to methods for identifying
compounds that modulate the activity or expression of NIP45 and methods for
identifying compounds that modulate an interaction between NIP45 and an NF-AT
family protein. Screening methods for identifying proteins that interact with NIP45 are
also encompassed by the invention.
.. ..... ...
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Yet another aspect of the invention pertains to compositions for expressing a maf
protein in a host cell, such as an immune cells, and host cells expressing maf proteins.
In one embodiment, these compositions include recombinant expression vectors that
encode a maf family protein, wherein the maf-encoding sequences are operatively linked
5 to regulatory sequences that direct expression of the maf family protein in a specific cell
type, such as Iymphoid cells (e.g., T cells or B cells) or hematopoietic stem cells. In
another embodiment, these compositions include host cells, such as host Iymphoid cells
(e.g, host T cells or host B cells) or host hematopoietic stem cells, into which a
recombinant expression vector encoding a maf family protein has been introduced.The invention further provides transgenic ~nim~ that express a c-Maf protein.
In a preferred embodiment, the transgenic animal is a mouse and the mouse
overexpresses c-Maf in T cells, preferably using a trangene that comprises a CD4promoter/enhancer operatively linked to a c-maf cDNA.
Brief Description of the Drawings
Figure 1 A is a schematic of the cell fusion approach used to demonstrate that
cytokine expression is not due to a repressor
Figure I B is a reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis of Il-2 and IL-4 cytokine, and control ~-actin, mRNA expressed by an unfused
Thl clone (Dl.l), an unfused Th2 clone (D10), Thl and Th2 homokaryons and Thl-Th2
heterokaryons.
Figure 2A is a Northern blot analysis depicting expression of an isolated cDNA
clone in Thl cells, Th2 cells or B Iymphoma cells. A control probe specific for GAPDH
was used to show equal loading of RNA.
Figure 2B is a Northern blot analysis depicting upregulated expression of the
isolated cDNA clone during in vitro differentiation of normal naive spleen cells into Th2
cells. Total RNA was isolated from cells harvested at the indicated time points. Culture
supernatant at the applo,ul;ate dilution was measured for cytokine (IL-I0) production by
ELISA to determine differentiation into the Thl or Th2 lineage.
Figure 3A is a bar graph depicting transactivation of the IL-4 promoter by c-Mafin a Thl clone (AE7). AE7 cells were cotransfected with a wild-type IL-4 CAT reporter
construct and either a control plasmid (pMEX-NeoI), a c-Maf expression plasmid
(pMEX-Maf) or a c-Fos expression plasmid (pMEX-c-Fos). Half of each sample was
stimulated 24 hours after transfection with antibodies to CD3. All samples were
harvested 48 hours after transfection and relative CAT activities were determined.
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Figure 3B is a photograph of a thin layer chromotography plate depicting the
relative CAT activity in Ml2 B Iymphoma cells cotransfected with a wild-type IL-4
CAT reporter construct and either two control plasmids (pMEX-NeoI and pREP4), a c-
Maf expression plasmid and a control plasmid (pMEX-Maf and pREP4), a c-Fos
5 expression plasmid and a control plasmid (pMEX-c-Fos and pREP4), a c-Jun expression
plasmid and a control plasmid (pMEX-c-Jun and pREP4), a control plasmid and an NF-
ATp expression plasmid (pMEX-NeoI and pREP-NF-ATp), a c-Maf expression plasmid
and an NF-ATp expression plasmid (pMEX-Maf and pREP-NF-ATp) or a c-Fos
expression plasmid and an NF-ATp expression plasmid (pMEX-c-Fos and pREP-NF-
10 ATp). Half of each sample was stimulated 24 hours after transfection with PMA andionomycin. All samples were harvested 48 hours after transfection and relative CAT
activities were determined.
Figure 4 is a bar graph depicting endogenous production of IL-4 in Ml2 cells by
ectopic expression of c-Maf and NF-ATp. Cells stably transfected with the indicated
15 control or expression plasmids were either unstimulated or stimulated with PMA and
ionomycin for 24 hours. 200 ~1 of supernatant from each sample was subjected to
ELISA for cytokine quantitation.
Figure SA is a photograph of a DNAse I footprint gel of the IL-4 promoter
performed using nuclear extracts from Th2 (DlO, CDC35) or Thl (AE7, S53) clones
20 harvested at the indicated time points after stimulation with anti-CD3 antibodies, which
depicts a Th2-specific footprint immediately downstream of the putative MARE site in
the IL-4 promoter. Two Th2-specific hypersensitive residues on the non-coding strand
of the IL-4 promoter are indicated by *. Five lanes of a DNAse I digestion of the IL-4
promoter probe (without nuclear extract) and a Maxam-Gilbert A/G ladder were run next
25 to the DNAse I treated samples.
Figure 5B is a schematic representation of the proximal regulatory region of themurine IL-4 promoter. The top portion shows the primary sequence of the murine IL-4
promoter. The numbers indicated are relative to the start site of transcription at +1.
Asterisks denote the Th2-specific hypersensitive residues seen on DNAse I footprint.
30 The bottom portion shows the sequence of the wild type (-59 to -28) oligonucleotide and
the 4 bp mutants used in EMSA and the functional assays shown in Figures 6 and 7.
Altered nucleotides are shown in lowercase bold and correspond to the numbering
system shown in the top portion.
Figure 6 is a photograph of an electrophoretic mobility shift assay (EMSA)
35 demonstrating that c-Maf but not c-Jun binds to the proximal IL-4 promoter and forms a
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- 10 -
complex with NF-ATp. EMSA was performed using the indicated proteins and labeleddouble-stranded oligonucleotides.
Figure 7A is a bar graph (top) and a photograph of a thin layer chromotography
plate (bottom) depicting the relative CAT activity in M12 cells co-transfected with a c-
5 Maf expression vector and either the wild-type IL-4 CAT reporter construct or one of the
4 bp mutants shown in Figure 5B, demonstrating that transactivation of the IL-4
promoter by c-Maf maps to the MARE and Th2-specific footprint. The average of three
independent experiments and one representative experiment are shown in the top and
bottom portions, respectively.
Figure 7B is a photograph of an EMSA, performed using recombinant c-Maf~ the
IL-4 promoter (-59 to -27) probe and the indicated unlabeled double-stranded
oligonucleotides as competitors, demonstrating that binding of recombinant c-Maf to the
IL-4 promoter maps to the MARE and Th2-specific footprint.
Figure 8 is photograph of yeast colonies, in triplicate, transformed with the
NIP45 plasmid and either NF-ATp-RHD as "bait" or control baits, Max, CDK2 or
pEG202, together with the LacZ reporter plasmid pSH 18, indicating that only those
colonies containing the NIP45 plasmid and the NF-ATp-RHD bait expressed the LacZreporter gene.
Figure 9 is a photograph of an immunoprecipitation/Western blot experiment
demonstrating that NIP45 and NF-ATp interact in HepG2 cells.
Figure 10 is a schematic diagram comparing the structures of the original NIP45
cDNA clone isolated from the yeast two-hybrid screen (top) and the longest NIP45cDNA clone isolated from a DIO.G4 lambda zap II library (bottom).
Figure 11 depicts the nucleotide and predicted amino acid sequences of the
original NIP45 cDNA isolate.
Figure 12 depicts the hydrophobicity plot of the NIP45 cDNA.
Figure 13 is a photograph of an RNA blot analysis of NIP45 transcript levels in
various tissues.
Figure 14A is a photograph of immunofluorescence analysis of BHK cells
transfected with an expression construct encoding an HA-epitope tagged NIP45 protein
and probed with a monoclonal antibody specific for the HA peptide as the primaryantibody and an indocarbocyanine labelled goat anti-mouse secondary reagent.
Figure 14B is a photograph of the sarne cells depicted in Figure 7A
counterstained with the DNA staining dye Hoechst 33258.
Figure 14C is a photograph of immunofluorescence analysis of unstimulated
BHK cells transfected with an expression construct encoding NF-AT4 and probed with
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an anti-NF-AT4 specific antibody as the primary antibody and an indocarbocyaninelabelled goat anti-mouse secondary reagent.
Figure 14D is a photograph of the same cells depicted in Figure 7C
counterstained with the DNA staining dye Hoechst 33258.
Figure 14E is a photograph of immunofluorescence analysis of ionomycin-
treated BHK cells transfected with an expression construct encoding NF-AT4 and
probed with an anti-NF-AT4 specific antibody as the primary antibody and an
indocarbocyanine labelled goat anti-mouse secondary reagent.
Figure 14F is a photograph of the same cells depicted in Figure 7D
counterstained with the DNA staining dye Hoechst 33258.
Figure 15 is a photograph of CAT assay results (left) and a bar graph quantitating
the relative fold induction of CAT activity (right) in HepG2 cells transfected with a 3X
NF-AT-CAT reporter gene construct (cont:~ining three NF-AT binding sites) and either a
control expression plasmid or an NF-AT family expression plasmid (NF-ATp, NF-ATc,
NF-AT3 or NF-AT4), alone (-) or in combination with a NIP45 expression plasmid (+).
Figure 16 is a photograph of CAT assay results (left) and a bar graph qu~,liL~ gthe relative fold induction of CAT activity (right) in HepG2 cells transfected with an IL-
4-CAT reporter gene construct (extending to -732 bp of the IL-4 promoter) and
combinations of NF-ATp, NIP45 and/or c-Maf expression constructs, as indicated.
Figure 17 is a bar graph depicting the level of IL-4 (in pg/ml) in the supernatants
of M12 B Iymphoma cells transiently cotransfected with expression plasmids for NF-
ATp, c-Maf and a pCI vector control (top bar) or expression plasmids for NF-ATp, c-
Maf and NIP45 (bottom bar).
Figure 18 is a Northern blot analysis of transcripts expressed on day 0, 1, 3, 5 or
7 during in vitro differentiation of norrnal naive spleen cells into Th2 cells, depicting
upregulated expression of c-maf over time and downregulated expression of pl 8 over
time.
Figure 19 is a photograph of a thin layer chromotography plate depicting the
relative CAT activity in M 12 cells transfected with an IL-4 promoter reporter gene
construct and either a c-Maf expression vector alone (5 ,ug), a pl 8 expression vector
alone (10 ~g) or a constant amount of c-Maf expression vector (5 ~lg) together with
increasing amounts of a pl 8 expression vector alone (2.5, 5 or 10 ~g), depicting
repression of IL-4 promoter activity by pl 8.
Figure 20 is a schematic diagram of a c-maf transgene for overexpression in T
cells using a CD4 promoter/enhancer to regulate expression of the c-maf cDNA.
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Figure 21 is a bar graph depicting total cell numbers (x million) in Iymph nodes(LN), spleen or thymus from either wild type mice or c-maf transfenic mice,
demonstrating that c-maf transgenic mice exhibit decreased cell numbers in lymphoid
organs.
Figure 22 is a bar graph depicting basal levels of serum IgE (ng/ml) in wild type
mice or c-maf transfenic mice, demonstrating that c-maf transgenic mice exhibit
increased basal levels of serum IgE.
Detailed Description of the Invention
In one aspect, this invention pertains to methods and compositions for regulating
cytokine gene expression and T cell subsets by modulating transcription factor activity.
The invention is based, at least in part, on the discovery that Th2-specific expression of
the interleukin-4 gene does not result from the action of a specific repressor protein (as
shown in Example 1 ) but rather from the action of a specific transactivator protein. As
described further herein, the transcription factor responsible for Th2-specific expression
of the interleukin-4 gene has now been identified as the c-Maf proto-oncoprotein, which
is selectively expressed in differenti~ting and mature Th2 cells and absent from Thl
cells (see Example 2). Ectopic expression of c-Maf in cells that do not normally express
it (such as Thl cells and B cells) leads to transactivation of the IL-4 promoter (see
Example 3) and, under appropriate conditions, to production of endogenous IL-4 (see
Example 4). Moreover, a protein present in nuclear extracts of Th2 cells, but not Thl
cells, footprints the IL-4 promoter in the region of a maf response element (MARE~ (see
Example 5) and recombinant c-Maf binds to the IL-4 promoter in vitro (see Example 6).
The ability of c-Maf to transactivate IL-4 maps to the MARE and Th2-specific footprint
in the IL-4 promoter (see Example 7).
The invention further is based, at least in part, on the discovery of a protein that
interacts with NF-AT and potentiates transcriptional activation by c-Maf and NF-AT.
This protein, NIP45, was identified based upon its interaction with the Rel Homology
Domain (RHD) of NF-AT (see Example 8). Coimmunoprecipitation experiments
demonstrated that NIP45 and NF-AT interact in vivo in m~mm~ n cells (see Example9). The cDNA encoding NIP45 has been sequenced and characterized (see Example 10).
Ex~min~tion of the tissue expression pattern of NIP45 mRNA revealed that the NIP45
transcript is preferentially expressed in spleen, thymus and testis (see Example 1 1).
Subcellular localization studies demonstrated that NIP45 protein is evenly distributed
throughout the cell nucleus (see Example 12). Functional studies showed that NIP45
synergizes with NF-AT to stimulate transcription from promoters cont~ining NF-AT
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binding sites and, moreover, synergizes with NF-AT and c-Maf to stimulate
transcription from the IL-4 promoter (see Example 13). Moreover, NIP45, NF-AT and
c-Maf can act in concert to induce expression of the endogenous IL-4 gene in cells that
do not norrnally express IL-4 (e.g., B cells.) (see Example 14).
The invention still further is based, at least in part, on the discovery that a small
maf protein, p 18, that lacks an activation domain, can repress cytokine gene expression
mediated by c-Maf. Differentiation of T helper cell precursors in vitro is associated with
upregulation of c-maf gene expression and downregulation of p l 8 gene expression (see
Example 15). Furthermore, coexpression of pl8 with c-Mafrepresses IL-4 promoter
activity, as compared to IL-4 promoter activity in the presence of c-Maf alone (see
Example 16).
The invention is still further based on the generation of c-maf transgenic :~nimz
that overexpress c-Maf protein in T cells (see Example 17). These ~nim~l.c exhibit a
phenotype very similar to transgenic ~nim~l~ that overexpress IL-4, namely smallthymus and spleen, dramatic decreases in numbers of CD+/CD8+ (double-positive)
thymocytes, decreased CD4+ positive T cells and increased basal levels of serum IgE.
So that the invention may be more readily understood, certain terms are first
defined.
As used herein, the term "Th2-associated cytokine" is intended to refer to a
cytokine that is produced preferentially or exclusively by Th2 cells rather than by Thl
cells. Examples of Th2-associated cytokines include IL-4, IL-5, IL-6 and IL- 13 . A
preferred Th2-associated cytokine whose production is modulated according to themethods of the invention is interleukin-4.
As used herein, the term "transcription factor" is intended to refer to a factor(e.g., a protein) that acts in the nucleus to regulate the transcriptional expression of a
gene. The term "transcription factor" is intended to include factors that directly regulate
transcription (e.g., have instrinsic transcriptional activation or inhibitory activity) and
factors that indirectly regulate transcription (e.g., through interaction with other factors
that have intrinsic transcriptional activation or inhibitory activity).
As used herein, a transcription factor that "cooperates with a Nuclear Factor ofActivated T cells family protein to regulate expression of a Th2-associated cytokine
gene" is intended to refer to a transcription factor that synergizes or acts in concert with
an NF-AT protein to regulate expression of a Th2-associated cytokine gene. That is, the
expression of the Th2-associated cytokine gene (e.g, IL-4) is greater in the presence of
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both NF-AT and the cooperative transcription factor than in the presence of either alone.
The cooperative transcription factor may or may not physically associate with NF-AT.
Examples of transcription factors that cooperate with an NF-AT family protein toregulate expression of a Th2-associated cytokine gene include maf family proteins (e.g.,
c-Maf) and NF-AT-interacting proteins (e.g., NIP45).
As used herein, a transcription factor that "contributes to the regulation of a Th2-
associated cytokine gene" is intended to refer to a transcription factor that participates in
the transcriptional regulation of a Th2-associated cytokine gene, regardless of whether it
cooperates with an NF-AT family protein. Transcription factors that cooperate with NF-
AT to regulate the expression of a Th2-associated cytokine gene also are factors that
contribute to the regulation of the Th2-associated cytokine gene. However, othertranscription factors that do not cooperate with NF-AT also can contribute to the
regulation of the Th2-associated cytokine gene. Examples of transcription factors that
are thought to contribute to the regulation of Th2-associated cytokine genes include NF-
AT family proteins, NF-AT-interacting proteins, maf family proteins, AP- I family
proteins, and Stat6 (Lederer, J. et al. ( 1996) J. Exp. Med. 184:397-406).
As used herein, the term "Th2-specific transcription factor" is intended to refer to
a transcription factor that is expressed preferentially or exclusively in Th2 cells rather
than in Thl cells.
As used herein, the term "contacting" (i. e., contacting a cell with an agent) is
intended to include incubating the agent and the cell together in vitro (e.g, adding the
agent to cells in culture) and ~lmini~tering the agent to a subject such that the agent and
cells of the subject are contacted in vivo.
As used herein, the various forms of the term "modulation" are intended to
include stimulation (e.g, increasing or upregulating a particular response or activity) and
inhibition (e.g., decreasing or downregulating a particular response or activity).
As used herein, the term "maf family protein" is intended to refer to a member of
a sub-family of AP-1/CREB/ATF proteins that includes v-Maf, c-Maf, mafB, Nrl, mafK,
maf~, mafG and pl 8. See e.g., Nishizawa, M. e~ al. (1989) Proc. Natl. Acad. Sci. USA
86:7711-7715; Kataoka, K. etal. (1993)J. Virol. 67:2133-2141; Swaroop, A. etal.
(1992) Proc. Natl. Acad. Sci. USA 89:266-270; Fujiwara, K.T. e~ al. (1993) Oncogene
8:2371 -2380; Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; Andrews, N.C. et
al. (1993) Proc. Natl. Acad. Sci. USA 90:11488-11492; and Kataoka, K. et al. (1995)
Mol. Cell. Biol. 15 :2180-2190.
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As used herein, the term "small maf protein" is intended to refer to a maf family
protein that lacks a domain corresponding to the amino-terminal activation domain of c-
Maf. Examples of small maf proteins include mafK, mafl~, mafG and pl 8.
- As used herein, the term "NF-AT family protein" (also referred to
interchangeably as simple "NF-AT") is intended to refer to a member of the family of
Nuclear Factors of Activated T cell transcription factors, including NF-ATp, NF-ATc,
NF-AT4/x/c3 and NF-AT3/c4.
As used herein, the term "Rel Homology Domain of an NF-AT family protein"
(abbreviated as RHD domain) is intended to refer to a domain within NF-AT familyproteins having approximately 70% sequence similarity within the RHD of the
Rel/NFKB family of transcription factors.
As used herein, the term "NF-AT-interacting protein" (used interchangeably with
"a protein that interacts with an NF-AT family protein") is intended to refer to a factor
that forms a physical association with an NF-AT family protein (e.g., co-
immunoprecipitates with an NF-AT family protein). Preferably, the NF-AT-interacting
protein interacts with the RHD of an NF-AT family protein. An example of an NF-AT-
interacting protein is NIP45.
As used herein, the term "NIP45" is intended to include proteins having the
amino acid sequence shown in SEQ ID NO: 6 (or encoded by the nucleotide sequenceshown in SEQ ID NO: 5), as well as m~rnm~ n homologues thereof (e.g., human
NIP45) and modified forms thereof (e.g., mutated or truncated forms) that retain the
ability to interact with the RHD of NF-AT.
As used herein, the term "AP-1 family protein" is intended to refer to a proteinthat is a member of the AP-I family of transcription factors, examples of which include
c-Jun, c-Fos, Fra- 1, Fra-2, Jun B and Jun D.
As used herein, the term "nucleic acid molecule" is intended to include DNA
molecules (e.g, cDNA or genomic DNA) and RNA molecules (e.g, mRNA). The
nucleic acid molecule may be single-stranded or double-stranded, but preferably is
double-stranded DNA.
An used herein, an "isolated nucleic acid molecule" refers to a nucleic acid
molecule that is free of gene sequences which naturally flank the nucleic acid in the
genomic DNA of the organism from which the nucleic acid is derived (i. e., gene
sequences that are located adjacent to the isolated nucleic molecule in the genomic DNA
of the organism from which the nucleic acid is derived). For example, in variousembodiments, the isolated NIP45 nucleic acid molecule may contain less than about 5
kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank
.
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the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, may
be free of other cellular material.
As used herein, the term "hybridizes under stringent conditions" is intended to
describe conditions for hybridization and washing under which nucleotide sequences at
least 60 % homologous to each other typically remain hybridized to each other.
Preferably, the conditions are such that at least sequences at least 65 %, more preferably
at least 70 %, and even more preferably at least 75 % homologous to each other typically
remain hybridized to each other. Such stringent conditions are known to those skilled in
10 the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization
conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45~C,
followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65~C.
As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA
15 or DNA molecule having a nucleotide sequence that occurs in nature (e.g, encodes a
natural protein).
As used herein, an "antisense" nucleic acid comprises a nucleotide sequence
which is complementary to a "sense" nucleic acid encoding a protein, e.g,
complementary to the coding strand of a double-stranded cDNA molecule,
complementary to an mRNA sequence or complementary to the coding strand of a gene.
Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
As used herein, the term "coding region" refers to regions of a nucleotide
sequence comprising codons which are tr~n~lAtecl into amino acid residues, whereas the
term "noncoding region" refers to regions of a nucleotide sequence that are not
translated into amino acids (e.g, 5' and 3' untranslated regions).
As used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into which additional
DNA segments may be ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome. Certain vectors are
capable of autonomous replication in a host cell into which they are introduced (e.g,
bacterial vectors having a bacterial origin of replication and episomal m~mm~ n
vectors). Other vectors (e.g., non-episomal m~mm~ n vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such vectors are referred to
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herein as "recombinant expression vectors" or simply "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the forrn of
plasmids. In the present specification, "plasmid" and "vector" may be used
interchangeably as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression vectors, such as viral
vectors (e.g, replication defective retroviruses, adenoviruses and adeno-associated
viruses), which serve equivalent functions.
As used herein, the term "host cell" is intended to refer to a cell into which anucleic acid of the invention, such as a recombinant expression vector of the invention,
has been introduced. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. It should be understood that such terms refer not only to the
particular subject cell but to the progeny or potential progeny of such a cell. Because
certain modifications may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be identical to the parent cell,
but are still included within the scope of the term as used herein.
As used herein, a nucleic acid molecule that is "in a form suitable for expression
of the nucleic acid molecule in a cell" is intended to means that the nucleic acid
molecule includes one or more regulatory sequences, selected on the basis of the host
cells to be used for expression and the level of expression desired, which is operatively
linked to the nucleic acid molecule to be expressed such that a protein encoded by the
nucleic acid molecule is expressed in the host cell. Examples of such nucleic acid
molecules include recombinant expression vectors cont:~ining nucleotide sequences
encoding the protein to be expressed in the host cell.
As used herein, a "transgenic animal" refers to a non-human animal, preferably am~mm~l, more preferably a mouse, in which one or more of the cells of the animalincludes a "transgene". The term "transgene" refers to exogenous DNA which is
integrated into the genome of a cell from which a transgenic animal develops and which
remains in the genome of the mature animal, for example directing the expression of an
encoded gene product in one or more cell types or tissues of the transgenic animal.
As used herein, a "homologous recombinant animal" refers to a type of
transgenic non-human animal, preferably a m~mm~l, more preferably a mouse, in which
an endogenous gene has been altered by homologous recombination between the
endogenous gene and an exogenous DNA molecule introduced into a cell of the animal,
e.g, an embryonic cell of the animal, prior to development of the animal.
As used herein, an "isolated protein" refers to a protein that is substantially free
of cellular material or culture medium when isolated from cells or produced by
.... . .. . . ~, . ......
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recombinant DNA techniques, or chemical precursors or other chemicals when
chemically synthesized.
As used herein, the term "antibody" is intended to include immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules, i. e.,
5 molecules that contain an antigen binding site which specifically binds (immunoreacts
with) an antigen, such as Fab and F(ab')2 fragments. The terms "monoclonal antibody"
and "monoclonal antibody composition", as used herein, refer to a population of
antibody molecules that contain only one species of an antigen binding site capable of
immunoreacting with a particular epitope of an antigen. A monoclonal antibody
10 composition thus typically displays a single binding affinity for a particular antigen with
which it immunoreacts.
As used herein, an agent that "acts intracellularly to modulate the expression or
activity of a transcription factor" is intended to refer to an agent that functions in an
intracellular region of a cell, e.g., the cytoplasm or nucleus, to modulate the expression
15 or activity of the transcription factor. Thus, an agent that binds to the cell surface, such
as an antibody, is not intended to be encompassed by the term "an agent that acts
intracellularly to modulate the expression or activity of a transcription factor."
Examples of agents that act intracellularly to modulate the expression or activity of a
transcription factor include nucleic acid molecules that encode the transcription factor,
20 antisense nucleic acid molecules, intracellular antibodies, dominant negative inhibitors
and chemical agents that enter a cell and modulate (i. e., stimulate or inhibit)transcription factor expression or activity.
As used herein, the terrn "intracellular binding molecule" is intended to include
agents that act intracellularly to inhibit the expression or activity of a target protein of
25 interest (e.g, a transcription factor) by binding to the protein itself or to a nucleic acid
(e.g, an mRNA molecule) that encodes the protein. Examples of intracellular binding
molecules include antisense nucleic acids, intracellular antibodies and dominantnegative inhibitors.
Various aspects of the present invention are described in further detail in the
following subsections.
I. Modulation of Th2-Associated Cytokine Production
The transcription factor responsible for the Th2-specific expression of the
interleukin-4 gene has now been identified as the c-Maf proto-oncogene. Modulation of
the expression and/or activity of c-Maf, therefore, provides a means to regulate the
~ . .
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production of interleukin-4. Since IL-4 itself serves an autoregulatory function in the
development of Th2 cells (see e.g., Paul, W.E. and Seder, R.A. (1994) Cell 76:241-251;
Seder, R.A. and Paul, W.E. (1994) Ann. Re-~. Immunol. 12:635-673), and thus
- production of IL-4 can lead to the production of additional Th2-associated cytokines
5 such as IL-5, IL-6, IL-10 and IL-13 through further Th2 differentiation, modulation of c-
Maf expression and/or activity provides a general approach for modulating production of
Th2-associated cytokines.
The maf farnily of proteins are a sub-family of AP- 1 /CREB/ATF proteins that
includes v-Maf, c-Maf, mafB, Nrl, mafK, mafF, mafG and p 18. The v-maf oncogene
10 was originally isolated from a spontaneous _usculo_poneurotic fibrosarcoma of chicken
and identified as the transforming gene of the avian retrovirus, AS42 (Nishizawa, M. et
al. (1989) Proc. Natl. Acad. Sci. USA 86:7711 -7715). V-maf encodes a 42 kd basic
region/leucine zipper (b-zip) protein with homology to the c-fos and c jun oncogenes.
Its cellular homologue, the c-maf proto-oncogene has only two structural changes in the
15 coding region from v-maf (Kataoka, K. et al. (1993) J. Virol. 67:2133-2141). The maf
family includes c-Maf, mafB, a human retina-specific protein Nrl (Swaroop, A. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:266-270), mafK, mafF, mafG and pl 8. The latter
four, mafK, mafF, mafG and pl8, each encode proteins that lack the amino terminal two
thirds of c-Maf that contains the transactivating domain ("small maf proteins")
20 (Fujiwara, K.T. et al. (1993) Oncogene 8:2371-2380; Igarashi, K. et al. (1995) .J. Biol.
Chem. 270:7615-7624; Andrews, N.C. et al. (1993) Proc. Natl. Acad. Sci. USA
90: 11488- 11492; Kataoka, K. et al. ( 1995) Mol. Cell. Biol. 15 :2180-2190). C-maf and
other maf family members form homodimers and heterodimers with each other and with
Fos and Jun, consistent with the known ability of the AP- 1 proteins to pair with each
25 other (Kerppola, T.K. and Curran, T. (1994) Oncogene 9:675-684; Kataoka, K. et al.
(1994) Mol. Cell. Biol. 14:700-712). The DNA target sequence to which c-Maf
homodimers bind, termed the c-Maf response element (MARE), is a 13 or 14 bp element
which contains a core TRE (T-MARE) or CRE (C-MARE) palindrome respectively.
Prior to the present invention, little was known about the function of maf family
30 members, although c-Maf has been shown to stimulate transcription from the Purkinje
neuron-specific promoter L7 (Kurscher, C. and Morgan, J.I. (1994) Mol. Cell. Biol.
15:246-254) and Nrl has been shown to drive expression of the QRl retina-specific gene
(Swaroop, A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:266-270). However, prior to
the present invention, there have been no reports implicating c-Maf or other maf family
35 members in the regulation of genes expressed in Iymphoid cells or in cytokine gene
expression in any tissue.
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The small mafs have been shown to function as repressors of a and ~-globin
transcription when bound as homodimers but are essential as heterodimeric partners with
the erythroid-specific factor p45NF-E2 to activate globin gene transcription (Kataoka,
K. et al. ( 1995) Mol. Cell. Biol. 15 :2180-2190; Igarashi, K. et al. ( 1994) Nature
367:568-572). MafK overexpression has been shown to induce erythroleukemia cell
differentiation (lgarashi, K. et al. (1995) Proc. Natl. Acad Sci. USA 92:7445-7449).
The present invention provides evidence that small mafproteins (e.g, pl8) can modulate
the expression of Th2-associated cytokine genes. Accordingly, modulation of the
expression and/or activity of a small maf protein also provides a means to regulate the
production of Th2-associated cytokine genes.
The present invention further provides an NF-AT-interacting protein, NIP45, thatbinds to and synergizes with NF-AT to regulate expression of a Th2-associated cytokine
gene. NIP45 was identified based upon its interaction with the Rel Homology Domain
of NF-ATp. NIP45 is described in further detail hereinbelow. Modulation of the
expression and/or activity of an NF-AT-interacting protein, such as NIP45, thus also
provides a means to regulate the production of Th2-associated cytokine genes.
Accordingly, this invention provides methods for mo~ ting production of a
Th2-associated cytokine by a cell by modulating the expression or activity of one or
more transcription factors involved in Th2-associated cytokine gene expression. In one
embodiment of the methods of the invention, a cell is contacted with an agent that
modulates the expression or activity of a transcription factor such that such that
production of the Th2-associated cytokine by a cell is modulated. In one embodiment,
the transcription factor to be modulated is characterized as a transcription factor that
cooperates with an NF-AT family protein to regulate expression of the Th2-associated
cytokine gene ~e.g, c-Maf or NIP45). In another embodiment, the transcription factor to
be modulated is a maf family protein (e.g., c-Maf or a small maf protein, such as pl 8).
ln yet another embodiment, the transcription is an NF-AT-interacting protein (e.g.,
NIP45). In preferred embodiments, the modulatory agents of the invention are
characterized by acting intracellularly to modulate the activity of a transcription factor.
In one embodiment, production of a Th2-associated cytokine by a cell is stimulated by
contacting the cell with a stimulatory agent that stimulates transcription factor
expression and/or activity. In another embodiment of the method of the invention,
production of a Th2-associated cytokine by a cell is inhibited by contacting the cell with
a inhibitory agent that inhibits transcription factor expression andlor activity.
As demonstrated in the Examples, although c-Maf is responsible for the tissue
specificity of IL-4 gene expression, c-Maf acts synergistically with one or more
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additional transcription factors to activate IL-4 gene transcription. In particular, c-Maf
acts synergistically with an NF-AT protein to stimulate IL-4 gene expression.
Moreover, NF-AT proteins and other members of the AP- I /CREB/ATF family of
- transcription factors have been demonstrated to be involved in regulating expression of
S both Thl- and Th2-associated cytokine genes. As further demonstrated in the Examples,
a protein that interacts with NF-AT, NIP45, acts synergistically with NF-AT to stimulate
expression from promoters cont~ining NF-AT sites. Moreover, expression of a Th2-associated cytokine gene is potentiated by the presence of all three factors, c-Maf, NF-
AT and NIP45. Accordingly, in another embodiment, the method of the invention for
10 mo~ ting Th2-associated cytokine production by a cell can comprises contacting the
cell with multiple agents that modulate the expression or activity of transcription factors.
Thus, in the methods of the invention in which a cell is contacted with a first agent, the
methods can further comprise contacting the cell with one or more additional agents that
modulate the activity of one or more additional transcription factors that contributes to
15 the regulation of the Th2-associated cytokine gene. Preferably, the additonal agent(s)
modulates the expression or activity or an additional transcription factor(s) selected from
the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family
proteins and AP-I family proteins.
As still further demonstrated in the Examples, a small maf protein (e.g, pl 8) can
20 repress Th2-associated cytokine gene expression me~ tçd by positive transactivators
(e.g., c-Maf). Accordingly, in yet another embodiment, the method of the invention for
mo(l~ ting Th2-associated cytokine production by a cell comprises contacting the cell
with an agent that modulates (i. e., stimulates or inhibits) the expression or activity of a
small maf protein, alone or in combination with agents that modulate the activity of
25 other transcription factors, such as other maf family proteins, NF-AT family proteins or
NF-AT-interacting proteins. Preferably, the small maf protein is pl 8. Other examples
of small maf proteins include mafK, mafF and mafG.
A. Stimulatory Agents
According to the method of the invention, to stimulate Th2-associated cytokine
production by a cell, the cell is contacted with a stimulatory agent that stimulates
expression and/or activity of a transcription factor (e.g, c-Maf, NIP45, p l 8) that
regulates expression of a Th2-associated cytokine gene. Th2-associated cytokine
35 production can be stimulated in cell types that do not normally express such cytokines,
such as Thl cells, B cells or non-lymphoid cells. Furthermore, Th2-associated cytokine
.
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production can be stimulated in helper precursor cells (Thp) to promote their
differentiation along the Th2 pathway instead of the Th I pathway.
A preferred stimulatory agent is a nucleic acid molecule encoding a transcription
factor that regulates expression of a Th2-associated cytokine gene, wherein the nucleic
5 acid molecule is introduced into the cell in a form suitable for expression of the
transcription factor in the cell. For example, a c-Maf cDNA is cloned into a
recombinant expression vector and the vector is transfected into the cell. As
demonstrated in Example 3, ectopic expression of a c-mafrecombinant expression
vector in Th l cells, B cells or non-lymphoid cells leads to activation of the IL-4
10 promoter. Additionally, under appropriate conditions (discussed in further detail
below), transcription of the endogenous IL-4 gene is stimulated, leading to IL-4production by cells that do not normally express this cytokine (see Exarnp}e 4).To express a maf family protein in a cell, typically a maf family cDNA is first
introduced into a recombinant expression vector using standard molecular biology15 techniques. A maf family cDNA can be obtained, for example, by amplification using
the polymerase chain reaction (PCR) or by screening an a~pl~p,;ate cDNA library. The
nucleotide sequences of maf family cDNAs are known in the art and can be used for the
design of PCR primers that allow for amplification of a cDNA by standard PCR
methods or for the design of a hybridization probe that can be used to screen a cDNA
20 library using standard hybridization methods. Preferably, the maf family cDNA is that
of the c-mafproto-oncogene. The nucleotide and predicted amino acid sequences of a
m~mm~ n (mouse) c-mafcDNA are disclosed in Kurscher C. and Morgan, J.I. (1995)
Mol. Cell. Biol. 15:246-254 and deposited in the GenBank database at accession number
S74567. This m~mm~ n c-maf is highly homologous to the avian v-maf sequence
25 (disciosed in Nishizawa, M.K. et al. (1989) Proc. Na~l. Acad. Sci. USA 86:7711 -7715
and GenBank accession numbers D28598 and D28596), indicating that c-maf is well
conserved among species. c-mafcDNAs from other m~mm~ n species, including
humans, can be isolated using standard molecular biology techniques (e.g., PCR or
cDNA library screening) and primers or probes designed based upon the mouse or avian
30 sequences. Human partial cDNA sequences homologous to the mouse c-maf cDNA are
also deposited in the GenBank database at accession numbers H24189 and N75504.
The sequences of other maf family members are also known in the art, for exampleMafB (Kataoka, K. et al. (1994) Mol. Cell Biol. 14:7581-91; GenBank accession
number D28600), MafG (Kataoka et al. (1994) Mol. Cell Biol. 14:7581-91; GenBank
35 accession numbers D28601 and D28602), MafF (GenBank accession number D16184)
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W O 97/39721 PCT~US97/06708
and MafK (Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; GenBank accession
numbers D 16187 and D42124).
Following isolation or amplification of a maf family cDNA, the DNA fragment
- is introduced into an expression vector. As used herein, the term "vector" refers to a
5 nucleic acid molecule capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments may be ligated. Another type of vector
is a viral vector, wherein additional DNA segments may be ligated into the viralgenome. Certain vectors are capable of autonomous replication in a host cell into which
10 they are introduced (e.g, bacterial vectors having a bacterial origin of replication and
episomal m~mm~ n vectors). Other vectors (e.g., non-episomal m:~mm~ n vectors)
are integrated into the genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they are operatively linked. Such
15 vectors are referred to herein as "recombinant expression vectors" or simply "expression
vectors". In general, expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used forrn of vector.
However, the invention is intended to include such other forms of expression vectors,
20 such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-
associated viruses), which serve equivalent functions
The recombinant expression vectors of the invention comprise a nucleic acid in aform suitable for expression of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory sequences, selected on
25 the basis of the host cells to be used for expression and the level of expression desired,
which is operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant expression vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a manner which allows for
expression of the nucleotide sequence (e.g, in an in vitro transcription/translation
30 system or in a host cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to includes promoters, enhancers and other expression
control elements (e.g, polyadenylation signals). Such regulatory sequences are
described, for example, in Goeddel; Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences
35 include those which direct constitutive expression of a nucleotide sequence in many
types of host cell, those which direct expression of the nucleotide sequence only in
... , , .. ~., ,
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- 24 -
certain host cells (e.g., tissue-specific regulatory se~uences) or those which direct
expression of the nucleotide sequence only under certain conditions (e.g., inducible
regulatory sequences).
It will be appreciated by those skilled in the art that the design of the expression
5 vector may depend on such factors as the choice of the host cell to be transformed, the
level of expression of protein desired, etc. When used in m~mm~ n cells, the
expression vector's control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma virus, adenovirus,cytomegalovirus and Simian Virus 40. Non-limiting examples of m~mm~
10 expression vectors include pCDM8 (Seed, B., (1987) Nafure 329:840) and pMT2PCufm~n et al. (1987), EMBO J. 6:1 87-195). A variety of m~mm~ n expression
vectors carrying different regulatory sequences are commercially available. For
constitutive expression of the nucleic acid in a m~mm~ n host cell, a preferred
regulatory element is the cytomegalovirus promoter/enhancer. Moreover, inducible15 regulatory systems for use in m:~mm~ n cells are known in the art, for example systems
in which gene expression is regulated by heavy metal ions (see e.g, Mayo et al. (1982)
Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985) Mol. Cell.
Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991) in Heat Shock Response,
e.d. Nouer, L., CRC, Boca Raton, FL, ppl67-220), hormones (see e.g., Lee et al.
20 (1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-
2042; Klock et al. (1987) Nature 329:734-736; Israel & ~Allfm~n (1989) Nucl. Acids
Res. 17:2589-2604; and PCT Publication No. WO 93/23431), FK506-related molecules(see e.g, PCT Publication No. WO 94/18317) or tetracyclines (Gossen, M. and Bujard,
H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science
25 268:1766-1769; PCT Publication No. WO 94/29442; and PCT Publication No. WO
96/01313). Still further, many tissue-specific regulatory sequences are known in the art,
including the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. I :268-
277), Iymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
30 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament
promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. .'Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916) and m~mm~ry
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and
35 European Application Publication No. 2647166). Developmentally-regulated promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990)
CA 022~2643 1998-10-22
wo 97t39721 PCT/US97/06708
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989)
GenesDev. 3:537-546).
Vector DNA can be introduced into m~mm~ n cells via conventional
- transfection techniques. As used herein, the various forms of the term "transfection"
5 are intended to refer to a variety of art-recognized techniques for introducing foreign
nucleic acid (e.g., DNA) into m~mm~ n host cells, including calcium phosphate co-
precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
Suitable methods for transfecting host cells can be found in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press
10 (1989)), and other laboratory manuals.
For stable transfection of m~mm~ n cells, it is known that, depending upon the
expression vector and transfection technique used, only a small fraction of cells may
integrate the foreign DNA into their genome. In order to identify and select these
integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is
15 generally introduced into the host cells along with the gene of interest. Preferred
selectable markers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable marker may be
introduced into a host cell on a separate vector from that encoding a maf family protein
or, more preferably, on the same vector. Cells stably transfected with the introduced
20 nucleic acid can be identified by drug selection (e.g., cells that have incorporated the
selectable marker gene will survive, while the other cells die).
Nucleic acid molecules encoding other transcription factors that regulate Th2-
associated cytokine gene expression, in form suitable for expression of the transcription
factor in a host cell, can be prepared as described above using nucleotide sequences
25 known in the art or disclosed herein. The nucleotide sequences can be used for the
design of PCR primers that allow for amplification of a cDNA by standard PCR
methods or for the design of a hybridization probe that can be used to screen a cDNA
library using standard hybridization methods. The nucleotide and predicted amino acid
sequence of NIP45 are disclosed in SEQ ID NOs: 5 and 6, respectively. The nucleotide
30 and predicted amino acid sequences of small maf proteins, including pl 8, mafK, mafF
and mafG, are known in the art (see e.g, Fujiwara, K.T. et al. (1993) C)ncogene 8:2371-
2380; Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; Andrews, N.C. et al.
(1993) Proc. Natl. Acad. Sci. USA 90: 11488-11492; Kataoka, K. et al. ( 1995) Mol. Cell.
Biol. 15:2180-2190). The nucleotide and predicted amino acid sequences of NF-AT
35 family proteins, including NF-ATp, NF-ATc, NF-AT4/x/c3 and NF-AT3/c4, are known
in the art. Four NF-AT family members have been identified (see e.g., Emmel, E.A. et
. .
CA 022~2643 1998-10-22
W O97/39721 PCT~US97/06708
-26-
al. ( I 989) Science 246: 1617- 1620; Flanagan, W.M. et al. ( 1991 ) Nature 352: 803-807,
Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P.G. et al. (1993) Science262:750-754; Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J.P. et al. (1994)
Nature 369:497). Preferably, the NF-AT cDNA is that of NF-ATp. The nucleotide and
5 predicted amino acid sequences of a m~mm~lian NF-ATp cDNA are disclosed in
McCaffrey, P.G. et al. ( 1993) Science 262:750-754. The nucleotide and predictedamino acid sequences of a m~mm~ n NF-ATc cDNA are disclosed in Northrop, J.P. etal. (1994) Na~ure 369:497 and deposited in the GenBank database at accession number
U08015. The nucleotide and predicted amino acid sequences of m~mm~ n NF-AT3
10 and NF-AT4 cDNAs are disclosed in Hoey, T. et al. (1995) Immunity 2:461-472. The
nucleotide and predicted amino acid se~uences of AP- I family proteins are known in the
art. For example, the nucleotide and predicted amino acid sequences of human c-fos are
disclosed in van Straaten, F. et al. (1983) Proc. Natl. Acad. Sci. USA 80:3183-3187.
The nucleotide and predicted amino acid sequences of human c jun are disclosed in
15 Bohmann, D. et al. (1987) Science 238:1386-1392. The nucleotide and predicted amino
acid sequences of human jun-B and jun-D are disclosed in Nomura, N. et al. (1990)
Nucl. Acids Res. 18:3047-3048. The nucleotide and predicted amino acid sequences of
humanJ~a-l and ~a-2 are disclosed in Matsui, M. et al. (1990) Oncogene 5:249-255.
Another form of a stimulatory agent for stimulating expression of a Th2-
20 associated cytokine in a cell is a chemical compound that stimulates the expression or
activity of an endogenous transcription factor that regulates expression of Th2-associated cytokine genes in the cell (e.g, a maf family protein, such as c-Maf or pl 8, or
a protein that interacts with NF-AT, such as NIP45). Such compounds can be identified
using screening assays that select for compounds that stimulate the expression or activity
25 of the transcription factor. Examples of suitable screening assays are described in
further detail in subsection V below.
In addition to use of a first agent that stimulates the expression or activity of a
first transcription factor that regulates Th2-associated cytokine gene expression, the
stimulatory methods of the invention can involve the use of one or more additional
30 agents that stimulate the expression or activity of one or more additional transcription
factors that contribute to regulating the expression of a Thl- or Th2-associated cytokine
gene. In Example 4. it is shown that stimulation of the expression of endogenous IL-4 in
M12 B Iymphoma cells required the introduction into the cells of both a c-Maf
expression vector and an NF-AT expression vector, thereby demonstrating that c-Maf
35 and NF-AT act synergistically to activate IL-4 transcription, with c-maf responsible for
the tissue-specificity of expression. In Example 14, it further is shown that stimulation
CA 022~2643 1998-10-22
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- 27 -
of the expression of endogenous IL-4 in M12 B Iymphoma cells is potentiated by
coexpression of c-Maf, NF-AT and NIP45. While the skilled artisan will appreciate that
certain cells may express sufficient amounts of endogenous c-Maf, NF-AT and/or NIP45
- such that use of a single agent alone may be sufficient to stimulate expression of a Th2-
associated cytokine gene, in certain situations and with certain cell types it may be
necessary to stimulate multiple transcription factors, such as both c-Maf and NF-AT,
both c-Maf and NIP45, or all three proteins (c-Maf, NF-AT and NIP45), to achieve the
desired stimulation of Th2-associated cytokine production.
Accordingly, in the stimulatory method of the invention in which a cell is
contacted with a first agent that stimulates the expression or activity of a first
transcription factor, the method can further comprise contacting the cell with at least one
additional agent that stimulates the expression or activity of at least additional
transcription factors that contribute to regulating the expression of a Thl- or Th2-
associated cytokine gene. Preferably, the at least one additional transcription factor
whose expression or activity is modulated is selected from the group consisting of NF-
AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-l family
proteins. For example, a stimulatory method of the invention can involve the use of a
first agent that stimulates the expression or activity of c-Maf and a second agent that
stimulates the expression or activity of either an NF-AT family protein or a protein that
interacts with an NF-AT family protein (e.g., NIP45). In another embodiment, thestimulatory methods of the invention involve the use of a first agent that stimulates the
expression or activity of c-Maf, a second agent that stimulates the expression or activity
of an NF-AT family protein and a third agent that stimulates the expression or activity of
a protein that interacts with an NF-AT family protein (e.g., NIP45). A preferred agent
for stimulating NF-AT or NIP45 activity in a cell is a recombinant expression encoding
NF-AT or NIP45, respectively, wherein the recombinant expression vector is introduced
into the cell and NF-AT or NIP45 is expressed in the cell. NF-AT- and NIP45-encoding
expression vectors can be prepared and introduced into cells as described above for c-
Maf expression vectors.
Alternative to use of an NF-AT or NIP45 cDNA to stimulate the activity of NF-
AT or NIP45 in a cell, one or more chemical compounds that stimulate NF-AT or NIP45
activity in a cell can be used as a second (or additional) agent in a stimulatory method of
the invention. Compounds that stimulate NF-AT activity in cells are known in the art
(for a review see Rao, A. (1994) Imm~nol. Today 15:274-281). For example,
stimulation of certain cells with the phorbol ester phorbol myristate acetate (PMA) and a
calcium ionophore (e.g, ionomycin) results in translocation of NF-ATs to the cell
CA 022~2643 1998-10-22
W O 97/39721 PCT~US97/06708
- 28 -
nucleus (see e.g., Fl~n~g~n, W.M. et al. (1991) Nafure 352:803-807; Jain, J. et al. (1993)
Nature 365:352-355). Additionally, stimulation of T cells through the T cell receptor
(TcR), for example with an anti-CD3 antibody, results in activation of NF-AT in the T
cells.
In addition to NF-AT proteins, AP- 1 family members, including c-Jun, c-Fos,
Fra- I, Fra-2, Jun B and Jun D, have been shown to be involved in regulating theexpression of both Thl- and Th2-associated cytokine genes (e.g, IL-2 and IL-4) (see
e.g., Rao, A. et al. (1994) Immunol. Today 15:274-281; Jain, J. et al. (1993) Nature
365:352-355; Boise, L.H. et al. (1993) Mol. Cell. Biol M3: 1911 -1919; Rooney, J. et al.
(1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310).
Although these factors are not responsible for the Thl/Th2 specificity of expression of
the cytokine genes, and these factors do not appear to synergize with c-Maf in regulating
IL-4 gene expression (see the Examples), AP-1 family members have been shown to
increase IL-4 expression in Th2 cells (see e.g, Rooney, J. et al. (1995) Immunity 2:545-
553). Accordingly, in certain circumstances it may be beneficial, in addition tostimulating c-Maf activity (and possibly NF-AT activity), also to stimulate the activity
of an AP-I family protein. Accordingly, in one embodiment, the stimulatory methods of
the invention involve the use of a first agent that stimulates the expression or activity of
c-Maf and a second agent that stimulates the expression or activity of an AP- 1 protein.
In another embodiment, the invention involves the use of a first agent that stimulates the
expression or activity of c-Maf, a second agent that stimulates the expression or activity
of an NF-AT protein and a third agent that stimulates the expression or activity of an
AP-I protein. NIP45 activity also can be modulated in combination with maf, AP-1and/or NF-AT family proteins.
A preferred agent for stimulating AP-l activity in a cell is a recombinant
expression encoding an AP- 1 protein, wherein the recombinant expression vector is
introduced into the cell and the AP-I protein is expressed in the cell. AP-l-encoding
expression vectors can be prepared and introduced into cells as described above for c-
Maf expression vectors. Alternatively, one or more chemical compounds that stimulate
AP-l activity in a cell can be used as additional agents in a stimulatory method of the
invention. Compounds that stimulate AP- 1 activity in cells are known in the art,
including PMA/calcium ionophore (e.g., ionomycin) and anti-CD3 antibodies.
B. Inhibitory Agents
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WO 97/39721 PCT/US97/06708
- 29 -
According to the method of the invention, to inhibit Th2-associated cytokine
production by a cell, the cell is contacted with an inhibitory agent that inhibits
expression and/or activity of a transcription factor (e.g., c-Maf, NIP45, pl 8) that
- regulates expression of a Th2-associated cytokine gene. In one embodiment, Th2-
associated cytokine production by a cell is inhibited by contacting the cell with an agent
that modulates the expression or activity of a transcription factor that cooperates with an
NF-AT family protein to regulate expression of a Th2-associated cytokine gene. In
another embodioment, Th2-associated cytokine production by a cell is inhibited by
contacting the cell with an agent that modulates the expression or activity of a Th2-
specific transcription factor, preferably c-Maf. In another embodiment, Th2-associated
cytokine production by a cell is inhibited by contacting the cell with an agent that
modulates the expression or activity of a protein that interacts with an NF-AT family
protein, preferably NIP45. In yet another embodiment, Th2-associated cytokine
production by a cell is inhibited by contacting the cell with an agent that modulates the
expression or activity of a small maf protein. As discussed above for stimulatory
methods, the inhibitory methods of the invention can comprise contacting the cell with
two or more agents that modulate the expression or activity of two or more transcription
factors that regulate Th2-associated cytokine gene expression, including maf family
proteins, NF-AT family proteins, NF-AT-interacting proteins and AP-1 farnily proteins.
Th2-associated cytokine production can be inhibited in, for example, Th2 cells or
in helper precursor cells (Thp) to promote their differentiation along the Thl pathway
instead of the Th2 pathway. Inhibitory agents of the invention can be, for example,
intracellular binding molecules that act to inhibit the expression or activity of the
transcription factor. As used herein, the term "intracellular binding molecule" is
intended to include molecules that act intracellularly to inhibit the expression or activity
of a protein by binding to the protein or to a nucleic acid (e.g., an mRNA molecule) that
encodes the protein. Exarnples of intracellular binding molecules, described in further
detail below, include antisense nucleic acids, intracellular antibodies and dominant
negative inhibitors.
In one embodiment, an inhibitory agent of the invention is an antisense nucleic
acid molecule that is complementary to a gene encoding a transcription factor (e.g., a
maf family protein, such as c-Maf or p 18, or an NF-AT-interacting protein, such as
NIP45), or to a portion of said gene, or a recombinant expression vector encoding said
~nti~n~e nucleic acid molecule. The use of antisense nucleic acids to downregulate the
expression of a particular protein in a cell is well known in the art (see e.g., Weintraub,
H. e~ al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
, . , , . . -- .. . . . . . . . .
CA 022~2643 1998-10-22
WO 97/39721 PCT/US97/06708
- 30 -
Genetics, Vol. I (1 ) 1986; Askari, F.K. and McDonnell, W.M. (1996) N. Eng ~ Med.
334:316-318; Bennett, M.R. and Schwartz, S.M. (1995) Circulation 92:1981-1993;
Mercola, D. and Cohen, J.S. (1995) Cancer Gene ~her. 2:47-59; Rossi, J.J. (1995) Br.
Med. Bull. 51:217-225; Wagner, R.W. (1994) Nature 372:333-335). An antisense
5 nucleic acid molecule comprises a nucleotide sequence that is complementary to the
coding strand of another nucleic acid molecule (e.g., an mRNA sequence) and
accordingly is capable of hydrogen bonding to the coding strand of the other nucleic
acid molecule. Antisense sequences complementary to a sequence of an mRNA can becomplementary to a sequence found in the coding region of the mRNA, the 5' or 3'10 untr~n.~l~ted region of the mRNA or a region bridging the coding region and an
untr~n.~l~ted region (e.g, at the junction of the 5' untranslated region and the coding
region). Furthermore, an antisense nucleic acid can be complementary in sequence to a
regulatory region of the gene encoding the mRNA, for instance a transcription initiation
sequence or regulatory element. Preferably, an antisense nucleic acid is designed so as
15 to be complementary to a region prece~ling or spanning the initiation codon on the
coding strand or in the 3' untranslated region of an mRNA. An antisense nucleic acid
for inhibiting in a cell the expression of a transcription factor discussed herein can be
designed based upon the nucleotide sequence of the transcription factor, as disclosed
herein or known in the art, constructed according to the rules of Watson and Crick base
20 pairing.
An antisense nucleic acid can exist in a variety of different forrns. For example,
the antisense nucleic acid can be an oligonucleotide that is complementary to only a
portion of a maf family gene. An antisense oligonucleotides can be constructed using
chemical synthesis procedures known in the art. An antisense oligonucleotide can be
25 chemically synthesized using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense and sense nucleic
acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used.
To inhibit transcription factor expression in cells in culture, one or more antisense
30 oligonucleotides can be added to cells in culture media, typically at 200 ~Ig oligonucleotide/ml .
Alternatively, an antisense nucleic acid can be produced biologically using an
expression vector into which a nucleic acid has been subcloned in an antisense
orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an
35 antisense orientation to a target nucleic acid of interest). Regulatory sequences
operatively linked to a nucleic acid cloned in the antisense orientation can be chosen
. . . ...
CA 022~2643 1998-10-22
wo 97/39721 PCT/US97/06708
which direct the expression of the antisense RNA molecule in a cell of interest, for
instance promoters and/or enhancers or other regulatory sequences can be chosen which
direct constitutive, tissue specific or inducible expression of antisense RNA. The
- antisense expression vector is prepared as described above for recombinant expression
5 vectors, except that the cDNA (or portion thereoi) is cloned into the vector in the
antisense orientation. The antisense expression vector can be in the forrn of, for
example, a recombinant plasmid, phagemid or attenuated virus. The antisense
expression vector is introduced into cells using a standard transfection technique, as
described above for recombinant expression vectors.
In another embodiment, an antisense nucleic acid for use as an inhibitory agent is a
ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region (for reviews on ribozymes see e.g., Ohkawa, J. et al. (1995) J.
Biochem. 118:251-258; Sigurdsson, S.T. and Eckstein, F. (1995) Trends Biotechnol.
13:286 289; Rossi, J.J. (1995) Trends Biotechnol. 13:301-306; Kiehntopf, M. et al. (1995)
J. Mol. Med. 73 :65-71). A ribozyme having specificity for mRNA encoding a transcription
factor discussed herein can be designed based upon the nucleotide sequence of the
transcription factor. For example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the base sequence of the active site is complementary to the base
sequence to be cleaved in a c-mafmRNA or other transcription factor mRNA. See for
example U.S. Patent Nos. 4,987,071 and 5,116,742, both by Cech et al. Alternatively, c-
mafmRNA (or other transcription factor mRNA) can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA molecules. See for example
Bartel, D. and Szostak, J.W. (1993) Science 261: 1411-1418.
Another type of inhibitory agent that can be used to inhibit the expression and/or
activity of a Maf protein in a cell is an intracellular antibody specific for a transcription
factor discussed herein. The use of intracellular antibodies to inhibit protein function in a
cell is known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646;
Biocca, S. et al. (1990) EMBO J. 9:101-108; Werge, T.M. et al. (1990) FEBSLetters
274:193-198; Carlson, J.R. (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco,
W.A. etal. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. e~al. (1994)
Bio/Technology 12:396-399; Chen, S-Y. et al. (1994) Human Gene Therapy 5:595-601;
Duan, L et al. (1994) Proc. Natl. Acad Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R.R. etal. (1994) J. Biol. Chem.
269:23931-23936; Beerli, R.R. et al. (1994) Biochem. Biophys. Res. Commun. 204:666-
672; ~h~chilk~r, A.M. et al. (1995) EMBO J. 14:1542 1551; Richardson, J.H. et al. (1995)
.... ~ .. . .. . . .
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Proc. Natl.Acad. Sci. USA 92:3137-3141;PCTPublicationNo. WO94/02610byMarasco
et al.; and PCT Publication No. WO 95/03832 by Duan et al.).
To inhibit protein activity using an intracellular antibody, a recombinant expression
vector is prepared which encodes the antibody chains in a form such that, upon introduction
5 of the vector into a cell, the antibody chains are expressed as a functional antibody in an
intracellular compartment of the cell. For inhibition of transcription factor activity
according to the inhibitory methods of the invention, preferably an intracellular antibody
that specifically binds the transcription factor is expressed within the nucleus of the cell.
Nuclear expression of an intracellular antibody can be accomplished by removing from the
10 antibody light and heavy chain genes those nucleotide sequences that encode the N-
terminal hydrophobic leader sequences and adding nucleotide sequences encoding anuclear localization signal at either the N- or C-terminus of the light and heavy chain genes
(see e.g, Biocca, S. et al. (1990) EMBO J. 2:101-108; Mh~hilk~r, A. M. et al. (1995)
~MBO J. 14: 1542- 1551). A preferred nuclear localization signal to be used for nuclear
15 targeting of the intracellular antibody chains is the nuclear localization signal of SV40
Large T antigen (see Biocca, S. et al. (1990) EMBO J. _:101-108; Mh~hilk~r, A. M. et al.
(1995)EMBOJ. 14:1542 1551).
To prepare an intracellular antibody expression vector, antibody light and heavychain cDNAs encoding antibody chains specific for the target protein of interest, e.g, a
20 Maf family protein or other transcription factor discussed herein, are isolated, typically
from a hybridoma that secretes a monoclonal antibody specific for the maf protein.
Preparation of antisera against Maf family proteins has been described in the art (see
e.g., Fujiwara, K.T. et al. (1993) Oncogene 8:2371-2380; Kataoka, K. et al. (1993) J.
Virol. 67:2133-2141; Kerppola, T.K. and Curran, T. (1994) Oncogene 2:675-684;
25 Igarashi, K et al. (1995) Proc. Natl. Acad. Sci. USA 92:7445-7449). Anti-Maf protein
antibodies can be prepared by immunizing a suitable subject, (e.g., rabbit, goat, mouse
or other m~mm~l ) with a Maf protein immunogen. An applol~l;ate immunogenic
preparation can contain, for examples, recombinantly expressed Maf protein or a
chemically synthesized Maf peptide. The plel)aldlion can further include an adjuvant,
30 such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Antibody-producing cells can be obtained from the subject and used to prepare
monoclonal antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also,
Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) JBiol Chem
35 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer
29:269-75). The technology for producing monoclonal antibody hybridomas is well
.. . . . .. .. .
CA 022~2643 1998-10-22
W O 97t39721 PCT~US97tO6708
known (see generally R. H. Kenneth, in Monoclonal An~ibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A.
Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell
- Genet., 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to
5 Iymphocytes (typically splenocytes) from a m~mm:~l immunized with a maf protein
- immunogen as described above, and the culture supernatants of the resulting hybridoma
cells are screened to identify a hybridoma producing a monoclonal antibody that binds
specifically to the Maf protein. Any of the many well known protocols used for fusing
Iymphocytes and immortalized cell lines can be applied for the purpose of generating an
anti-Maf protein monoclonal antibody (see, e.g, G. Galfre et al. (1977) Nature 266:550-
52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinary skilled
worker will appreciate that there are many variations of such methods which also would
be useful. Typically, the immortal cell line (e.g, a myeloma cell line) is derived from
the same m~mm~ n species as the lymphocytes. For example, murine hybridomas can
be made by fusing Iymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized mouse cell line. Preferred
immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium
cont~ining hypox~nthine, aminopterin and thymidine ("HAT medium"). Any of a
number of myeloma cell lines may be used as a fusion partner according to standard
techniques, e.g, the P3-NSl/l-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines.These myeloma lines are available from the American Type Culture Collection (ATCC),
Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse
splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the
fusion are then selected using HAT medium, which kills unfused and unproductively
fused myeloma cells (unfused splenocytes die after several days because they are not
transformed). Hybridoma cells producing a monoclonal antibody that specifically binds
the maf protein are identified by screening the hybridoma culture supernatants for such
antibodies, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody that binds to a transcription factor discussed herein can beidentified and isolated by screening a recombinant combinatorial immunoglobulin
library (e.g, an antibody phage display library) with the protein, or a peptide thereof, to
thereby isolate immunoglobulin library members that bind specifically to the protein.
Kits for generating and screening phage display libraries are commercially available
(e.g, the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and
. .
CA 022~2643 1998-10-22
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-34-
the Stratagene Su~PTM Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, Ladner et al. U.S.
Patent No. 5,223,409; Kang et al. International Publication No. WO 92/1~619; Dower et
al. International Publication No. WO 91/17271; Winter et al. International Publication
W O 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et
al. International Publication WO 93/01288; McCafferty et al. International Publication
No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Fuchs et
al. (1991) Bio/Tec~nology 9:1370-1372; Hay et al. (1992) HumAntibod Hybridomas
3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J
12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc~cid ~es 19:4133-4137;
Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990)348:552-
554.
Once a monoclonal antibody specific for the transcription factor of interest hasbeen identified (e.g, either a hybridoma-derived monoclonal antibody or a recombinant
antibody from a combinatorial library), DNAs encoding the light and heavy chains of
the monoclonal antibody are isolated by standard molecular biology techniques. For
hybridoma derived antibodies, light and heavy chain cDNAs can be obtained, for
example, by PCR amplification or cDNA library screening. For recombinant antibodies,
such as from a phage display library, cDNA encoding the light and heavy chains can be
recovered from the display package (e.g., phage) isolated during the library screening
process. Nucleotide sequences of antibody light and heavy chain genes from whichPCR primers or cDNA library probes can be prepared are known in the art. For
example, many such sequences are disclosed in Kabat, E.A.? et al. ( 1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242 and in the "Vbase" human germline
sequence ~l~t~b~e
Once obtained, the antibody light and heavy chain sequences are cloned into a
recombinant expression vector using standard methods. As discussed above, the
sequences encoding the hydrophobic leaders of the light and heavy chains are removed
and sequences encoding a nuclear localization signal (e.g, from SV40 Large T antigen)
are linked in-frame to sequences encoding either the amino- or carboxy terminus of both
the light and heavy chains. The expression vector can encode an intracellular antibody
in one of several different forms. For example, in one embodiment, the vector encodes
CA 022~2643 1998-10-22
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- 35 -
full-length antibody light and heavy chains such that a full-length antibody is expressed
intracellularly. In another embodiment, the vector encodes a full-length light chain but
only the VH/CH I region of the heavy chain such that a Fab fragment is expressed- intracellularly. In the most preferred embodiment, the vector encodes a single chain
5 antibody (scFv) wherein the variable regions of the light and heavy chains are linked by
a flexible peptide linker (eg., (Gly4Ser)3) and expressed as a single chain molecule. To
inhibit transcription factor activity in a cell, the expression vector encoding the
transcription factor-specific intracellular antibody is introduced into the cell by standard
transfection methods, as discussed hereinbefore.
Yet another form of an inhibitory agent of the invention is an inhibitory form of
a transcription factor discussed herein (e.g., a maf protein), also referred to herein as a
dominant negative inhibitor. The maf family of proteins are known to homodimerize
and to heterodimerize with other AP-1 family members, such as Fos and Jun (see e.g,
Kerppola, T.K. and Curran, T. (1994) Oncogene 9:675-684; Kataoka, K. e~al. (1994)
15 Mol. Cell. Biol. 14:700-712). One means to inhibit the activity of transcription factors
that form dimers is through the use of a dominant negative inhibitor that has the ability
to dimerize with functional transcription factors but that lacks the ability to activate
transcription (see e.g, Petrak, D. et al. (1994) J. Immunol. 153 :2046-2051). Bydimerizing with functional transcription factors, such dominant negative inhibitors can
20 inhibit their functional activity. This process may occur naturally as a means to regulate
gene expression. For example, there are a number of "small" maf proteins, such as
map~, maJ~, mafG andpl8, which lack the amino terminal two thirds of c-Maf that
contains the transactivating domain (Fujiwara, K.T. et al. (1993) Oncogene 8:2371-
2380; Igarashi, K. et al. (1995) J. Biol. Chem. 270:7615-7624; Andrews, N.C. et al.
25 (1993) Proc. Natl. Acad. Sci. USA 90:11488-11492; Kataoka, K. et al. (1995) Mol. Cell.
Biol. 15:2180-2190). Homodimers of the small maf proteins act as negative regulators
of transcription (Igarashi, K. et al. (1994) Nature 367:568-572) and three of the small
maf proteins (MafK, MafF and MafG) have been shown to competitively inhibit
transactivation mediated by the v-Maf oncoprotein (Kataoka, K. et al. ( 1996) Oncogene
30 12:53-62). Additionally, MafB has been identified as an interaction partner of Ets-l and
shown to inhibit Ets- 1 -mediated transactivation of the transferrin receptor and to inhibit
erythroid differentiation (Sieweke, M.H. et al. (1996) Cell 85:49-60).
Accordingly, an inhibitory agent of the invention can be a form of a Maf proteinthat has the ability to dimerize with c-Maf but that lacks the ability to activate
35 transcription. This dominant negative form of a Maf protein may be, for example, a
small Maf protein (e.g., MafK, MafF, MafG) that naturally lacks a transactivation
.. . . .
CA 022~2643 l998-l0-22
W O97/39721 PCT~US97/06708
-36-
domain~ MafB or a mutated form of c-Maf in which the transactivation domain has been
removed. Such dominant negative Maf proteins can be expressed in cells using a
recombinant expression vector encoding the Maf protein, which is introduced into the
cell by standard transfection methods. To express a mutant form of c-Maf lacking a
5 transactivation domain, nucleotide sequences encoding the amino terminal
transactivation domain of c-Maf are removed from the c-mafcDNA by standard
recombinant DNA techniques. Preferably, at least amino acids 1-122 are removed.
More preferably, at least amino acids 1-187, or amino acids 1-~57, are removed.
Nucleotide sequences encoding the basic-leucine zipper region are maintained. The
10 truncated cDNA is inserted into a recombinant expression vector, which is then
introduced into a cell to allow for expression of the truncated c-maf, lacking atransactivation domain, in the cell.
Yet another type of inhibitory agent that can be used to inhibit the expression
and/or activity of a maf protein in a cell is chemical compound that inhibits the
15 expression or activity of an endogenous maf family protein in the cell. Such compounds
can be identified using screening assays that select for compounds that inhibit the
expression or activity of a maf family protein. Examples of suitable screening assays are
described in further detail in subsection V below.
As discussed above with regard to stimulatory agents, the inhibitory methods of
20 the invention can involve the use of one or more additional inhibitory agents that inhibit
the expression or activity of one or more additional transcription factors that contributes
to regulating the expression of a Thl- or Th2-associated cytokine gene. For example, in
one embodiment, the inhibitory method of the invention comprises contacting a cell with
a first agent that inhibits the expression or activity a maf farnily protein and a second
25 agent that inhibits the expression or activity of an NF-AT family protein or an NF-AT-
interacting protein (e.g, NIP45). In another embodiment, the inhibitory method of the
invention comprises contacting a cell with a first agent that inhibits the expression or
activity a maf family protein and a second agent that inhibits the expression or activity
of an AP- I farnily protein. In yet another embodiment, the inhibitory method of the
30 invention comprises contacting a cell with a first agent that inhibits the expression or
activity a maf family protein, a second agent that inhibits the expression or activity of an
NF-AT family protein and a third agent that inhibits the expression or activity of an NF-
AT-interacting protein (e.g, NIP45). Examples of types of inhibitory agents for
inhibiting NF-AT, NF-AT-interacting and AP-I proteins include antisense nucleic acids,
35 intracellular antibodies, dominant negative inhibitors and chemical compounds that
inhibit the endogenous proteins, as described above. Regarding the latter, it is known in
CA 022~2643 1998-10-22
W O97/39721 PCTAJS97/06708
the art that the nuclear translocation of NF-ATp is inhibited by the immunosuppressive
drugs cyclosporin A and FK506 (see e.g, Rao, A. (1994) Immunol. Today 15:274 281;
Rao, A. (1995)J. Leukoc. Biol. 57:536-542). Accordingly, in one embodiment ofthe- inhibitory method, an immunosuppressive drug such as cyclosporin A or FK506 (or
5 other related drug that inhibits the calcineurin pathway) is used in combination with an
agent that inhibits the expression or activity of c-Maf.
The method of the invention for modulating production of Th2-associated
cytokines by a cell can be practiced either in vitro or in vivo (the latter is discussed
10 further in the following subsection). For practicing the method in vitro, cells can be
obtained from a subject by standard methods and incubated (i. e., cultured) in vitro with a
stimulatory or inhibitory agent of the invention to stimulate or inhibit, respectively, the
production of Th2-associated cytokines. For example, peripheral blood mononuclear
cells (PBMCs) can be obtained from a subject and isolated by density gradient
15 centrifugation, e.g., with Ficoll/Hypaque. Specific cell populations can be depleted or
enriched using standard methods. For example, monocytes/macrophages can be isolated
by adherence on plastic. T cells or B cells can be enriched or depleted, for example, by
positive and/or negative selection using antibodies to T cell or B cell surface markers,
for example by incubating cells with a specific primary monoclonal antibody (mAb),
20 followed by isolation of cells that bind the mAb using magnetic beads coated with a
secondary antibody that binds the primary mAb. Peripheral blood or bone marrow
derived hematopoietic stem cells can be isolated by similar techniques using stem cell-
specific mAbs (e.g., anti-CD34 mAbs). Specific cell populations can also be isolated by
fluorescence activated cell sorting according to standard methods. Monoclonal
25 antibodies to cell-specific surface markers known in the art and many are commercially
available.
When a stimulatory agent is used in vitro, resulting in stimulation of the
production of Th2-associated cytokines, in particular IL-4, the cytokine(s) can be
recovered from the culture supernatant for further use. For example, the culture30 supernatant, or a purified fraction thereof, can be applied to T cells in culture to
influence the development of Thl or Th2 cells in vitro. Alternatively, the culture
supernatant, or a purified fraction thereof, can be a~lmini~tered to a subiect to influence
the development of Thl vs. Th2 responses in the subject.
Moreover, cells treated in vitro with either a stimulatory or inhibitory agent can
35 be ~lmini~tered to a subject to influence the development of a Thl vs. Th2 response in
the subject. Accordingly, in another embodiment, the method of the invention for
.. .... . . . . .
CA 022~2643 1998-10-22
W O 97139721 PCT~US97/06708
modulating the production of Th2-associated cytokines by a cell further comprises
~lmini.~tering the cell to a subject to thereby modulate development of Thl or Th2 cells
in a subject. Preferred cell types for ex vivo modification and re~llmini~tration include T
cells, B cells and hematopoietic stem cells. For ~lmini~tration to a subject, it may be
5 preferable to first remove residual agents in the culture from the cells before
~lmini~tering them to the subject. This can be done for example by a Ficoll/Hypaque
gradient centrifugation of the cells. For further discussion of ex vivo genetic
modification of cells followed by re~dmini.~tration to a subject, see also U.S. Patent No.
5,399,346 by W.F. Anderson et al.
II. Methods for Mod~ ting Development of Thl or Th2 Cells in a Subject
Another aspect of the invention pertains to a method for moclul~ting
development of Thl or Th2 cells in a subject. The term "subject" is intended to include
living org~ni~m~ in which an immune response can be elicited. Preferred subjects are
15 m~mm~l~. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows,
horses, goats and sheep. As discussed above, one way to modulate Thl/Th2 ratios in a
subject is to treat cells (e.g, T cells, B cells or hematopoietic stem cells) ex vivo with
one or more modulatory agents of the invention, such that production of a Th2-
associated cytokine by the cells is modulated, followed by ~lmini~tration of the cells to
20 the subject. In another embodiment, Thl/Th2 ratios are modulated in a subject by
~lmini~tering to the subject an agent that modulates the activity of a transcription factor
that regulates expression of a Th2-associated cytokine gene such that development of
Thl or Th2 cells in the subject is modulated. In a preferred embodiment, the
transcription factor is a maf family protein, preferably a c-Maf protein or a small maf
25 protein (e.g., pl8). ln another preferred embodiment, the transcription factor is a protein
that interacts with an NF-AT family protein, preferably NIP45. Preferably, the Th2-
associated cytokine is I~-4. Development of a Th2 response in the subject can bepromoted by a-lmini~tration of one or more stimulatory agents of the invention, whereas
development of a Thl response in the subject can be promoted by ~dmini.~tration of one
30 or more inhibitory agents of the invention. As discussed above, in certain situations it
may be desirable, in addition to modulating the activity of multiple transcription factors
(e.g, combinations of a maf family protein, an NF-AT family protein, an NF-AT-
interacting protein and/or an AP-l family protein).
For stimulatory or inhibitory agents that comprise nucleic acids (including
35 recombinant expression vectors encoding transcription factors. antisense RNA,intracellular antibodies or dominant negative inhibitors), the agents can be introduced
CA 022~2643 1998-10-22
WO97/39721 PCTrUSg7/06708
- 39 -
into cells of the subject using methods known in the art for introducing nucleic acid
(e.g, DNA) into cells in vivo. Examples of such methods include:
Direct Injection: Naked DNA can be introduced into cells in vivo by directly
injecting the DNA into the cells (see e.g, Acsadi et al. (1991) Nature 332:815-818;
5 Wolff et al. (1990) Science 247:1465-1468). For example, a delivery apparatus (e.g, a
"gene gun") for injecting DNA into cells in vivo can be used. Such an apparatus is
commercially available (e.g., from BioRad).
Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into cells
in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a
10 ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol.
Chem. 263:14621;Wilsonetal. (1992)J. Biol. Chem. 267:963-967;andU.S.PatentNo.
5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the
DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus
capsids which naturally disrupt endosomes, thereby releasing material into the
15 cytoplasm can be used to avoid degradation of the complex by intracellular Iysosomes
(see for exarnple Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al.
(1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
Retroviruses: Defective retroviruses are well characterized for use in gene
transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
20 A recombinant retrovirus can be constructed having a nucleotide sequences of interest
incorporated into the retroviral genome. Additionally, portions of the retroviral genome
can be removed to render the retrovirus replication defective. The replication defective
retrovirus is then packaged into virions which can be used to infect a target cell through
the use of a helper virus by standard techniques. Protocols for producing recombinant
25 retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in
Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well
known to those skilled in the art. Examples of suitable packaging virus lines include ~4
30 Crip, ~yCre, w2 and ~Am. Retroviruses have been used to introduce a variety of genes
into many different cell types, including epithelial cells, endothelial cells, Iymphocytes,
myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example
Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.
Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-
35 3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.
(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad.
CA 022~2643 1998-10-22
Wo 97/39721 PCTtUSg7/06708
- 40 -
Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992)
Human Gene Therapy 3:641-647; Dai e~ al. (1992) Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwuetal. (1993)J. Immunol. 150:4104-4115; U.S. PatentNo.
5 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT
Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573). Retroviral vectors require target cell division in order for the retroviral
genome (and foreign nucleic acid inserted into it) to be integrated into the host genome
to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate
10 replication of the target cell.
Adenoviruses: The genome of an adenovirus can be manipulated such that it
encodes and expresses a gene product of interest but is inactivated in terms of its ability
to replicate in a normal Iytic viral life cycle. See for example Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431 -434; and Rosenfeld et al.
15 (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain
Ad type 5 dl324 or other strains of adenovirus (e.g, Ad2, Ad3, Ad7 etc.) are well known
to those skilled in the art. Recombinant adenoviruses are advantageous in that they do
not require dividing cells to be effective gene delivery vehicles and can be used to infect
a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited
20 supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-
6486), hepatocytes (Herz and Gerard (1993) Proc. NaJI. Acad. Sci. USA 90:2812-2816)
and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).
Additionally. introduced adenoviral DNA (and foreign DNA contained therein) is not
integrated into the genome of a host cell but remains episomal, thereby avoiding25 potential problems that can occur as a result of insertional mutagenesis in situations
where introduced DNA becomes integrated into the host genome (e.g, retroviral DNA).
Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up
to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-
Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral
30 vectors currently in use are deleted for all or parts ofthe viral E1 and E3 genes but retain
as much as 80 % of the adenoviral genetic material.
Adeno-Associated r~iruses: Adeno-associated virus (AAV) is a naturally
occurring defective virus that requires another virus, such as an adenovirus or a herpes
virus, as a helper virus for efficient replication and a productive life cycle. (For a review
35 see Muzyczka et al. Curr. Topics in Micro. and lmmunol. (1992) 158:97 129). It is also
one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a
,, ~
CA 022~2643 1998-10-22
wo 97/39721 PCT/US97/06708
- 41 -
high frequency of stable integration (see for example Flotte et al. ( 1992) ~m. J. Respir.
Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McT .~lghlin et al. (1989) J. Virol. 62: 1963-1973). Vectors containing as little as 300
base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is
5 limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985)
Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of
nucleic acids have been introduced into different cell types using AAV vectors (see for
example Hermonat et al. ( 1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et
al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol.
10 _:32-39; Tratschin et al. ( 1984) J. Virol. 51 :611 -619; and Flotte et al. ( 1993) J. Biol.
Chem. 268:3781-3790).
The efficacy of a particular expression vector system and method of introducing
nucleic acid into a cell can be assessed by standard approaches routinely used in the art.
For example, DNA introduced into a cell can be detected by a filter hybridization
15 technique (e.g., Southern blotting) and RNA produced by transcription of introduced
DNA can be detected, for example, by Northern blotting, RNase protection or reverse
transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected
by an ~plol,liate assay, for example by immunological detection of a produced protein,
such as with a specific antibody, or by a functional assay to detect a functional activity
20 of the gene product, such as an enzymatic assay.
A modulatory agent, such as a chemical compound that stimulates or inhibits
endogenous transcription factor activity, can be allmini~tered to a subject as apharrn~cel-tical composition. Such compositions typically comprise the modulatory
agent and a ph~ ceutically acceptable carrier. As used herein the term
25 "pharmaceutically acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical atlmini.~tration. The use
of such media and agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is incompatible with the active
30 compound, use thereof in the compositions is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is forrnulated to be compatible
with its intended route of,q(lmini~tration. For example, solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
35 components: a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
CA 022~2643 1998-10-22
WO 97/39721 PCT/US97/06708
- 42 -
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as ethylene~ minetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases,
5 such as hydrochloric acid or sodium hydroxide. The parenteral plepald~ion can be
enclosed in ampoules, disposable syringes or multiple dose vials made of glass or
plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the
10 extemporaneous preparation of sterile injectable solutions or dispersion. Forintravenous ~(lmini~tration, suitable carriers include physiological saline, bacteriostatic
water, Cremophor ELTM (BASF, Parsil,pally, NJ) or phosphate buffered saline (PBS).
In all cases, the composition must be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of manufacture and storage
15 and must be preserved against the cont~min~ting action of microorg~nism~ such as
bacteria and fungi. The carrier can be a solvent or dispersion medium cont~ining, fcr
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity
can be m~int~ined, for example, by the use of a coating such as lecithin, by the20 maintenance of the required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorg~ni~m~ can be achieved by various
antibacterial and antifungal agents, for exarnple, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium
25 chloride in the composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an ~ppropl ;ate solvent with one or a combination of
30 ingredients enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into a sterile vehicle
which contains a basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-drying
35 which yields a powder of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
.
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Oral compositions generally include an inert diluent or an edible carrier. They
can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral
therapeutic ~(1mini.~tration, the active compound can be incorporated with excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also be prepared
5 using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder such as
10 microcrystalline cellulose, gum trag~nth or gelatin; an excipient such as starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
In one embodiment, the active compounds are prepared with carriers that will
protect the compound against rapid elimin~tion from the body, such as a controlled
release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
20 Methods for plepaldlion of such formulations will be ~pa~ to those skilled in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected
cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically
acceptable carriers. These may be prepared according to methods known to those skilled
25 in the art, for example, as described in U.S. Patent No. 4,522,81 1.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of ~1mini~tration and uniformity of dosage. Dosage unit form
as used herein refers to physically discrete units suited as unitary dosages for the subject
to be treated; each unit cont~ining a predetermined quantity of active compound
30 calculated to produce the desired therapeutic effect in association with the required
pharrnaceutical carrier. The specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique characteristics of the active
compound and the particular therapeutic effect to be achieved, and (b) the limitations
inherent in the art of compounding such an active compound for the treatment of
35 individuals.
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III. Applications of the Methods of the Invention
Identification of transcription factors that control the production of IL-4, andhence continued formation of Th2 cells, allows for selective manipulation of T cell
subsets in a variety of clinical situations using the modulatory methods of the invention.
5 The stimulatory methods of the invention (i.e., methods that use a stimulatory agent)
result in production of Th2-associated cytokines, with concomitant promotion of a Th2
response and downregulation of a Thl response. In contrast, the inhibitory methods of
the invention ~i. e., methods that use an inhibitory agent) inhibit the production of Th2-
associated cytokines, with concomitant downregulation of a Th2 response and
10 promotion of a Th 1 response. Thus, to treat a disease condition wherein a Th2 response
is beneficial, a stimulatory method of the invention is selected such that Th2 responses
are promoted while downregulating Thl responses. Alternatively, to treat a disease
condition wherein a Thl response is beneficial, an inhibitory method of the invention is
selected such that Th2 responses are downregulated while promoting Thl responses.
15 Application of the methods of the invention to the treatment of disease conditions may
result in cure of the condition, a decrease in the type or number of symptoms associated
with the condition, either in the long term or short term (i. e., amelioration of the
condition) or simply a transient beneficial effect to the subject.
Numerous disease conditions associated with a predominant Thl or Th2-type
20 response have been identified and could benefit from modulation of the type of response
mounted in the individual suffering from the disease condition. Application of the
immunomodulatory methods of the invention to such diseases is described in further
detail below.
A. Allergies
Allergies are mediated through IgE antibodies whose production is regulated by
the activity of Th2 cells and the cytokines produced thereby. In allergic reactions, IL-4
is produced by Th2 cells, which further stimulates production of IgE antibodies and
30 activation of cells that mediate allergic reactions, i.e., mast cells and basophils. IL-4
also plays an important role in eosinophil mediated infl~mm~tory reactions.
Accordingly, the inhibitory methods of the invention can be used to inhibit the
production of Th2-associated cytokines, and in particular IL-4, in allergic patients as a
means to downregulate production of pathogenic IgE antibodies. An inhibitory agent
35 may be directly ~lmini~tered to the subject or cells (e.g, Thp cells or Th2 cells) may be
obtained from the subject, contacted with an inhibitory agent ex vivo, and re~-lmini~tered
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to the subject. Moreover, in certain situations it may be beneficial to co~flmini~ter to the
subject the allergen together with the inhibitory agent or cells treated with the inhibitory
agent to inhibit (e.g., desensitize) the allergen-specific response. The treatment may be
further enhanced by ~(lmini~tering other Thl-promoting agents, such as the cytokine IL-
5 12 or antibodies to Th2-associated cytokines (e.g., anti-IL-4 antibodies), to the allergic
subject in amounts sufficient to further stimulate a Thl-type response.
B. C'ancer
The expression of Th2-promoting cytokines has been reported to be elevated in
cancerpatients (see e.g., Yarnamura, M., etal. (1993) J. Clin. Invest. 91:1005-1010;
Pisa, P., et al. (1992) Proc. Na~l. Acad. Sci. USA 89:7708-7712) and malignant disease
is often associated with a shift from Thl type responses to Th2 type responses along
with a worsening of the course of the disease. Accordingly, the inhibitory methods of
15 the invention can be used to inhibit the production of Th2-associated cytokines in cancer
patients, as a means to counteract the Thl to Th2 shift and thereby promote an ongoing
Th 1 response in the patients to arneliorate the course of the disease. The inhibitory
method can involve either direct af~mini~tration of an inhibitory agent to a subject with
cancer or ex vivo treatment of cells obtained from the subject (e.g, Thp or Th2 cells)
20 with an inhibitory agent followed by re~-lmini~tration of the cells to the subject. The
treatment may be fùrther enhanced by ~(lmini~tering other Thl-promoting agents, such
as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g, anti-IL-4
antibodies), to the recipient in amounts sufficient to further stimulate a Thl-type
response.
C. Infectious Diseases
The expression of Th2-promoting cytokines also has been reported to increase
during a variety of infectious diseases, including HIV infection, tuberculosis,
30 lei~hm:~ni~.~is, schistosomiasis, filarial nematode infection and intestinal nematode
infection (see e.g; Shearer, G.M. and Clerici, M. (1992) Prog Chem. Immunol. 54:21-
43; Clerici, M and Shearer, G.M. (1993) Immunology Today 14:107-111; Fauci, A.S.(1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992) lmmunoparasitology
Today l:A58 A61; Pearce, E.J., etal. (l991)J. Exp. Med. 173:159 166; Grzych, J-M., et
35 al. (1991) J. Immunol. 141 :1322-1327; Kullberg, M.C., et al. (1992) J. Immunol.
148:3264 3270; Bancroft, A.J., et al. (1993).~. Immunol. 150:1395-1402; Pearlman, E.,
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et al. (1993) Infect. Immun. 61 :1105-1112; Else, K.J., et al. (1994) J. Exp. Med
179:347-351) and such infectious diseases are also associated with a Thl to Th2 shift in
the immune response. Accordingly, the inhibitory methods of the invention can be used
to inhibit the production of Th2-associated cytokines in subjects with infectious
5 diseases, as a means to counteract the Thl to Th2 shift and thereby promote an ongoing
Thl response in the patients to ameliorate the course of the infection. The inhibitory
method can involve either direct ~(1ministration of an inhibitory agent to a subject with
an infectious disease or ex vivo treatment of cells obtained from the subject (e.g, Thp or
Th2 cells) with an inhibitory agent followed by re~lministration of the cells to the
10 subject. The treatment may be further enhanced by a-lmini.~tering other Thl-promoting
agents, such as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g, anti-
IL-4 antibodies), to the recipient in amounts sufficient to further stimulate a Thl-type
response.
D. AutoimmuneDiseases
The stimulatory methods of the invention can be used therapeutically in the
treatment of autoimmune diseases that are associated with a Th2-type dysfunction.
Many autoimmune disorders are the result of inapplopl.ate activation of T cells that are
20 reactive against self tissue and that promote the production of cytokines andautoantibodies involved in the pathology of the diseases. Modulation of T helper-type
responses can have an effect on the course of the autoimmune disease. For exarnple, in
experimental allergic encephalomyelitis (EAE), stimulation of a Th2-type response by
~lministration of IL-4 at the time of the induction of the disease ~limini~hes the intensity
25 ofthe autoimmune disease (Paul, W.E., et al. (1994) Cell 76:241-251). Furthermore,
recovery of the ~nim~1~ from the disease has been shown to be associated with anincrease in a Th2-type response as evidenced by an increase of Th2-specific cytokines
(Koury, S. J., et al. (1992) ~ Exp. Med 176:1355-1364). Moreover, T cells that can
suppress EAE secrete Th2-specific cytokines (Chen, C., et al. (1994) Immunity I :147-
30 154). Since stimulation of a Th2-type response in EAE has a protective effect against
the disease, stimulation of a Th2 response in subjects with multiple sclerosis (for which
EAE is a model) is likely to be beneficial therapeutically.
Similarly, stimulation of a Th2-type response in type I diabetes in mice provides
a protective effect against the disease. Indeed, treatment of NOD mice with IL-4 (which
35 promotes a Th2 response) prevents or delays onset of type I diabetes that norrnally
develops in these mice (Rapoport, M.J., et al. (1993) J. Exp. Med. 178:87-99). Thus,
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stimulation of a Th2 response in a sub~ect suffering from or susceptible to diabetes may
ameliorate the effects of the disease or inhibit the onset of the disease.
Yet another autoimmune disease in which stimulation of a Th2-type response
- may be beneficial is rheumatoid arthritis (RA). Studies have shown that patients with
rheumatoid arthritis have predominantly Thl cells in synovial tissue (Simon, A.K., et
al., (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566). By stimulating a Th2 response
in a subject with RA, the detrimental Thl response can be concomitantly
downmodulated to thereby ameliorate the effects of the disease.
Accordingly, the stimulatory methods of the invention can be used to stimulate
production of Th2-associated cytokines in subjects suffering from, or susceptible to, an
autoimmune disease in which a Th2-type response is beneficial to the course of the
disease. The stimulatory method can involve either direct ;~mini.~tration of a
stimulatory agent to the subject or ex vivo treatment of cells obtained from the subject
(e.g., Thp, Thl cells, B cells, non-lymphoid cells) with a stimulatory agent followed by
re~dmini~tration ofthe cells to the subject. The treatment may be further enhanced by
~mini.ctering other Th2-promoting agents, such as IL-4 itself or antibodies to Thl-
associated cytokines, to the subject in amounts sufficient to further stimulate a Th2-type
response.
In contrast to the autoimmune diseases described above in which a Th2 response
is desirable, other autoimmune diseases may be arneliorated by a Thl-type response.
Such diseases can be treated using an inhibitory agent of the invention (as described
above for cancer and infectious diseases). The treatment may be further enhanced by
~mini.ctrating a Thl-promoting cytokine (e.g, IFN-~) to the subject in amounts
sufficient to further stimulate a Thl-type response.
The efficacy of agents for treating autoimmune diseases can be tested in the
above described animal models of human diseases (e.g, EAE as a model of multiplesclerosis and the NOD mice as a model for diabetes) or other well characterized animal
models of human autoimmune ~ e~ces Such animal models include the mrl/lpr/lpr
mouse as a model for lupus erythematosus, murine collagen-induced arthritis as a model
for rheumatoid arthritis, and murine experimental myasthenia gravis (see Paul ed.,
Flmdamental Immunology, Raven Press, New York, 1989, pp. 840-856). A modulatory
(i.e., stimulatory or inhibitory) agent of the invention is ~1mini~tered to test ~nim~l~ and
the course of the disease in the test ~nim~l~ is then monitored by the standard methods
for the particular model being used. Effectiveness of the modulatory agent is evidenced
by amelioration of the disease condition in ~nim~l~ treated with the agent as compared to
untreated ~nim~l~ (or ~nim~l~ treated with a control agent).
...... .. ..
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Non-limiting examples of autoimmune diseases and disorders having an
autoimmune component that may be treated according to the invention include diabetes
mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus
5 erythematosis, autoimmune thyroiditis? dermatitis (including atopic dermatitis and
eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis
sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to
arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus
10 erythematosus, scleroderma, vaginitis, proctitis, drug eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis,
15 Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial
lung fibrosis.
E. Transplan~ation
While graR rejection or graR acceptance may not be attributable exclusively to
the action of a particular T cell subset (i.e., Thl or Th2 cells) in the graR recipient (for a
discussion see Dallman, M.J. (1995) Curr. Opin. Immunol. 7:632-638), numerous
studies have implicated a predominant Th2 response in prolonged graR survival or a
25 predominant Th2 response in graR rejection. For example, graR acceptance has been
associated with production of a Th2 cytokine pattern and/or graR rejection has been
associated with production of a Thl cytokine pattern (see e.g, Takeuchi, T. et al. (1992)
Transplantation 53:1281-1291; Tzakis, A.G. etal. (1994)J. Pediatr. Surg. 29:754-756;
Thai, N.L. et al. (1995) Transplantation 59:274-281). Additionally, adoptive transfer of
30 cells having a Th2 cytokine phenotype prolongs skin graR survival (Maeda, H. et al.
(1994) Int. Immunol. 6:855-862) and reduces graft-versus-host disease (Fowler, D.H. et
al. (1994) Blood 84:3540-3549; Fowler. D.H. et al. (1994) Prog Clin. Biol. Res.
389:533-540). Still further, :~(lmini~tration of IL-4, which promotes Th2 differentiation,
prolongs cardiac allograR survival (Levy, A.E. and Alexander, J.W. (1995)
35 Transplantation 60:405-406), whereas ~lmini~tration of IL-12 in combination with anti-
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IL-I0 antibodies, which promotes Thl differentiation, enhances skin allograft rejection
(Gorczynski, R.M. et al. (1995) Transplantation 60:1337-1341).
Accordingly, the stimulatory methods of the invention can be used to stimulate
- production of Th2-associated cytokines in transplant recipients to prolong survival of the
5 graR. The stimulatory methods can be used both in solid organ transplantation and in
bone marrow transplantation (e.g, to inhibit graft-versus-host disease). The stimulatory
method can involve either direct a~lmini~tration of a stimulatory agent to the transplant
recipient or ex vivo treatment of cells obtained from the subject (e.g., Thp, Thl cells, B
cells, non-lymphoid cells) with a stimulatory agent followed by re~(lmini~tration of the
10 cells to the subject. The treatment may be further enhanced by ~-lministering other Th2-
promoting agents, such as
IL-4 itself or antibodies to Thl-associated cytokines, to the recipient in amounts
sufficient to further stimulate a Th2-type response.
In addition to the foregoing disease situations, the modulatory methods of the
invention also are useful for other purposes. For example, the stimulatory methods of
the invention (i.e., methods using a stimulatory agent) can be used to stimulateproduction of Th2-promoting cytokines (e.g, IL-4) in vitro for commercial production
of these cytokines (e.g., cells can be contacted with the stimulatory agent in vitro to
20 stimulate IL-4 production and the IL-4 can be recovered from the culture supernatant,
further purified if necessary, and packaged for commercial use).
Furthermore, the modulatory methods of the invention can be applied to
vaccinations to promote either a Thl or a Th2 response to an antigen of interest in a
subject. That is, the agents of the invention can serve as adjuvants to direct an immune
25 response to a vaccine either to a Thl response or a Th2 response. For example, to
stimulate an antibody response to an antigen of interest (i. e., for vaccination purposes),
the antigen and a stimulatory agent of the invention can be coallmini~tered to a subject
to promote a Th2 response to the antigen in the subject, since Th2 responses provide
efficient B cell help and promote IgG1 production. Alternatively, to promote a cellular
30 immune response to an antigen of interest, the antigen and an inhibitory agent of the
invention can be co~lmini~tered to a subject to promote a Thl response to the antigen in
a subject, since Thl responses favor the development of cell-mediated immune
responses (e.g., delayed hypersensitivity responses). The antigen of interest and the
modulatory agent can be formulated together into a single pharmaceutical composition
35 or in separate compositions. In a preferred embodiment, the antigen of interest and the
modulatory agent are ~-lministered simultaneously to the subject. Alternatively, in
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- 50-
certain situations it may be desirable to ~lmini~ter the antigen first and then the
modulatory agent or vice versa (for example, in the case of an antigen that naturally
evokes a Thl response, it may be beneficial to first allmini~ter the antigen alone to
stimulate a Thl response and then ~lmini~ter a stimulatory agent, alone or together with
5 a boost of antigen, to shift the immune response to a Th2 response).
IV. Maf Compositions
Another aspect of the invention pertains to compositions that can be used to
modulate Th2-associated cytokine production by a cell or Thl/Th2 development in a
10 subject in accordance with the methods of the invention. The invention provides
recombinant expression vectors comprising a nucleotide sequence encoding a maf
family protein operatively linked to regulatory sequences that direct expression of the
maf family protein specifically in certain cell types. In a preferred embodiment, the
regulatory sequences direct expression of the maf family protein specifically in15 Iymphoid cells (e.g., T cells or B cells). In one embodiment, the Iymphoid cells are T
cells. T cell specific regulatory elements are known in the art, such as the promoter
regulatory region of T cell receptor genes (see e.g, Winoto and Baltimore (1989) EMBO
J. 8:729-733; Leiden, J.M. (1994) Annu. Rev. lmmunol. 1 1:539-570; Hettman, T. and
Cohen, A. (1994) Mol. Immunol. 31 :315-322, Redondo, J.M. et al. (1991) Mol. Cell.
20 Biol. I 1:5671-5680). Other examples of T cell specific regulatory elements are those
derived from the CD3 gene (see e.g., Clevers, H. et al. (1988) Proc. Natl. Acad. Sci.
USA 85:8623-8627; Clevers, H.C. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8156-
8160; Georgopoulos, K. et al. (1988) EMBO J: 7:2401-2407), the CD4 gene (see e.g,
Sawada, S. and Littman, D.R. (1991) Mol. Cell. Biol. 11:5506-5515; Salmon, P. et al.
25 (1993) Proc. Natl. Acad. Sci. USA 90:7739-7743; Hanna, Z. et al. (1994) Mol. Cell.
Biol. 14:1084-1094; see also GenBank accession numbers U01066 and S68043 for
human CD4 promoter sequences) and the CD2 gene (see e.g, Zhumabekov, T. et al.
(1995)~ Immunol. Methods 185:133 140). ADNAfragmentcomprisingoneormoreT
cell specific regulatory elements, such as a promoter and enhancer of a T cell receptor
30 gene, can be obtained by standard molecular biology methods, such as by PCR using
oligonucleotide primers corresponding to the S' and 3' ends of the desired region and
genomic DNA from T cells as the template. Once the DNA fragment comprising T cell
specific regulatory elements is obtained, it can be operatively linked to a cDNAencoding a maf protein (e.g, the two DNA fragments can be ligated together such that
35 the regulatory elements are located 5' of the maf sequences) and introduced into vector,
such as a plasmid vector, using standard molecular biology techniques.
CA 022~2643 1998-10-22
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In another embodiment, the lymphoid cells are B cells (i.e., within the
recombinant expression vector the nucleotide sequences encoding a maf family protein
are operatively linked to regulatory sequences that direct expression of the maf family
- specifically in B cells). B cell specific regulatory elements are known in the art, such as
5 the promoter regulatory region of immunoglobulin genes (see e.g., Banerji et al. (1983)
Cell 33 :729-740; Queen and Baltimore (1983) Cell 33:741 -748). Other examples of B
cell specific regulatory elements are those derived from the CD20 (B1) gene (see e.g.,
Thevenin, C. etal. (1993)J. Biol. Chem. 268:5949-5956; Rieckmann, P. etal. (1991)J.
Immunol. 147:3994 3999), the Fc epsilon RIIa gene (see e.g, Suter, U. et al. (1989) J.
10 Immunol. 143:3087-3092) and major histocompatibility class II genes (see e.g,Glimcher, L.H. and Kara, C.J. (1992) Annu. Rev. Immunol. 10:13-49; Benoist, C. and
Mathis, D. (1990) Annu. Rev. Immunol. 8:681 -715). A DNA fragment comprising B
cell specific regulatory elements, such as a promoter and enhancer of an
immunoglobulin gene, can be obtained by standard molecular biology methods, such as
15 by PCR using oligonucleotide primers corresponding to the S' and 3' ends of the desired
region and genomic DNA from B cells as the template. Once the DNA fragment
comprising B cell specific regulatory elements is obtained, it can be operatively linked
to a cDNA encoding a maf protein (e.g., the two DNA fragments can be ligated together
such that the regulatory elements are located 5' of the maf sequences) and introduced
20 into vector, such as a plasmid vector, using standard molecular biology techniques.
In yet another embodiment, the invention provides recombinant expression
vectors comprising a nucleotide sequence encoding a maf family protein operatively
linked to regulatory sequences that direct expression of the maf family protein
specifically in hematopoietic stem cells. Hematopoietic stem cell specific regulatory
25 elements are known in the art. Preferably regulatory elements derived from the CD34
gene are used (see e.g, Satterthwaite, A.B. et rll. (1992) Genomics 12:788 794; Burn, T.
C. et al. ( 1992) Blood 80:3051 -3059).
Another aspect of the invention pertains to recombinant host cells that express a
maf family protein. Such host cells can be used to produce a Th2-associated cytokine
30 (e.g., IL-4). Such host cells also can be ~mini.~tered to a subject to produce a Th2-
associated cytokine in the subject as a means to manipulate Thl :Th2 ratios in the
subject. The terms "host cell" and "recombinant host cell" are used interchangeably
herein to refer to a cell into which a recombinant expression vector has been introduced
It is understood that such terms refer not only to the particular subject cell but to the
35 progeny of such a cell. Because certain modifications may occur in sllccee.1ing
generations due to either mutation or environmental influences. such progeny may not,
CA 022~2643 1998-10-22
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in fact, be identical to the parent cell, but as long as these progeny cells retain the
recombinant expression vector, these progeny are still intended to be included within the
scope of the term "host cell" as used herein.
In one embodiment, the invention provides a host lymphoid cell into which a
recombinant expression vector encoding a maf family protein has been introduced. The
host Iymphoid cell can be a T cell or a B cell. A host T cell of the invention can be, for
example a T cell clone that is cultured in vi~ro (such as those described in the Examples)
or, alternatively, a normal T cell that is isolated from a subject (e.g, a peripheral blood T
cell or a splenic T cell). Standard methods for preparing and culturing T cell clones in
vi~ro, or isolating T cells (e.g, from peripheral blood) are known in the art, for example
through the use of mAbs that bind T cell specific cell surface markers (e.g, CD3) or
surface markers for specific subsets of T cells (e.g, CD4 or CD8). The recombinant
expression vector can be introduced into the T cell by one of a variety of knowntransfection methods for introducing DNA into m~mm~ n cells, including calcium
phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, or electroporation. Suitable methods for transforming or transfecting host
cells can be found in Sambrook et al. (Molec~lar Cloning. A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
In another embodiment, the host Iymphoid cell of the invention is a host B cell
into which a recombinant expression vector encoding a maf family protein has been
introduced. The B cell can be, for example a B Iymphoma cell that is cultured in vitro
(such as M12 cells as described in the Examples) or, alternatively, a normal B cell that is
isolated from a subject (e.g, a peripheral blood B cell or a splenic B cell). Various B
Iymphoma cell lines are available in the art and standard methods for culturing such
cells in vi~ro are known. Additionally, standard methods for isolating normal B cells
(e.g, from peripheral blood) are known in the art, for example through the use of mAbs
that bind B cell specific cell surface markers (e.g, membrane immunoglobulin, B7-1,
CD20). The recombinant expression vector can be introduced into the B cell by
standard methods, as described above for T cells.
In yet another embodiment, the invention provides a host hematopoietic stem cellinto which a recombinant expression vector encoding a maf family protein has been
introduced. Hematopoietic stem cells can be isolated from a subject (e.g., from
peripheral blood or bone marrow of the subject) using standard methods known in the
art for isolating such stem cells, for example through the use of mAbs that bindhematopoietic stem cell specific cell surface markers, preferably CD34 (for further
descriptions of isolation of stem cells, see e.g, Wagner, J.E. et al. (1995) Blood 86:512-
. _ t
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523; Murray, L. e~ al. (1995) Blood 85:368-378; Bernardi, A.C. et al. (1995) Scrence
267:104-108; Bernstein, I.D. et al. (1994) Blood Cells 20:15-24; Angelini, A. et al.
(1993) Int. J. Artif~ Organs 16 Suppl. 5: 13-18; Kato, K. and Radburch, A. (1993)
- Cytometry 14:384 392; Lebkowski, J.S. et al. (1992) Transplantation 53:1011-1019;
5 Lebkowski, J. et al. (1993) J. Hematother. 2:339-342). The recombinant expression
vector can be introduced into the hematopoietic stem cell by standard methods, as
described above for T cells.
The skilled artisan will appreciate that the compositions described above with
regard to maf family proteins can be prepared for various different maf family proteins,
10 such as c-Maf and small mafs (e.g., pl 8).
The invention further provides transgenic ~nim~l~ carrying a transgene encoding
a maf family protein. In a preferred embodiment, the maf family transgene is expressed
preferentially or exclusively in T cells of the animal. Tissue-specific expression of maf
family proteins can be achieved through linkage of the maf coding sequences in the
15 transgene to regulatory sequences that direct expression of the encoded protein in a
particular cell type. Such tissue-specific regulatory elements are known in the art and
are described further herein. A preferred regulatory element for T cell-specificexpression of a maf family transgene is the CD4 promoter/enhancer. A preferred maf
family protein for use in the transgene is c-maf. A schematic diagram of a preferred
20 c-maf transgenic construct is shown in Figure 20. In this construct, the CD4
promoter/enhancer is operatively linked to the first intron of the mouse c-maf gene,
which in turn is operatively linked to the mouse c-maf cDNA, which in turn is
operatively linked to an SV40 polyadenylation sequence. Transgenic ~nim~l~ can be
prepared by microinjecting a c-maf transgene construct into fertilized oocytes according
25 to standard procedures for making transgenic ~nim~ls, described in further detail herein.
The phenotype of c-maf transgenic mice that overexpress c-Maf protein in T cells is
described further in Example 17. In brief, these ;~nim~l~ exhibit a phenotype very
similar to IL-4 overexpressing transgenic mice, such as small spleen and thymus,decreased numbers of double positive thymocytes and single positive CD4+ thymocytes,
30 and increased basal levels of serum IgE. Transgenic ;~nim~l~ expressing a maf family
protein can be used, for example, to evaluate test compounds for their ability to
modulate the activity of the maf family protein. For example, a test compound can be
:l~lmini~ered to transgenic animal expressing a maf family protein and the effect of the
test compound in the transgenic animal can be determined by comparing the phenotype
35 of the transgenic animal in the presence of the test compound to the phenotype of the
transgenic animal in the absence of the test compound.
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V. NIP45 Compositions and Methods of Use Thereof
A. Isolated Nucleic Acid Molecules
S One aspect of the invention pertains to isolated nucleic acid molecules that
encode NIP45, or fragments thereof. In a l~le~ell~d embodiment, an isolated nucleic
acid molecule of the invention comprises the nucleotide sequence shown in SEQ IDNO: 5. The sequence of SEQ ID NO: 5 corresponds to the mouse NIP45 cDNA. This
cDNA comprises sequences encoding the NIP45 protein ~i. e., "the coding region", from
nucleotides 13-1248), as well as 5' untr~n.~l~ted sequences (nucleotides 1-12) and 3'
untranslated sequences (nucleotides 1249-1946). Alternatively, the nucleic acid
molecule may comprise only the coding region of SEQ ID NO: 5 (i.e., nucleotides 13-
1248).
Moreover, the nucleic acid molecule of the invention can comprise only a
portion of the coding region of SEQ ID NO: 5, for example a fragment encoding a
biologically active portion of NIP45. The term "biologically active portion of NIP45" is
intended to include portions of NIP45 that retain the ability to interact with the RHD of
NF-AT family proteins. The ability of portions of NIP45 to interact with an NF-AT
RHD can be determined in standard in vitro interaction assays, for example using a NF-
AT RHD fusion protein. Nucleic acid fragments encoding biologically active portions
of NIP45 can be prepared by isolating a portion of SEQ ID NO: 5, expressing the
encoded portion of NIP45 protein or peptide (e.g, by recombinant expression in a host
cell) and assessing the ability of the portion to interact with NF-AT, in particular the
NF-AT RHD, for example using a glutathione-S-transferase (GST)-NF-AT RHD fusion
protein.
The invention further encompasses nucleic acid molecules that differ from SEQ
ID NO: 5 (and fragments thereof) due to degeneracy of the genetic code and thus encode
the same NIP45 protein as that encoded by SEQ ID NO: 5. Accordingly, in another
embodiment, an isolated nucleic acid molecule of the invention has a nucleotide
sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 6.Moreover, the invention encompasses nucleic acid molecules that encode portions of
SEQ ID NO: 6, such as biologically active portions thereof.
A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 5, or a
portion thereof, can be isolated using standard molecular biology techniques and the
sequence information provided herein. For example, a NIP45 cDNA can be isolated
from a cDNA library using all or portion of SEQ ID NO: 5 as a hybridization probe and
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standard hybridization techniques (e.g., as described in Sambrook, J., e~ al. Molecular
Cloning. A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, 1989). Moreover, a nucleic acid molecule encompassing all or a portion of
- SEQ ID NO: 5 can be isolated by the polymerase chain reaction using oligonucleotide
5 primers designed based upon the sequence of SEQ ID NO: 5. For example, mRNA can
be isolated from cells (e.g, by the guanidinium-thiocyanate extraction procedure of
Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using
reverse transcriptase (e.g, Moloney MLV reverse transcriptase, available from
Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku
l O America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for PCR
amplification can be designed based upon the nucleotide sequence shown in SEQ IDNO: 5. A nucleic acid of the invention can be amplified using cDNA or, alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. The nucleic acid so amplified can be cloned
15 into an a~,plol),iate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to a NIP45 nucleotide sequence can be prepared bystandard synthetic techniques, e.g, using an automated DNA synthesizer.
In addition to the NIP45 nucleotide sequence shown in SEQ ID NO: 5, it will be
appreciated by those skilled in the art that DNA sequence polymorphisms that lead to
20 changes in the amino acid sequences of NIP45 may exist within a population. Such
genetic polymorphism in the NIP45 gene may exist among individuals within a
population due to natural allelic variation. Such natural allelic variations can typically
result in 1-5 ~/0 variance in the nucleotide sequence of the a gene. Any and all such
nucleotide variations and resulting amino acid polymorphisms in NIP45 that are the
25 result of natural allelic variation and that do not alter the functional activity of NIP45 are
intended to be within the scope of the invention. Moreover, nucleic acid molecules
encoding NIP45 proteins from other species, and thus which have a nucleotide sequence
that differs from the mouse sequence of SEQ ID NO: 5 but that is related to the mouse
sequence, are intended to be within the scope of the invention. Nucleic acid molecules
30 corresponding to natural allelic variants and human and other m~mm~ n homologues
of the mouse NIP45 cDNA of the invention can be isolated based on their homology to
the mouse NIP45 nucleic acid molecule disclosed herein using the mouse cDNA, or a
portion thereof, as a hybridization probe according to standard hybridization techniques
under stringent hybridization conditions. Accordingly, in another embodiment, an35 isolated nucleic acid molecule of the invention hybridizes under stringent conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 5. ln
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certain embodiment, the nucleic acid is at least 15, 30, 50, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000 or 1500 nucleotides in length. Preferably, an isolated nucleic acid
molecule of the invention that hybridizes under stringent conditions to the sequence of
SEQ ID NO: 5 corresponds to a naturally-occurring nucleic acid molecule. In on
embodiment, the nucleic acid encodes natural human NIP45 protein. In another
embodiment, the nucleic acid molecule encodes a murine NIP45 protein, such as mouse
NIP45 protein.
In addition to naturally-occurring allelic variants of the NIP45 sequence that may
exist in the population, the skilled artisan will further appreciate that changes may be
introduced by mutation into the nucleotide sequence of SEQ ID NO: 5, thereby leading
to changes in the amino acid sequence of the encoded protein, without altering the
functional activity of the NIP45 protein. For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues may be made in the
sequence of SEQ ID NO: 5. A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of NIP45 (e.g, the sequence of SEQ ID NO: 6)
without altering the functional activity of NIP45, such as its ability to interact with an
NF-AT RHD or its ability to synergize with NF-AT and c-Maf in stimulating gene
transcription, whereas an "essential" amino acid residue is required for functional
activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules
encoding NlP45 proteins that contain changes in amino acid residues that are notessential for NIP45 activity. Such NIP45 proteins differ in amino acid sequence from
SEQ ID NO: 6 yet retain NIP45 activity. In one embodiment, the isolated nucleic acid
molecule comprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least 60 % homologous to the amino acid sequence
of SEQ ID NO: 6 and interacts with the RHD of an NF-AT family protein. Preferably,
the protein encoded by the nucleic acid molecule is at least 70 % homologous to SEQ ID
NO: 6, more preferably at least 80 % homologous to SEQ ID NO: 6, even more
preferably at least 90 % homologous to SEQ ID NO: 6, and most preferably at least 95
% homologous to SEQ ID NO: 6.
To determine the percent homology of two amino acid sequences (e.g, SEQ ID
NO: 6 and a mutant form thereof), the sequences are aligned for optimal comparison
purposes (e.g, gaps may be introduced in the sequence of one protein for optimalalignment with the other protein). The amino acid residues at corresponding amino acid
positions are then compared. When a position in one sequence (e.g, SEQ ID NO: 6) is
occupied by the same amino acid residue as the corresponding position in the other
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sequence (e.g., a mutant form of NIP45), then the molecules are homologous at that
position (i. e., as used herein amino acid "homology" is equivalent to amino acid
"identity"). The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., % homology = # of identical
5 positions/total # of positions x 100).
An isolated nucleic acid molecule encoding a NIP45 protein homologous to the
protein of SEQ ID NO: 6 can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 5 such
that one or more amino acid substitutions, additions or deletions are introduced into the
10 encoded protein. Mutations can be introduced into SEQ ID NO: 5 by standard
techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue having a similar
15 side chain. Families of amino acid residues having similar side chains have been
defined in the art, including basic side chains (e.g., Iysine, arginine, histidine), acidic
side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g,
~l~nine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),
20 beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g, tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue in
NIP45 protein is preferably replaced with another amino acid residue from the same side
chain family. Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a NIP45 coding se~uence, such as by saturation
25 mutagenesis, and the resultant mutants can be screened for their ability to interact with
an NF-AT RHD (e.g, using a GST-NF-AT-RHD fusion protein) to identify mutants that
retain NF-AT-interacting ability. Following mutagenesis of SEQ ID NO: 5, the encoded
mutant protein can be expressed recombinantly in a host cell and the ability of the
mutant protein to interact with NF-AT can be determined using an in vitro interaction
30 assay. For example, a recombinant NIP45 protein (e.g, a mutated or truncated form of
SEQ ID NO: 6) can be radiolabeled and incubated with a GST-NF-AT RHD fusion
protein. Glutathione-sepharose beads are then added to the mixture to precipitate the
NIP45-GST-NF-AT RHD complex, if such a complex is formed. After washing the
beads to remove non-specific binding, the amount of radioactive protein associated with
35 the beads is determined and compared to the amount of radioactive protein rem~ining in
.
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the eluate to thereby determine whether the NIP45 protein is capable of interacting with
the RHD of NF-AT.
Another aspect of the invention pertains to isolated nucleic acid molecules thatare antisense to the coding strand of a NIP45 mRNA or gene. An antisense nucleic acid
5 of the invention can be complementary to an entire NIP45 coding strand, or to only a
portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a
coding region of the coding strand of a nucleotide sequence encoding NIP45 (e.g., the
entire coding region of SEQ ID NO: 5 comprises nucleotides 13-1248). In another
embodiment, the antisense nucleic acid molecule is antisense to a noncoding region of
10 the coding strand of a nucleotide sequence encoding NIP45. In certain embodiments, an
antisense nucleic acid of the invention is at least 15, 30, 50, 100, 200, 300, 400, 500,
600, 700, 800? 900, 1000 or 1500 nucleotides in length.
Given the coding strand sequences encoding NIP45 disclosed herein (e.g, SEQ
ID NO: 5), antisense nucleic acids of the invention can be designed according to the
15 rules of Watson and Crick base pairing. The antisense nucleic acid molecule may be
complementary to the entire coding region of NIP45 mRNA, or alternatively can be an
oligonucleotide which is ~nti.cen.~e to only a portion of the coding or noncoding region of
NIP45 mRNA. For example, the antisense oligonucleotide may be complementary to
the region surrounding the translation start site of NIP45 mRNA. An antisense
20 oligonucleotide can be, for example. about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in
length. An antisense nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g, an antisense oligonucleotide) can be chemically
synthesized using naturally occurring nucleotides or variously modified nucleotides
25 designed to increase the biological stability of the molecules or to increase the physical
stability of the duplex formed between the antisense and sense nucleic acids, e.g,
phosphorothioate derivatives and acridine substituted nucleotides can be used.
Alternatively, the antisense nucleic acid can be produced biologically using an
expression vector into which a nucleic acid has been subcloned in an antisense
30 orientation (i. e., RNA transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest, described further in the following
subsection).
In another embodiment, an antisense nucleic acid of the invention is a ribozyme.Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of
35 cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. A ribozyme having specificity for a NIP45-encoding nucleic
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acid can be designed based upon the nucleotide sequence of a NIP45 cDNA disclosed
herein (i. e., SEQ ID NO: 5). For example, a derivative of a Tetrahymena L- 19 IVS
RNA can be constructed in which the base sequence of the active site is complementary
to the base sequence to be cleaved in a NIP45-encoding mRNA. See for example Cech
5 et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No.5,116,742.
Alternatively, NIP45 mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and
Szostak, J.W. (1993) Science 261: 1411-1418.
Yet another aspect of the invention pertains to isolated nucleic acid molecules
10 encoding NIP45 fusion proteins. Such nucleic acid molecules, comprising at least a first
nucleotide sequence encoding a NIP45 protein, polypeptide or peptide operatively linked
to a second nucleotide sequence encoding a non-NIP45 protein, polypeptide or peptide,
can be prepared by standard recombinant DNA techniques. NIP45 fusion proteins are
described in further detail below in subsection C.
B. Recombinant Expression Vectors and Hos~ Cells
Another aspect of the invention pertains to vectors, preferably recombinant
expression vectors, cont~ining a nucleic acid encoding NIP45 (or a portion thereof). The
expression vectors of the invention comprise a nucleic acid of the invention in a form
20 suitable for expression of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory sequences, selected on
the basis of the host cells to be used for ~x~re~ion, which is operatively linked to the
nucleic acid sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence of interest is linked
25 to the regulatory sequence(s) in a manner which allows for expression of the nucleotide
sequence (e.g, in an in vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory sequence" is intended to
includes promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described, for example, in
30 Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, CA (1990). Regulatory sequences include those which direct constitutive
expression of a nucleotide sequence in many types of host cell and those which direct
expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific
regulatory sequences). It will be appreciated by those skilled in the art that the design of
35 the expression vector may depend on such factors as the choice of the host cell to be
transforrned, the level of expression of protein desired, etc. The expression vectors of
.. . . .. , . ... , . . , . . . . ~ .. ~ . .. . . . ..
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the invention can be introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g.,
NIP45 proteins, mutant forms of NIP45 proteins, NIP45 fusion proteins and the like).
The recombinant expression vectors of the invention can be designed for
S expression of NIP45 protein in prokaryotic or eukaryotic cells. For example, NIP45 can
be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression
vectors) yeast cells or m~mm~ n cells. Suitable host cells are discussed further in
Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, CA (1990). Altematively, the recombinant expression vector may be
10 transcribed and translated in vitro, for example using T7 promoter regulatory sequences
and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with
vectors containing constitutive or inducible promotors directing the expression of either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein
15 encoded therein, usually to the amino terminus of the recombinant protein. Such fusion
vectors typically serve three purposes: 1) to increase expression of recombinant protein;
2) to increase the solubility of the recombinant protein; and 3) to aid in the purification
of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion
expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion
20 moiety and the recombinant protein to enable sel)araLion of the recombinant protein from
the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B.
and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA)25 and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET 1 ld (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
30 California (1990) 60-89). Target gene expression from the pTrc vector relies on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene
expression from the pET 1 ld vector relies on transcription from a T7 gnlO-lac fusion
promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral
polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
35 prophage harboring a T7 gnl gene under the transcriptional control of the lacUV S
promoter.
t
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One strategy to maximize recombinant protein expression in E. coli is to expressthe protein in a host bacteria with an impaired capacity to proteolytica]ly cleave the
recombinant protein (Gottesm~n, S., Gene Expression Technology: Methods in
- Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in E. coli (Wada et al., (1992) Nuc. Acids ~es. 20:2111-2118).
Such alteration of nucleic acid sequences of the invention can be carried out by standard
DNA synthesis techniques.
In another embodiment, the NIP45 expression vector is a yeast expression vector.Examples of vectors f'or expression in yeast S. cerivisae include pYepSecl (Baldari. et
al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen
Corporation, San Diego, CA).
Alternatively, NIP45 can be expressed in insect cells using baculovirus
expression vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells (e.g, Sf 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) ~irology
170:31 -39).
In yet another embodiment, a nucleic acid of the invention is expressed in
m~mm~ n cells using a m~mm~ n expression vector. Examples of m~mm~ n
expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987) Nature 329:840) and
pMT2PC (K~llfm~n et al. ( 1987), EMBO J. 6: 187- 195). When used in m~mm~ n cells,
the expression vector's control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40.
In another embodiment, the recombinant m~mm~ n expression vector is
capable of directing expression of the nucleic acid preferentially in a particular cell type
(e.g, tissue-specific regulatory elements are used to express the nucleic acid). Tissue-
specific regulatory elements are kno~vn in the art. Non-limiting examples of suitable
tissue-specific promoters include lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43 :235-275), in particular promoters of T cell receptors (Winoto
and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983)
Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), the albumin promoter
(liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), neuron-specific promoters
(e.g, the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA
,. ~, ~ . . .. . .
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86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),
and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No.
4,873,316 and European Application Publication No. 264,166). Developmentally-
regulated promoters are also encompassed, for example the murine hox promoters
5 (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter
(Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation.
That is, the DNA molecule is operatively linked to a regulatory sequence in a manner
10 which allows for expression (by transcription of the DNA molecule) of an RNA molecule
which is antisense to NIP45 mRNA. Regulatory sequences operatively linked to a nucleic
acid cloned in the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell types, for instance viral
promoters and/or enhancers, or regulatory sequences can be chosen which direct
15 constitutive, tissue specific or cell type specific expression of antisense RNA. The
antisense expression vector can be in the form of a recombinant plasmid, phagemid or
attenuated virus in which antisense nucleic acids are produced under the control of a high
efficiency regulatory region, the activity of which can be determined by the cell type into
which the vector is introduced. For a discussion of the regulation of gene expression using
20 antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic
analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to recombinant host cells into which a
vector, preferably a recombinant expression vector, of the invention has been
introduced. A host cell may be any prokaryotic or eukaryotic cell. For example, NIP45
25 protein may be expressed in bacterial cells such as E coli, insect cells, yeast or
m~mm~ n cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other
suitable host cells are known to those skilled in the art. Vector DNA can be introduced
into prokaryotic or eukaryotic cells via conventional transformation or transfection
techniques. As used herein, the terms "transformation" and "transfection" are intended
30 to refer to a variety of art-recognized techni~ues for introducing foreign nucleic acid
(e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-
precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
Suitable methods for transforming or transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
35 Laboratory press (1989)), and other laboratory manuals.
......
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For stable transfection of m~mm~ n cells, it is known that, depending upon the
expression vector and transfection technique used, only a small fraction of cells may
integrate the foreign DNA into their genome. In order to identify and select these
- integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is
S generally introduced into the host cells along with the gene of interest. Preferred
selectable markers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable marker may be
introduced into a host cell on the same vector as that encoding NIP45 or may be
introduced on a separate vector. Cells stably transfected with the introduced nucleic
10 acid can be identified by drug selection (e.g., cells that have incorporated the selectable
marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) NIP45 protein. Accordingly, the invention
further provides methods for producing NIP45 protein using the host cells of the15 invention. In one embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding NIP45 has been
introduced) in a suitable medium until NIP45 is produced. In another embodiment, the
method further comprises isolating NIP45 from the medium or the host cell. In its
native form NIP45 protein is an intracellular protein and, accordingly, recombinant
20 NIP45 protein can be expressed intracellularly in a recombinant host cell and then
isolated from the host cell, e.g, by Iysing the host cell and recovering the recombinant
NIP45 protein from the Iysate. Alternatively, recombinant NIP45 protein can be
prepared as a extracellular protein by operatively linking a heterologous signal sequence
to the amino-terminus of the protein such that the protein is secreted from the host cells.
25 In this case, recombinant NIP45 protein can be recovered from the culture medium in
which the cells are cultured.
Certain host cells of the invention can also be used to produce nonhuman
transgenic ~nim~l~ For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or an embryonic stem cell into which NIP45-coding sequences have
30 been introduced. Such host cells can then be used to create non-human transgenic
~nim~1s in which exogenous NIP45 sequences have been introduced into their genome
or homologous recombinant ~nim~l.c in which endogenous NIP45 sequences have beenaltered. Such ~nim~l~ are useful for studying the function and/or activity of NIP45 and
for identifying and/or evaluating modulators of NIP45 activity. Accordingly, another
35 aspect of the invention pertains to nonhuman transgenic ~nim~l~ which contain cells
carrying a transgene encoding a NIP45 protein or a portion of a NIP45 protein. In a
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subembodiment, of the transgenic zlnimzll.s of the invention, the transgene alters an
endogenous gene encoding an endogenous NIP45 protein (e.g., homologous
recombinant ~nim~ls in which the endogenous NIP45 gene has been functionally
disrupted or "knocked out", or the nucleotide sequence of the endogenous NIP45 gene
5 has been mutated or the transcriptional regulatory region of the endogenous NIP45 gene
has been altered).
A transgenic animal of the invention can be created by introducing NIP45-
encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g, by
microinjection, and allowing the oocyte to develop in a pseudopregnant female foster
10 animal. The mouse NIP45 cDNA sequence of SEQ ID NO: 5 can be introduced as a
transgene into the genome of a non-human animal (e.g., a mouse). Alternatively, a
m~mm~ n homologue of the mouse NIP45 gene, such as a human NIP45 gene, can be
isolated based on hybridization to the mouse NIP45 cDNA and used as a transgene.Intronic sequences and polyadenylation signals can also be included in the transgene to
15 increase the efficiency of expression of the transgene. A tissue-specific regulatory
sequence(s) can be operably linked to the NIP45 transgene to direct expression of NIP45
protein to particular cells. Methods for generating transgenic :~nimz~ls via embryo
manipulation and microinjection, particularly ~nim~ls such as mice, have become
conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and
20 4,870,009, both by Leder et ~l., U.S. Patent No. 4,873,191 by Wagner et al. and in
Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other
transgenic ~nim~ A transgenic founder animal can be identified based upon the
presence of the NIP45 transgene in its genome and/or expression of NIP45 mRNA in25 tissues or cells of the ~nim~l.s. A transgenic founder animal can then be used to breed
additional ~nim~ carrying the transgene. Moreover, transgenic ~nim~l.s carrying a
transgene encoding NIP45 can further be bred to other transgenic ~nim~ls carrying other
transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
30 at least a portion of a NIP45 gene into which a deletion, addition or substitution has
been introduced to thereby alter, e.g, functionally disrupt, the endogenous NIP45 gene.
The NIP45 gene preferably is a mouse NIP45 gene. For example, a mouse NIP45 genecan be isolated from a mouse genomic DNA library using the mouse NIP45 cDNA of
SEQ ID NO: 5 as a probe. The mouse NIP45 gene then can be used to construct a
35 homologous recombination vector suitable for altering an endogenous NIP45 gene in the
mouse genome. In a preferred embodiment, the vector is designed such that, upon
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homologous recombination, the endogenous NIP45 gene is functionally disrupted (i. e.,
no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination,
the endogenous NIP45 gene is mutated or otherwise altered but still encodes functional
5 protein (e.g, the upstream regulatory region can be altered to thereby alter the
expression of the endogenous NIP45 protein). ln the homologous recombination vector,
the altered portion of the NIP45 gene is flanked at its 5' and 3' ends by additional nucleic
acid of the NIP45 gene to allow for homologous recombination to occur between the
exogenous NIP45 gene carried by the vector and an endogenous NIP45 gene in an
10 embryonic stem cell. The additional fl~nkin~ NIP45 nucleic acid is of sufficient length
for successful homologous recombination with the endogenous gene. Typically, several
kilobases of fl~nking DNA (both at the 5' and 3' ends) are included in the vector (see
e.g., Thomas, K.R. and Capecchi, M. ~. (1987) Cell 51 :503 for a description of
homologous recombination vectors). The vector is introduced into an embryonic stem
15 cell line (e.g., by electroporation) and cells in which the introduced NIP45 gene has
homologously recombined with the endogenous NIP45 gene are selected (see e.g., Li, E.
et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an
animal (e.g, a mouse) to form aggregation chimeras (see e.g., Bradley, A. in
Terafoca~cinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson,20 ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a
suitable pseudopregnant female foster animal and the embryo brought to term. Progeny
harboring the homologously recombined DNA in their germ cells can be used to breed
~nim~l~ in which all cells of the animal contain the homologously recombined DNA by
germline tr~n~mi~ion of the transgene. Methods for constructing homologous
25 recombination vectors and homologous recombinant ~nim~l~ are described further in
Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT
International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by
Smithies e~ al.; WO 92/096~ by Zijlstra et al.; and WO 93/04169 by Berns et al.
C. Isolated NIP45 Proteins and Anti-NIP45 Antibo~ s
Another aspect of the invention pertains to isolated NIP45 proteins, and portions
thereof, such as biologically active portions, as well as peptide fragments suitable as
immunogens to raise anti-NIP45 antibodies. In one embodiment, the invention provides
an isolated pl~d~ion of NIP45 protein. Preferably, the NIP45 protein has an amino
acid sequence shown in SEQ ID NO: 6. In other embodiments, the NIP45 protein is
substantially homologous to SEQ ID NO: 6 and retains the functional activity of the
.. . , . , .. , . .. , " .. , , ., ~ . ,
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protein of SEQ ID NO: 6 yet differs in amino acid sequence due to natural allelic
variation or mutagenesis, or is a m~mm~ n homologue of the protein of SEQ ID NO: 6
(e.g., a human homologue), as described in detail in subsection A above. Accordingly,
in another embodiment, the NIP45 protein is a protein which comprises an amino acid
5 sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 6 and
that interacts with the RHD of an NF-AT family protein. Preferably, the protein is at
least 70 % homologous to SEQ ID NO: 6, more preferably at least 80 % homologous to
SEQ ID NO: 6, even more preferably at least 90 % homologous to SEQ ID NO: 6, andmost preferably at least 95 % homologous to SEQ ID NO: 6.
In other embodiments, the invention provides isolated portions of the NIP45
protein. For example, the invention further encompasses a portion of a NIP45 protein
that interacts with NF-AT. As demonstrated in the examples, NIP45 protein interacts
with the RHD of NF-AT. An in vitro interaction assay (such as that described above in
subsection A lltili7ing a GST-NF-AT RHD fusion protein) can be used to determine the
15 ability of NIP45 peptide fragments to interact with the NF-AT Rel Homology Domain to
thereby identify peptide fragments that interact with NF-AT.
NIP45 proteins are preferably produced by recombinant DNA techniques. For
example, a nucleic acid molecule encoding the protein is cloned into an expression
vector (as described above), the expression vector is introduced into a host cell (as
20 described above) and the NIP45 protein is expressed in the host cell. The NIP45 protein
can then be isolated from the cells by an applop,iate purification scheme using standard
protein purification techniques. Alternative to recombinant expression, a NIP45
polypeptide can be synthesized chemically using standard peptide synthesis techniques.
Moreover, native NIP45 protein can be isolated from cells (e.g, from T cells), for
25 example by irnmunoprecipitation using an anti-NIP45 antibody.
The invention also provides NIP45 fusion proteins. As used herein, a NIP45
"fusion protein" comprises a NIP45 polypeptide operatively linked to a non-NIP45polypeptide. A "NIP45 polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to NIP45 protein, or a peptide fragment thereof, whereas a
30 "non-NIP45 polypeptide" refers to a polypeptide having an amino acid sequencecorresponding to another protein. Within the fusion protein, the term "operatively
linked" is intended to indicate that the NIP45 polypeptide and the non-NIP45
polypeptide are fused in-frame to each other. The non-NIP45 polypeptide may be fused
to the N-terminus or C-terminus of the NIP45 polypeptide. For example, in one
35 embodiment, the fusion protein is a GST-NIP45 fusion protein in which the NIP45
sequences are fused to the C-terminus of the GST sequences. In another embodiment,
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the fusion protein is a NIP45-HA fusion protein in which the NIP45 nucleotide sequence
is inserted in to the pCEP4-HA vector (Herrscher, R.E;. et al. (1995) Genes I~ev. 9:3067-
3082) such that the NIP45 sequences are fused in frame to an influenza hemagglutinin
- epitope tag. Such fusion proteins can facilitate the purification of recombinant NIP45.
Preferably, a NIP45 fusion protein of the invention is produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance with conventional
techniques, for example employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as applol)liate, alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primers which give rise
to complementary overhangs between two consecutive gene fragments which can
subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for
example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley &
Sons: 1992). Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g, a GST polypeptide or an HA epitope tag). A
NIP45-encoding nucleic acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the NIP45 protein.
An isolated NIP45 protein, or fragment thereof, can be used as an immunogen to
generate antibodies that bind NIP45 using standard techniques for polyclonal andmonoclonal antibody preparation. The NIP45 protein can be used to generate antibodies
or, alternatively, an antigenic peptide fragment of NIP45 can be used as the immunogen.
An antigenic peptide fragment of NIP45 typically comprises at least 8 amino acidresidues of the amino acid sequence shown in SEQ ID NO: 6 and encompasses an
epitope of NIP45 such that an antibody raised against the peptide forms a specific
immune complex with NIP45. Preferably, the antigenic peptide comprises at least 10
amino acid residues, more preferably at least 15 amino acid residues, even more
preferably at least 20 amino acid residues, and most preferably at least 30 amino acid
residues. Preferred epitopes encompassed by the antigenic peptide are regions of NIP45
that are located on the surface of the protein, e.g., hydrophilic regions. A
hydrophobicity analysis of the NIP45 protein sequence of SEQ ID NO: 6 is shown in
Figure 12.
A NIP45 immunogen typically is used to prepare antibodies by immunizing a
suitable subject, (e.g., rabbit, goat, mouse or other m~mm~l) with the immunogen. An
. .
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-68-
appropriate immunogenic plc~aldlion can contain, for examples, recombinantly
expressed NIP45 protein or a chemically synthesized NIP45 peptide. The preparation
can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or
similar immunostimulatory agent. Immunization of a suitable subject with an
5 immunogenic NIP45 preparation induces a polyclonal anti-NIP45 antibody response.
Accordingly, another aspect of the invention pertains to anti-NIP45 antibodies.
Polyclonal anti-NIP45 antibodies can be prepared as described above by immunizing a
suitable subject with a NIP45 immunogen. The anti-NIP45 antibody titer in the
immunized subject can be monitored over time by standard techniques, such as with an
10 enzyme linked immunosorbent assay (ELISA) using immobilized NIP45. If desired, the
antibody molecules directed against NIP45 can be isolated from the m~mm~l (e.g, from
the blood) and further purified by well known techniques, such as protein A
chromatography to obtain the IgG fraction. At an appropriate time after immunization,
e.g., when the anti-NIP45 antibody titers are highest, antibody-producing cells can be
15 obtained from the subject and used to prepare monoclonal antibodies by standard
techni~ues, such as the hybridoma technique originally described by Kohler and
Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol 127:539-
46; Brown et al. (1980) JBiol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31;
and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell
20 hybridoma technique (Kozbor et al. ( 1983) Immunol Today 4:72), the EBV-hybridoma
technique (Cole et al. ( 1985), Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal
antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal
Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New25 York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.
Gefter e~ al. (1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell line
(typically a myeloma) is fused to Iymphocytes (typically splenocytes) from a m~mm~l
immunized with a NIP45 immunogen as described above, and the culture supern~t~nt~
of the resulting hybridoma cells are screened to identify a hybridoma producing a
30 monoclonal antibody that binds NIP45.
Any of the many well known protocols used for fusing Iymphocytes and
immortalized cell lines can be applied for the purpose of generating an anti-NIP45
monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Gene~., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth,
35 Monoclonal Antibodies, cited supra). Moreover, the ordinary skilled worker will
appreciate that there are many variations of such methods which also would be useful.
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Typically, the immortal cell line (e.g, a myeloma cell line) is derived from the same
m~mm~ n species as the Iymphocytes. For example, murine hybridomas can be made
by fusing Iymphocytes from a mouse immunized with an immunogenic preparation of
- the present invention with an immortalized mouse cell line. Preferred immortal cell
5 lines are mouse myeloma cell lines that are sensitive to culture medium cont~inin~;
hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of
myeloma cell lines may be used as a fusion partner according to standard techniques,
e.g., the P3-NSl/l-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These
myeloma lines are available from the American Type Culture Collection (ATCC),
10 Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse
splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the
fusion are then selected using HAT medium, which kills unfused and unproductively
fused myeloma cells (unfused splenocytes die after several days because they are not
transformed). Hybridoma cells producing a monoclonal antibody of the invention are
15 detected by screening the hybridoma culture supernatants for antibodies that bind
NIP45, e.g, using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal anti-NIP45 antibody can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody phage display
20 library) with NIP45 to thereby isolate immunoglobulin library members that bind
NIP45. Kits for generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-
9400-01; and the Stratagene SurJ~APTM Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly amenable for use in
25 generating and screening antibody display library can be found in, for example, Ladner
et al. U.S. Patent No. 5~223,409; Kang et al. International Publication No. WO
92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al.
International Publication WO 92/20791; Markland et al. International Publication No.
WO 92/15679; Breitling et al. Int~rn~tional Publication WO 93/01288; McCafferty et al.
30 International Publication No. WO 92/01047; Garrard et al. International Publication No.
WO 92/09690; Ladner e~ al. lnternational Publication No. WO 90/02809; Fuchs et al.
(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-
85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-
734; Hawkins et al. (1992) JMol Biol 226:889-896; Clarkson et al. (1991) Nature
35 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) NucAcid Res 19:4133-4137;
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W O97/39721 PCTrUS97/06708
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Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-
554.
Additionally, recombinant anti-NIP45 antibodies, such as chimeric and
hnm~ni7ed monoclonal antibodies, comprising both human and non-human portions,
which can be made using standard recombinant DNA techniques, are within the scope of
the invention. Such chimeric and hl-m~ni7~d monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using methods described in
Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT
Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science 240: 1041 - 1043; Liu
et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. l~es. 47:999- 1005; Wood eJ
al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst. 80:1553-
1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques
4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141 :4053-4060.
An anti-NIP45 antibody (e.g, monoclonal antibody) can be used to isolate
NIP45 by standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-NIP45 antibody can facilitate the purification of natural NIP45 from cells and of
recombinantly produced NIP45 expressed in host cells. Moreover, an anti-NIP45
antibody can be used to detect NIP45 protein (e.g, in a cellular Iysate or cell
supernatant). Detection may be facilitated by coupling (i. e., physically linking) the
antibody to a detectable substance. Accordingly, in one embodiment, an anti-NIP45
antibody of the invention is labeled with a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, ~Ik~line phosphatase, ,B-galactosidase~ or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rho~mine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
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D. P/tur.. n~eJ~ti~nl Compositions
The NIP45 proteins and anti-NIP45 antibodies of the invention can be
incorporated into pharmaceutical compositions suitable for administration. Such
- compositions typically comprise the protein or antibody and a pharmaceutically
acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like, compatible with
pharmaceutical ~(lmini~tration The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the compositions is
contemplated. Supplementary active compounds can also be incorporated into the
compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its intended route of ~tlmini.~tration. For example, solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as ethylene(li~minetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases,
such as hydrochloric acid or sodium hydroxide. The parenteral preparation can beenclosed in ampoules, disposable syringes or multiple dose vials made of glass or
plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous plepald~ion of sterile injectable solutions or dispersion. For
intravenous a(lmini~tration, suitable carriers include physiological saline, bacteriostatic
water, ~remophor ELTM (BASF, P~ippally, NJ) or phosphate buffered saline (PBS).
In all cases, the composition must be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of manufacture and storage
and must be preserved against the cont~min:~tinE action of microor~;~ni~m.c such as
bacteria and fungi. The carrier can be a solvent or dispersion medium cont~ininp, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity
can be maintained, for example, by the use of a coating such as lecithin, by the
~ ... ...
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maintenance of the required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorg~nism~ can be achieved by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include
5 isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
10 compound (e.g., a NIP45 protein or anti-NIP45 antibody) in the required amount in an
appropriate solvent with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those enumerated above. In
15 the case of sterile powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They
20 can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral
therapeutic a~lmini~tration, the active compound can be incorporated with excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically
25 compatible binding agents, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tr~g~c~nth or gelatin; an excipient such as starch or
lactose, a ~li.cintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant
30 such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
In one embodiment, the active compounds are prepared with carriers that will
protect the compound against rapid elimin~tion from the body, such as a controlled
35 release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
, .. .. ~ ,.
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polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations will be apl)arellt to those skilled in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected
5 cells with monoclonal antibodies to viral antigens) can also be used as ph~ ceutically
acceptable carriers. These may be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
E. Methods of Detecting NIP45 Activity
Another aspect of the invention pertains to a method of using the various NIP45
compositions of the invention. For example, the invention provides a method for
detecting the presence of NIP45 protein or mRNA in a biological sample. The method
involves contacting the biological sample with an agent capable of detecting NIP45
protein or mRNA such that the presence of NIP45 protein or mRNA is detected in the
15 biological sample. A preferred agent for detecting NIP45 mRNA is a labeled nucleic
acid probe capable of hybridizing to NIP45 mRNA. The nucleic acid probe can be, for
example, the NIP45 cDNA of SEQ ID NO: 5, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 nucleotides in length and sufficient to specifically hybridize under stringent
20 conditions to NIP45 mRNA. A preferred agent for detecting NIP45 protein is a labeled
antibody capable of binding to NIP45 protein. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2)
can be used. The term "labeled", with regard to the probe or antibody, is intended to
encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking)
25 a detectable substance to the probe or antibody, as well as indirect labeling of the probe
or antibody by reactivity with another reagent that is directly labeled. Examples of
indirect labeling include detection of a primary antibody using a fluorescently labeled
secondary antibody and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The term "biological sample" is
30 intended to include tissues, cells and biological fluids. For example, techniques for
detection of NIP45 mRNA include Northern hybridizations and in situ hybridizations.
Techniques for detection of NIP45 protein include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations and immunofluorescence.
.... . ...
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VI.. Combination Compositions and Kits
Compositions comprising combinations of modulatory agents are also provided
by the invention. For example, two or more nucleotide sequences encoding transcription
factors that regulate Th2-associated cytokine gene expression can be incorporated into a
5 recombinant expression vector and introduced into a host cell. For example, the
invention provides recombinant vectors, and host cells into which such vectors have
been introduced, comprising a first nucleotide sequence encoding a first transcription
factor that cooperates with an NF-AT family protein to regulate expression of the Th2-
associated cytokine gene and a second nucleotide sequence encoding a second
10 transcription factor that contributes to the regulation of the Th2-associated cytokine
gene. Preferably, the first nucleotide sequence encodes a maf family protein (e.g., c-
Maf) or an NF-AT-interacting protein (e.g., NIP45). Preferably, the second nucleotide
sequence encodes a transcription factor selected from the group consisting of NF-AT
family proteins, NF-AT-interacting proteins, maf family proteins and AP-I family1 5 proteins.
Kits for mo~ ting Th2-associated cytokine production or Thl/Th2 subset
development are also encompassed by the invention. In one embodiment, a kit of the
invention comprises at least one modulatory agent of the invention packaged withinstructions for using the modulatory agent to modulate Th2-associated cytokine
20 production or Thl/Th2 subset development. In one embodiment, the kit comprises at
least one stimulatory agent for use in stimulating Th2-associated cytokine production or
upregulating Th2 subset development (or downregulating Thl subset development). In
another embodiment, the kit comprises at least one inhibitory agent for use in inhibiting
Th2-associated cytokine production or downregulating Th2 subset development (or
25 upregulating Thl subset development). Combination kits, comprising two or more of
the modulatory (e.g, stimulatory or inhibitory) agents of the invention are also provided.
VII. Screenin~ Assays
Another aspect of the invention pertains to screening assays for identifying
30 compounds that modulate the activity of a transcription factor that regulates expression
of a Th2-associated cytokine gene. In various embodiments, these screening assays can
identify, for example, compounds that modulate the expression or functional activity of
the transcription factor, proteins that interact with the transcription factor, as well as
compounds that modulate these protein-protein interactions, and compounds that
35 modulate the interaction of the transcription factor with a cis-acting target site (e.g., a
MARE) within a Th2-associated cytokine gene.
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In one embodiment, the invention provides a method for identifying a compound
that modulates the activity of a transcription factor that cooperates with a Nuclear Factor
of Activated T cells (NF-AT) family protein to regulate expression of a Th2-associated
- cytokine gene, comprising
providing a indicator composition having activity of a transcription factor thatcooperates with an NFAT family protein to regulate expression of the Th2-associated
cytokine gene;
contacting the indicator composition with a test compound; and
deterrnining the effect of the test compound on the activity of the transcription
factor in the indicator composition to thereby identify a compound that modulates the
activity of a transcription factor that cooperates with an NFAT family protein to regulate
expression of a Th2-associated cytokine gene.
The transcription factor can be, for example, a maf family protein, such as c-Maf
or a small maf protein (e.g., p l 8). Alternatively, the transcription factor can be a factor
interacts with an NF-AT family protein, such as NIP45.
The indicator composition can be a cell-free composition or it can be a cellularcomposition. For example, in one embodiment, the indicator composition is a Iymphoid
cell, such as a Th2 cell. In another embodiment, the indicator composition is a yeast
cell.
In a preferred embodiment, the indicator composition comprises an indicator
cell, wherein said indicator cell comprises: (i) the transcription factor and (ii) a reporter
gene responsive to the transcription factor. Preferably, the indicator cell contains:
i) a recombinant expression vector encoding the transcription factor; and
ii) a vector comprising regulatory sequences of a Th2-associated cytokine
gene operatively linked a reporter gene; and
said method comprises:
a) contacting the indicator cell with a test compound;
b) determining the level of expression of the reporter gene in the indicator cell in
the presence of the test compound; and
c) comparing the level of expression of the reporter gene in the indicator cell in
the presence of the test compound with the level of expression of the reporter gene in the
indicator cell in the absence of the test compound to thereby identify a compound that
modulates the activity of the transcription factor.
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In another preferred embodiment, the indicator composition comprises a
preparation of: (i) the transcription factor and (ii) a DNA molecule to which the
transcription factor binds, and
said method comprises:
a) contacting the indicator composition with a test compound;
b) determining the degree of interaction of the transcription factor and the DNAmolecule in the presence of the test compound; and
c) comparing the degree of interaction of the transcription factor and the DNA
molecule in the presence of the test compound with the degree of interaction of the
transcription factor and the DNA molecule in the absence of the test compound tothereby identify a compound that modulates the activity of the transcription factor.
In one embodiment of the foregoing method, the transcription factor is a maf
family protein and the DNA molecule comprises a maf response element (MARE).
In another preferred embodiment, the method identifies a protein from Th2 cells
that interacts with the transcription factor. In this embodiment,
the indicator composition is an indicator cell, which indicator cell comprises:
i) a reporter gene operably linked to a transcriptional regulatory
sequence; and
ii) a first chimeric gene which encodes a first fusion protein, said first
fusion protein including the transcription factor that cooperates with
an NFAT family protein to regulate expression of a Th2-associated
cytokine gene;
the test compound comprises a librarv of second chimeric genes, which library
encodes second fusion proteins, the second fusion proteins including proteins derived
from Th2 cells;
expression of the reporter gene being sensitive to interactions between the first
fusion protein, the second fusion protein and the transcriptional regulator,v sequence; and
wherein the effect of the test compound on the transcription factor in the
indicator composition is determined by determining the level of expression of the
reporter gene in the indicator cell to thereby identify a test compound comprising a
protein from Th2 cells that interacts with the transcription factor.
The invention further provides a method for identifying a compound that
modulates an interaction between NIP45 and an NF-AT family protein, comprising:
a) combining:
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(i) NIP45, or an NF-AT-interacting portion thereof; and
(ii) an NF-AT family protein, or a NIP45-interacting portion thereof;
in the presence and absence of a test compound;
b) determining the degree of interaction between (i) and (ii) in the presence and
5 absence of the test compound; and
c) identifying a compound that modulates an interaction between NIP45 and an
NF-AT family protein.
Preferably, the NIP45-interacting portion of the NF-AT family protein comprises
the Rel Homology Domain of the NF-AT family protein. In one embodiment, the
10 degree of interaction between (i) and (ii) is determined by labeling (i) or (ii) with a
detectable substance, isolating non-labeled (i) or (ii) and quantitating the amount of
labeled (i) or (ii) that has become associated with non-labeled (i) or (ii). The method can
be used to identify compounds that either increase or decrease the interaction between
NIP45 and an NF-AT family protein.
In a preferred embodiment of the screening assays of the invention, once a test
compound is identified as mocl~ tin~ the activity of a transcription factor of interest, the
effect of the test compound on an immune response is then tested. Accordingly, the
screening methods of the invention can further comprise determining the effect of the
20 compound on an immune response to thereby identify a compound that modulates an
immune response. In one embodiment, the effect of the compound on an immune
response is determined by determining the effect of the compound on expression of a
Th2-associated cytokine gene, such as an interleukin-4 gene. In another embodiment,
the effect of the compound of interest on an immune response is determined by
25 determining the effect of the compound on development of T helper type I (Th 1 ) or T
helper type 2 (Th2) cells.
Recombinant expression vectors that can be used for expression of a
transcription factor in the indicator cell are known in the art (see discussions above and
30 also the Examples). In one embodiment, within the expression vector the transcription
factor-coding sequences are operatively linked to regulatory sequences that allow for
constitutive expression of the transcription factor in the indicator cell (e.g., viral
regulatory sequences, such as a cytomegalovirus promoter/enhancer, can be used). Use
of a recombinant expression vector that allows for constitutive expression of the
35 transcription factor in the indicator cell is preferred for identification of compounds that
enhance or inhibit the activity of the transcription factor. In an alternative embodiment,
, . ,, . ~ .. . .
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within the expression vector the transcription factor-coding se4uences are operatively
linked to regulator,v sequences of the endogenous corresponding transcription factor
gene (i. e., the promoter regulatory region derived from the endogenous gene). Use of a
recombinant expression vector in which transcription factor expression is controlled by
the endogenous regulatory sequences is preferred for identification of compounds that
enhance or inhibit the transcriptional expression of the transcription factor.
In methods in which a Th2-associated cytokine gene is utilized, preferably, the
Th2-associated cytokine is interleukin-4. It has previously shown that Th2-specific,
inducible IL-4 expression can be directed by as little as 157 bp of the proximal IL-4
promoter in Th2 cells (Hodge, M. et al. (1995) J. Immunol M 54:6397-6405).
Accordingly, in one embodiment, a method of the invention utilizes a reporter gene
construct containing this region of the proximal IL-4 promoter, most preferably
nucleotides -157 to +58 (relative to the start site of transcription at + 1) of the IL-4
promoter. Alternatively, stronger reporter gene expression can be achieved using a
longer portion of the IL-4 upstream regulatory region, such as about 3 kb of upstream
regulatory sequences. Suitable reporter gene constructs are described in Todd, M; et al.
(1993) J. Exp. Med M77:1663 1674. See also the Examples for descriptions of IL-4reporter gene constructs.
A variety of reporter genes are known in the art and are suitable for use in thescreening assays of the invention. Examples of suitable reporter genes include those
which encode chloramphenicol acetyltransferase, beta-galactosidase, alkaline
phosphatase or luciferase. Standard methods for measuring the activity of these gene
products are known in the art.
A variety of cell types are suitable for use as an indicator cell in the screening
assay. Preferably a cell line is used which does not normally express c-Maf, such as a B
cell (e.g., the M12 B Iymphoma cell line) or a Thl cell clone (e.g., AE7 cells).Nonlymphoid cell lines can also be used as indicator cells, such as the HepG2 hepatoma
cell line.
In one embodiment, the level of expression of the reporter gene in the indiGatorcell in the presence of the test compound is higher than the level of expression of the
reporter gene in the indicator cell in the absence of the test compound and the test
compound is identified as a compound that stimulates the expression or activity of the
transcription factor. In another embodiment, the level of expression of the reporter gene
in the indicator cell in the presence of the test compound is lower than the level of
expression of the reporter gene in the indicator cell in the absence of the test compound
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and the test compound is identified as a compound that inhibits the expression or
activity of the transcription factor.
Alternative to the use of a reporter gene construct, compounds that modulate theexpression or activity of a transcription factor can be identified by using other "read-
5 outs." For example, an indicator cell can be transfected with a transcription factorexpression vector, incubated in the presence and in the absence of a test compound, and
Th2-associated cytokine production can be assessed by detecting cytokine mRNA (e.g,
IL-4 mRNA) in the indicator cell or cytokine secretion (i.e., IL-4 secretion) into the
culture supernatant. Standard methods for detecting cytokine mRNA, such as reverse
10 transcription-polymerase chain reaction (RT-PCR) are known in the art. Standard
methods for detecting cytokine protein in culture supernatants, such as enzyme linked
immunosorbent assays (ELISA) are also known in the art. For further descriptions of
methods for detecting cytokine mRNA and/or protein, see also the Examples.
As described above, the invention provides a screening assay for identifying
proteins (e.g, proteins in Th2 cells) that interact with a transcription factor of interest,
e.g., c-Maf or NF-AT or NIP45. These assays can be designed based on the two-hybrid
assay system (also referred to as an interaction trap assay) known in the art (see e.g,
Field U.S. Patent No. 5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al.
20 (1993)J. Biol. Chem. 268:12046-12054;Barteletal. (1993)Biofec~niques 14:920-924;
and Iwabuchi et al. (1993) Oncogene 8:1693-1696). The two-hybrid assay is generally
used for identifying proteins that interact with a particular target protein. The assay
employs gene fusions to identify proteins capable of interacting to reconstitute a
functional transcriptional activator. The transcriptional activator consists of a DNA-
25 binding domain and a transcriptional activation domain, wherein both domains arerequired to activate transcription of genes downstream from a target sequence (such as
an upstream activator sequence (UAS) for GAL4). DNA sequences encoding a target
"bait" protein are fused to either of these domains and a library of DNA sequences is
fused to the other domain. "Fish" fusion proteins (generated from the fusion library)
30 capable of binding to the target-fusion protein (e.g, a target GAL4-fusion "bait') will
generally bring the two domains (DNA-binding domain and transcriptional activation
domain) into close enough proximity to activate the transcription of a reporter gene
inserted downstream from the target sequence. Thus, the "fish" proteins can be
identified by their ability to reconstitute a functional transcriptional activator (e.g., a
35 functional GAL4 transactivator).
... .. , .. . ,~ _ . ~ .
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This general two-hybrid system can be applied to the identification of proteins in
Th2 cells that interact with c-Maf (or, using similar methods, with other transcription
factors of interest) by construction of a target c-Maf fusion protein (e.g., a c-Maf/GAL4
binding domain fusion as the "bait") and a cDNA library of "fish" fusion proteins (e.g., a
5 cDNAIGAL4 activation domain library), wherein the cDNA library is prepared from
mRNA of Th2 cells, and introducing these constructs into a host cell that also contains a
reporter gene construct linked to a regulatory sequence responsive to c-Maf (e.g., a
MARE sequence, for examp}e a region of the IL-4 promoter, as discussed above).
cDNAs encoding proteins from Th2 cells that interact with c-Maf can be identified
10 based upon transactivation of the reporter gene construct.
Alternatively, a "single-hybrid" assay, such as that described in Sieweke, M.H. et
al. (1996) Cell 85:49-60, can be used to identify proteins from Th2 cells that interact
with c-Maf. This assay is a modification of the two-hybrid system discussed above. In
this system, the "bait" is a transcription factor from which the transactivation domain has
15 been removed (e.g, c-Maf from which the amino-terminal transactivation domain has
been removed) and the "fish" is a non-fusion cDNA library (e.g, a cDNA library
prepared from Th2 cells). These constructs are introduced into host cells (e.g., yeast
cells) that also contains a reporter gene construct linked to a regulatory sequence
responsive to the transcription factor (e.g, a MARE sequence, for example a region of
20 the IL-4 promoter, responsive to c-Maf). cDNAs encoding proteins from Th2 cells that
interact with c-Maf (or other transcription factor of interest) can be identified based upon
transactivation of the reporter gene construct.
As described above, the invention provides a screening assay for identifying
25 compounds that modulate the interaction of a transcription factor of the invention with a
DNA molecule to which the transcription factor binds, such as c-Maf and a MARE in an
IL-4 gene regulatory region, respectively. Assays are known in the art that detect the
interaction of a DNA binding protein with a target DNA sequence (e.g., electrophoretic
mobility shift assays, DNAse I footprinting assays and the like; for further descriptions
30 see the Examples). By performing such assays in the presence and absence of test
compounds, these assays can be used to identify compounds that modulate (e.g, inhibit
or enhance) the interaction of the DNA binding protein with its target DNA sequence.
In one embodiment, the amount of binding of the transcription factor to the DNA
fragment in the presence of the test compound is greater than the amount of binding of
3 5 the transcription factor to the DNA fragment in the absence of the test compound, in
which case the test compound is identified as a compound that enhances binding of the
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transcription factor. In another embodiment, the amount of binding of the transcription
factor to the DNA fragment in the presence of the test compound is less than the amount
of binding of the transcription factor to the DNA fragment in the absence of the test
compound, in which case the test compound is identified as a compound that inhibits
5 binding of the transcription factor.
In the methods of the invention for identifying agents that modulate an
interaction between NIP45 and an NF-AT family protein, isolated NIP45 and/or NF-AT
family proteins may be used in the method, or, alternatively, only portions of NIP45
10 and/or an NF-AT family protein may be used. For example, an isolated NF-AT Rel
Homology Domain (or a larger subregion of NF-AT that includes the RHD) can be used
as the NIP45-interacting portion of NF-AT. Likewise, a portion of NIP45 capable of
binding to the NF-AT RHD may be used. In a preferred embodiment, one or both of (i)
and (ii) are fusion proteins, such as GST fusion proteins (e.g., GST-NF-AT RHD can be
15 used as the NIP45-interacting portion of NF-AT). The degree of interaction between (i)
and (ii) can be deterrnined, for example, by labeling one of the proteins with a detectable
substance (e.g., a radiolabel), isolating the non-labeled protein and quantitating the
amount of detectable substance that has become associated with the non-labeled protein.
The assay can be used to identify agents that either stimulate or inhibit the interaction
20 between NIP45 and an NF-AT family protein. An agent that stimulates the interaction
between NIP45 and an NF-AT family protein is identified based upon its ability to
increase the degree of interaction between (i) and (ii) as compared to the degree of
interaction in the absence of the agent, whereas an agent that inhibits the interaction
between NIP45 and an NF-AT family protein is identified based upon its ability to
25 decrease the degree of interaction between (i) and (ii) as compared to the degree of
interaction in the absence of the agent. Assays systems for identifying agents that
modulate SH2 domain-ligand interactions as described in U.S. Patent No. 5,352,660 by
Pawson can be adapted to identifying agents that modulate the NIP45/NF-AT RHD
interaction.
This invention is further illustrated by the following examples which should notbe construed as limiting. The contents of all references, patents and published patent
applications cited throughout this application are hereby incorporated by reference.
Nucleotide and amino acid sequences deposited in public databases as referred to herein
35 are also hereby incorporated by reference.
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EXAMPLE 1: Cytokine Specificity is Due to a Positive Transacting Factor
and Not to a Repressor
Tissue specificity can be achieved through the action of repressor or silencer
proteins. Thus it was possible that the IL-2 and IL-4 genes were actively repressed in
Th2 and Thl cells respectively. To test for the existence of repressor proteins, somatic
cell fusions were performed between a Th 1 (D I . I ) and a Th2 (D 10) clone of differing
MHC Class I haplotypes. The Thl clone Dl.l (Kd) and the Th2 clone D10 (Kk) were
fused according to the "suspension cell fusion" procedure (Lane, R.D. et al. (1986)
Methods Enzymol. 121 :183-192). After fusion, the cells were allowed to recover for 8
hours and then double- stained using PE-conjugated anti-Kk and FITC-conjugated anti-
Kd antibodies (Ph~nningen, La Jolla, CA). Cells were then sorted on the basis of size to
distinguish unfused cells from hetero and homokaryons and by fluorescence to identify
single-positive and double-positive cells. As indicated in the schematic of this approach
shown in Figure I A, three populations were sorted for: large PE-positive cells (D1.1 x
Dl.1), large FITC-positive cells (D10 x D10), and large PE and FITC positive cells
(Dl.1 x D10). Cells ~x~res~ing both MHC class I Kb and Kk markers were
heterokaryons while cells expressing only Kb or Kk represented homokaryons and
served as controls.
The three populations were then stimulated in culture with antibodies to CD3 to
activate cytokine gene expression and RNA prepared for RT-PCR and Northern blot
analysis. Approximately SxlO5 cells were obtained for each population. Routinely, 5-
10% of the cells had undergone fusion. Each of these three populations was then split in
half, one half transferred to pre-rinsed anti-CD3 coated plates, the rem~ining half to
uncoated plates. After four hours, the cells were harvested, and poly(A+) RNA isolated
using the Micro-FastTrackTM kit (Stratagene, La Jolla, CA). cDNA was made using
the SuperScript kit (Gibco/BRL, Bethesda, MD), and used for PCR analysis using
commercially available primers specific for murine IL-2, IL-4 and ~-actin according to
the manufacturer's instructions (Stratagene, La Jolla, CA). PCR reactions included 0.5
Ci a-32P-dCTP (3000 Ci/mmol, NEN Dupont). PCR products were ethanol
precipitated, separated by nondenaturing PAGE and dried and Vis~ i7~cl by
autoradiography .
The results of the RT-PCT analysis of cytokine mRNA expression are shown in
Figure lB. The Thl and Th2 clones and the Th homokaryons transcribed only IL-2
(Thl) or IL-4 (Th2) respectively, while the Thl/Th2 heterokaryons produced both
cytokines. In contrast, the existence of repressor protein(s) should have resulted in the
.,
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extinction of both cytokines in the heterokaryons. From these experiments, it was
concluded that cytokine specificity in Thl vs. Th2 cells was mediated by Th-specific
positive transacting factors rather than by selective silencer proteins.
5 EXAMPLE 2: Isolation of a Th2-Specific c-maf Gene from a cDNA Library
Prepared from an Anti-CD3 Activated Th2 Clone
In the course of screening a cDNA library prepared from an anti-CD3 activated
Th2 clone, D 10, for NF-AT-interacting proteins by the yeast two-hybrid system (for
10 descriptions of this system, see e.g., Field U.S. Patent No. 5,283,173; Zervos et al.
(1993) Cell 72:223-232; Madura et al. (1993) J: Biol. Chem. 268:12046-12054; Bartel e~
al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696), multiple cDNAs were isolated, all of which were extremely weak interactors. All
cDNAs obtained in this screen were next evaluated for Th-specific expression by
15 Northern blot analysis using a panel of Thl and Th2 clones. One such cDNA, which
was repeatedly isolated (60 of 140) detected transcripts only in RNA prepared from Th2
clones (Dl0, CDC35) and not from either Thl clones (AR5, OS6, D1) or from a B cell
lymphoma, Ml2, as illustrated in the Northern blot analysis depicted in Figure 2A.
Further, the levels oftranscripts detected in Dl0 Th2 cells were substantially increased
20 upon activation by ligation of the T cell receptor with anti-CD3 antibody. No induction
of the transcript detected by this cDNA clone occurred in Th l clones upon anti-CD3
treatment. A control probe, GAPDH, demonstrated approximately equal loading of
RNA in all lanes. Thus, the expression of this cDNA clone in the Iymphoid lineage
appeared to be Th2-specific and sensitive to signals transmitted through the T cell
25 receptor. For these Northern blots, total RNA was prepared by using Trizol
(GIBCO/BRL) according to manufacturer's instructions. 10 ~lg oftotal RNA from each
sample was fractionated on a formaldehyde agarose gel and transferred to a nylonmembrane. A 300 bp DraI fragment derived from the 3 ' untr~n~l~ted region of theisolated clone was labeled with a-32P-dCTP using Random Primed DNA Labeling Kit
30 (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed using
QuikHyb (Stratagene, La Jolla, CA) according to m~nllf~turer's instructions.
To determine whether the expression of this gene was tissue-specific and
regulated during the course of normal Th cell development, the following experiment
was performed. Naive spleen cells (Th precursor (Thp) cells) were driven along a Thl
35 or Th2 pathway by treatment with anti-CD3 in the presence of cytokines and anti-
cytokine antibodies (IFN~ and anti-IL-4 for Thl, IL-4 and anti-IFNg for Th2). Splenic
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cell suspensions were prepared from 6-8 wee~-old Balb/c mice, cultured in RPMI 1640
supplemented with 10% FCS at a density of lo6 cells/ml, and stimulated with plate
bound anti-CD3 antibody in the presence of 5 ~lg/ml of anti-IL4 antibody ( I I B 1 1 ) for
the Th 1 lineage, or 5 ~g/ml of anti-IFN~ antibody (XMG- 1 ) for the Th2 lineage. 24
S hours after stimulation, 50 U/ml IL2 was added to all cultures, and 500 U/ml IL4
(Genzyme) was added to Th2 cultures. 7 days after the primary stimulation, all cells
were harvested, washed and restimulated with plate bound anti-CD3 antibody. Northern
blot analysis of differentiating cells harvested at various time points after stimulation in
a primary (day 0-8) and secondary (0-20 hours) response was performed, using the10 methodology described above, and identification of differentiating Thp cells as Thl or
Th2 was determined by analyzing culture supernatants by ELISA for IL-I0 and IFNy.
ELISA for cytokine quantitation was performed as follows. All anti-cytokine antibodies
were purchased from Pharmingen. ELISA was performed according to Pharmingen's
instructions with the exception that Avidin-Alkaline Phosphatase (Sigma) at 1:500
15 dilution in PBS/BSA was used in place of avidin-peroxidase. P-nitrophenyl phosphate
(GIBCO BRL) at 4 mg/ml in substrate buffer (10% diethanolamine, 0.5 mM MgC12,
0.02% sodium azide, pH 9.8) was used as substrate.
In two independent experiments, representative results of which are shown in
Figure 2B, this analysis revealed low level or undetectable expression of this cDNA in
20 naive spleen cells at baseline at day 0. In cultures differentiating along a Th2 pathway,
substantial induction of transcripts occurred by day 8 in a primary stimulation and by 20
hr in a secondary stimulation. In contrast, no induction occurred in cells being driven
along a Thl pathway. A control probe (GAPDH) showed approximately equal loading
of RNA in all lanes. The low level of transcripts present in cells being driven along a
25 Thl pathway likely reflects the presence of residual Th2 cells since complete skewing
does not occur in this in vitro differentiation system.
Together, these experiments revealed that the isolated cDNA is selectively
expressed in Th2 clones, where it is induced upon T cell activation, and that it is absent
from Thl clones and a B Iymphoma. Further, this gene is induced in normal Thp when
30 they are driven towards the Th2 lineage, but is not induced during Thl development.
The cDNA obtained from the yeast two-hybrid screen was used as a probe to
isolate a full-length cDNA from a Dl 0 Th2 cell cDNA library by standard hybridization
methods. A 4.3 kb cDNA clone was isolated from the Th2 cell library and sequenced by
standard methods. Sequence analysis revealed that this Th2-specific gene corresponded
35 in sequence to the c-mafproto-oncogene.
-
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EXAMPLE 3: Ectopic Expression of c-Maf in Thl and B Cells Results in
Activation of the IL-4 Promoter
The identification of the isolated cDNA described in Example 2 as a member of
S the AP-I/CREB/ATF gene family, together with its selective expression in Th2 cells
raised the possibility that c-Maf controlled the tissue-specific transcription of the IL-4
gene. Additionally, the presence of transcripts encoding c-maf correlated well with IL-4
expression in Th2 cells and in three of four transformed mast cell lines examined. To
test whether c-Maf could transactivate the IL-4 promoter, cotransfection experiments
10 were performed.
Thl clones and the B Iymphoma M12.4.C3 (M12) neither express c-mafnor
transcribe the IL-4 gene. If c-Maf is the transcription factor critical for controlling IL-4
gene expression, then forced expression in these cells should permit IL-4 gene
expression. To test this, the full-length (4.3 kb) c-maf cDNA clone was inserted into the
15 SalI site of the
pMex-NeoI m~mm~ n expression vector, which utilizes the CMV enhancer to drive
expression of the inserted sequence. The c-Maf expression vector was then
cotransfected with an IL-4 promoter reporter construct into the Thl clone AE7 and the B
Iymphoma M 12. The generation of the wild type IL4 CAT reporter construct,
20 cont~ining an IL4 promoter fragment from -157 to +68 operatively linked to a
chloramphenicol acetyltransferase gene is described in Hodge, M. et al. (1995) J.
Immunol. 154:6397 6405. The Thl clone was cultured in RPMI 1640 supplemented
with 10% FCS and 10% Con-A stimulated rat splenocyte supernatant, and maintainedby bi-weekly stimulation with appropriate antigen and APCs. M12 cells were cultured
25 in RPMI 1640 supplemented with 10% FCS.
The Thl clone AE7 or M12 B Iymphoma cells were transiently transfected by
preincubating 0.4 ml of cells, containing 2x107 cells/ml AE7 or 3X106 cells/ml M 12
cells in serum-free RPMI 1640 with 20 llg (AE7) or 5 llg (M 12) of each plasmid for 10
minutes at room temperature. The samples were then electroporated using a BIO-RAD
30 Gene Pulser (BIO-RAD, Richmond, CA) set at 975 ,uF, 280 V, and immediately placed
on ice for 10 minutes. The transfected cells were allowed to recover overnight in
complete media and stimulated with plate bound anti-CD3 antibody ({Ph~rmingen, San
Diego, CA} 1 ~g/ml in lXPBS overnight at 4 ~C) or with 50 ng/ml PMA (Sigma, St.
Louis, MO) and 1 IlM Ionomycin (Calbiochem Corp., La Jolla, California). for 24 hours
35 Cell Iysate was prepared by freeze-thaw lysis in 0.25 M Tris-CI, pH 7.8. Equal amounts
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- 86 -
of protein (between S-20 ~g) were used for CAT assays. CAT assays were performed as
described in Todd, M. et al. ( 1993) J. Exp. Med 177: 1663- 1674.
It has previously shown that Th2-specific, inducible IL-4 expression can be
directed by as little as 157 bp of the proximal IL-4 promoter in Th2 cells (Hodge, M. et
5 al. (1995) J. Immunol M54:6397-6405). In cotransfection experiments, the results of
which are summarized in Figure 3A, it is demonstrated that ectopic expression of c-Maf
in the Th l clone AE7 results in substantial activity of the IL-4 promoter reporter after
stimulation through the T cell receptor. The fold induction observed was approximately
5 fold over that observed with the control empty vector alone. Although expression of a
10 reporter construct cont~ining proximal (-157 to ~58) IL-4 promoter sequences in the
subclone of AE7 cells utilized here has not been previously observed, it has been
demonstrated that small amounts of IL-4 mRNA can be detected by RT-PCR in other
subclones of AE7. To more rigorously test the ability of c-Maf to transactivate the IL-4
promoter in a non-IL-4 producing cell, the same experiment was performed in the B
15 Iymphoma cell line, M12. Norrnal B cells and B Iymphoma cells do not produce IL-4.
Representative results of the cotransfection experiments are depicted in Figure 3B and a
summary of three independent experiments is shown below in Table l .
Table 1
CAT Activity (fold induction)
Plasmids PMA/iono. Exp. I* Exp. II Exp. III
pMEX-NeoI/pREP4
+ 7.6 l 1.4
pMEX-Maf/pREP4 - 95 5 18.6
+ 186 7 37
pMEX-c-Fos/pREP4 - 2.7 1 0.8
+ 7.6 1.2
pMEX-JunD/pREP4 - ND** 0.9 0.5
+ ND 1.4 l.9
pMEX-NeoI/pREP4-NF-ATp - 14.2 1.6 0.3
+ 41.2 3.5 0.3
pMEX-Maf/pREP4-NF-ATp - 136 54 26.3
+ 138 lO0 54.7
pMEX-c-Fos/pREP4-NF-ATp - 7.4 1.6 3
+ 15.4 l.9 6.1
20 * In experiment I, 20 mg of cell Iysate was incubated f'or 2 hours. In experiments II and
III, only 5 mg of cell Iysate was incubated for l hour in order to reveal synergy between
c-Maf and NF-ATp
**ND = not done
SUBSTITUTE SHEET (RULE 26)
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Ihe results in M12 B Iymphoma cells confirmed the findings in the Thl clone.
Ectopic expression of c-Maf resulted in substantial activity of the IL-4 promoter in M12
cells, either unstimulated or stimulated with PMA/Ca++ ionophore. The fold induction
5 observed when compared to transfection of a control vector averaged approximately 50
in unstimulated M12 cells. Stimulation of M12 cells with PMA/Ca++ ionophore, which
should result in translocation of NF-ATs to the nucleus and induction of other AP- 1
family members (Fl~n~g~n, W.M. (1991) Nature 352:803-807; Jain, J. et al. (1993)Nat~re 365:352-355), increased the basal activity of the IL-4 promoter, but a marked
10 induction in promoter activity by c-Maf was still present (average of approximately 25
fold). C-Maf did not transactivate a control reporter driven by NF-AT multimers,demonstrating the specificity of transactivation.
As a control for the specificity of c-Maf as opposed to other AP- 1 family
members, the c-Fos and c-Jun proteins were also overexpressed in M12 cells utili7ing
15 murine full-length cDNAs encoding c-Fos and JunD in the m~mm~ n expression
vector of pMEX-NeoI together with the IL-4 reporter plasmid. No IL-4 promoter
activity could be achieved by overexpression of either of these two AP- l familymembers in M 12 cells. Thus, c-Maf has a unique ability to drive IL-4 gene transcription
in M12 B cells. Further, forced expression of c-Maf in the hepatoma cell line HepG2
20 also resulted in IL-4 promoter transactivation. These experiments demonstrate that the
provision of c-Maf to c-Maf negative Thl or B cells, or to non-lymphoid cells (e.g., a
hepatoma cell line), permits the cells to transactivate the IL-4 promoter.
NF-AT proteins have been shown to be critically important in the regulation of
both the IL-4 and IL-2 cytokines. NF-ATp was the first member of this family to be
25 isolated (McCaffrey, P.G. et al. (1993) Science 262:750-754). Both AE7 and M12 cells
have endogenous NF-ATp protein, but nevertheless do not transcribe IL-4. Although
NF-ATp could not therefore account for selective IL-4 gene transcription, it was of
interest to test whether overexpression of NF-ATp in unstimulated or stimulated Ml2
cells would further increase the transactivation of the IL-4 promoter by c-maf. M 12
30 cells were cotransfected with the IL-4 reporter construct and either an NFAPpexpression vector (pREP4-NF-ATp, which also carries a hygromycin resistance gene)
alone or the NFAPp expression vector together with the c-Maf expression vector.
Overexpression of NF-ATp alone in M12 cells resulted in some modest transactivation
of the IL-4 promoter. This transactivation was markedly increased by ectopic
35 expression of c-Maf, an increase which was not just additive but was synergistic (see
Fig. 3B and Table 1). In contrast, c-Fos overexpression did not further increase the
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modest transactivation achieved by NF-ATp. These results indicate that c-maf and NF-
ATp interact to achieve maximal induction of the IL-4 promoter, the tissue-specificity
being provided by
c-Maf.
EXAMPLE 4: Ectopic Expression of C-Maf Activates Transcription
of the Endogenous IL-4 Gene in a B Lymphoma
As demonstrated in Example 3, c-Maf transactivates the IL-4 promoter in
10 transient transfection assays in Thl, B and non-lymphoid cells. To test whether
expression of c-maf in non-IL-4 producing cells can activate the transcription of
endogenous IL-4, the B Iymphoma M12 was stably transfected with expression vectors
encoding c-maf, NF-ATp or both, or junD with and without NF-ATp as a control. For
stable transfection, M12 cells were transfected as described above in Example 3. The
15 transfected cells were allowed to recover in complete media for 48 hours before the
addition of Neomycin (GIBCO/BRL, Gaithersburg, MD) and Hygromycin (C~albiochem,
Corp.) at a concentration of 400 ~Lg/ml of each antibiotic. The transfected cells were
supplemented with fresh media every other day.
Stably transfected M12 cells were plated at equal density supernatants harvested20 24 hours later to measure cytokines by ELISA. ELISAs were performed as described in
Example 2. The results, shown in Figure 4, demonstrate that in these experiments M12
cells transfected with c-maf, junD or NF-ATp alone did not produce measurable IL-4 by
ELISA. However, M12 cells stably transfected with both c-maf and NF-ATp did
produce detectable, but low level, IL-4 by ELISA. These results were confirmed by RT-
25 PCR on RNA from these transfected cells. In contrast, these cells did not producedetectable IL-2. The requirement for both c-maf and NF-ATp is consistent with the
synergistic effect of these factors in the transactivation of the IL-4 promoter noted in the
transient transfection experiments in M12 cells. In contrast, transfection of junD, an
AP-l family member which can increase IL-4 expression in Th2 cells, alone or together
30 with NF-ATp, did not result in IL-4 production. These results demonstrate the essential
and selective role of c-Maf in directing tissue-specific endogenous IL-4 production.
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EXAMPLE 5: A Site in the IL-4 Promoter is Footprinted by
Extracts from Th2 but not Thl Clones
~ The experiments described in Examples 3 and 4 demonstrated a clear functional
5 role for c-maf in controlling tissue-specific expression of IL-4. Further, c-maf
transcripts were expressed in Th2 but not Thl cells. However, DNA-protein complexes
were not detected by electrophoretic mobility shift assays (EMSA) when using nuclear
extracts prepared from Th2 cells. To further examine whether a protein in Th2 nuclear
extracts might bind to the MARE, or nearby sequences, the more sensitive technique of
10 DNAseI footprinting was used. Two Th2 clones (D10, CDC35) and two Thl clones
(AE7, S53) were activated by ligation of the T cell receptor with plate-bound anti-CD3
antibody, and nuclear extracts prepared at time 0 (unstimulated), 2 hours and 6 hours
later. DNAseI footprinting analysis was then performed according to standard methods
using a Klenow end-labeled IL-4 promoter fragment (-157 to +68). The results are15 shown in Figure 5A. Stimulated extracts from both Thl and Th2 cells footprinted the
two NF-AT sites and the AP-1 site upstream of the distal NF-AT site as describedpreviously (Rooney, J. et al. (1995) Immunity 2:545-553), consistent with the
demonstrated function of NF-AT and AP- 1 proteins in regulating both the IL-2 and the
IL-4 promoters (Rooney, J. et al. (1995) Immuni~y 2:545-553; Rooney, J. et al. (1995)
20 Mol. Cell. Biol. 15:6299 6310). Furtherrnore, inspection ofthe autoradiograph revealed
an area of hypersensitivity on the non-coding strand at residues -28 and -29 when
extracts from stimulated Th2 but not stimulated Thl cells were used. Unstimulated Th
cell extracts did not footprint this region. The Th2 footprint observed was subtle, but
reproducible in two experiments and is located in a site that has previously been
25 demonstrated to be critical for IL-4 promoter activation in Th2 cells (Hodge, M. et al.
(1995) J. Immunol. 154:6397-6405). A schematic summary of sites occupied in the IL-4
promoter as detected by footprint analysis is shown in Figure 5B. These results indicate
that a site in the proximal IL-4 promoter, previously shown to be functionally important,
is occupied in activated Th2 but not in activated Thl cells.
EXAMPLE 6: Recombinant c-maf Binds to a MARE Site in the IL-4 Promoter
The Th2-specific footprint does not contain a c-maf response element (MARE).
However, ex:~rnin~tion of the proximal IL-4 promoter revealed a half c-maf binding site
35 (MARE) ( residues -42 to -37) immediately downstream of the proximal NF-AT site
(residues -56 to -51) (shown schematically in Fig SB). It has previously been
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demonstrated that mutation of this site abolished activity of the IL-4 promoter in Th2
cells (Hodge, M. et al. (1995) J. Immunol. 154:6397-6405). To determine if c-Mafbound this site, a truncated
c-Maf recombinant protein containing the b-zip domain(amino acids 171 -371) was
5 expressed from E. coli, purified on an S-Tag agarose column and used in electrophoretic
mobility shift assays with radiolabeled MARE oligonucleotide.
The expression vector for recombinant c-Maf was constructed by inserting a
cDNA fragment encoding a.a. residues 171 to 371 of c-Maf (disclosed in Kurschner C.
and Morgan, J.I. (1995) Mol. Cell. Biol. 15:246 254) into the NotI site of pET2910 (Novagen, Inc. Madison, WI). The truncated c-Maf protein was expressed using T7
polymerase in the BL21(DE3)strain. Cells were induced by the addition of I mM IPTG
and incubated at 37~ C for 3 hours. The induced cells were Iysed in I X Bind/Wash
buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.1% Triton X-100) followed by
sonication. The c-Maf protein was then purified from the soluble fraction by using the
15 S-Tag Purification Kit (Novagen) according to manufacturer's instructions. Two
additional proteins, NF-ATp and c-Jun, were also used in EMSA assays. The
recombinant NF-ATp, cont~ining the Rel domain of murine NF-ATp, was expressed
using an in vitro transcription/translation vector TP7-N~-ATp, which contains a cDNA
fragment encoding the Rel domain of murine NF-ATp. The c-Jun expression vector,
20 pGEM-c-Jun, was constructed by inserting a full-length cDNA of murine c-Jun into the
PstI site of pGEM4. 1 ~lg of each plasmid DNA was transcribed from the T7 promoter
and translated in rabbit reticulocyte Iysate by using the TnT Coupled
Transcription/Translation Kit (Promega, Madison, WI).
Electrophoretic mobility shift assays (EMSA) were perforrned as follows. 100
25 ng of double-stranded oligonucleotides were end-labeled with ~-32P-dATP (DuPont
NEN Research Product, Wilmington, DE) using T4 polynucleotide kinase (Pharmacia
LKB Biotechnology, Inc., Piscataway, NJ). The labeled ds-oligonucleotides were
fractionated on 15-20% polyacrylamide gels, eluted overnight at 37~ C in lX TE and
precipitated in ethanol. Binding assays were performed at room temperature for 20
30 minutes using 0.5 llg of recombinant proteins or 4 ~LI of in vitro translated products, 500
ng poly(dI-dC), and 20,000 cpm of probe in a 15 ~I volume of 20 mM HEPES (pH 7.9),
100 mM KCl, 5% glycerol, lmM EDTA, SmM DTT, 0.1% NP-40, and 0.5 mg/ml BSA.
The samples were then fractionated in 4% non-denaturing polyacrylamide gel containing
0.5X TBE at room temperature.
35 Oligonucleotides derived from the murine IL4 promoter used in EMSA were:
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-59 to -27: 5'-CTCATTTTCCCTTGGTTTCAGCAACTTTAACTC-3' (SEQ ID NO:
l);
-79 to -60: 5'-ATAAAATTTTCCAATGTAAA-3' (SEQ ID NO: 2); and
-88 to -61: 5'-TGGTGTAATAAAATTTTCCAATGTAAA-3' (SEQ ID NO: 3).
The sequence of the MARE oligonucleotide used in EMSA was:
S'-GGAATTGCTGACTCAGCATTACT-3'(SEQ ID NO: 4).
All oligonucleotides were annealed with their respective reverse-complementary strands
to form double-stranded oligonucleotides.
The results of EMSA with recombinant c-Maf are shown in Figure 6. The
recombinant c-Maf protein bound well to both a consensus MARE oligonucleotide and
to a 33 bp oligonucleotide cont~ining the NF-AT site and MARE present in the IL-4
promoter. Binding was specifically competed by unlabeled homologous but not control
probe. Further, c-Maf did not bind to an oligonucleotide cont:~ining only the NF-AT
target sequence to which recombinant NF-ATp bound well. The ability of c-Maf to bind
to the IL-4 promoter probe was specific since in vitro translated c-Jun protein did not
bind to this oligonucleotide. The c-Jun protein was functional since it could bind to the
consensus MARE which contains a core TRE site. These results indicate that c-Maf, but
not another AP-1 family member (c-Jun), can bind to the MARE site within the
proximal IL-4 promoter.
NF-AT proteins interact cooperatively with AP-1 family member proteins to
form higher mobility complexes on IL-2 and IL-4 promoter DNA on EMSA (Jain, J.
(1993) Nature 365:353-355; Rooney, J. et al. (1995) Immunity _:545-553). That NF-AT
proteins might interact with c-maf was suggested by the functional studies described in
the previous examples. To determine if c-Maf interacted with NF-AT in the presence of
DNA, recombinant NF-ATp and c-Maf proteins were used separately or together in
EMSA with the 33 bp oligonucleotide cont~inin~ both the NF-AT and adjacent MARE
sites. The results are shown in Figure 6. Each protein alone bound to IL-4 promoter
DNA. Recombinant c-Maf plus recombinant NF-ATp protein produced these
complexes and in addition formed a higher mobility complex. No higher mobility
complex was observed when c-Jun and NF-ATp proteins were used, consistent with the
failure of c-Jun to bind this site. These results indicate that c-Maf can specifically bind
in vitro to a sequence located in the proximal IL-4 promoter, previously shown to be
functionally critical in Th2 cells, and that, like other AP-I proteins, c-Maf can interact i~z
vitro with NF-AT proteins.
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EXAMPLE 7: The Ability of c-Maf to Transactivate the IL-4 Promoter
Maps to the MARE and Th-2 Specific Footprint
An essential region of the IL-4 promoter located immediately upstream of the
TATA element has been characterized by high resolution mutagenesis (Hodge, M. et al.
(1995) J. Immunol. 154:6397-6405). Mutagenesis of this 33 bp region (-59 to -28)demonstrated multiple sites required for inducible IL-4 transcription in Th2 cells. These
sites included an NF-AT target sequence, the region footprinted by Th2 extracts, and
what is now recognized as a MARE. A series of IL-4 reporter gene constructs
comprising 4 base pair linker-sc~nning mutants generated across this region were used
to map the target sequence utilized by c-Maf in vivo in M12 cells. These cells were
cotransfected with the c-maf expression vector and this series of mutant IL-4 promoter
constructs. The results are shown in Figure 7A. Mutation of the MARE (muts 3 and 4),
or the site defined by the Th2 footprint (mut 2), abrogated (muts 2 and 4) or partially
abrogated (mut 3) the ability of transfected c-maf to drive IL-4 transcription. A modest
effect in reducing c-maf transactivation was also observed for mutant 8 which disrupts
the NF-AT sequence, consistent with the presence in M 12 cells of endogenous NF-ATp
and with the synergy between NF-ATp and c-maf demonstrated in the previous
examples. Mutants 6 and 7 had no significant effect while mutant 5 had enhanced
transactivation ability, consistent with previous observations in Th2 cells (Hodge, M. et
al. (1995) J. Immunol. 154:6397 6405). The transactivation data is consistent with
EMSA performed with recombinant c-Maf protein using as probe an oligonucleotide
which contains this 33 bp region, and this same series of mutant oligonucleotides as cold
competitors. The results of these EMSA experiments are shown in Figure 7B. Theseexperiments indicate that c-Maf specifically binds to and transactivates the MARE in the
proximal IL-4 promoter and that the adjacent Th2-specific element is intimately
involved in both the binding and function of c-Maf.
EXAMPLE 8: Isolation of a NIP45 cDNA Using a Yeast
Two-Hybrid Interaction Trap Assay
A yeast two-hybrid interaction trap assay was used to isolate proteins that could
directly bind to the RHD of NF-ATp. An NF-ATp(RHD)-Gal4 fusion protein was
prepared for use as the "bait" in the yeast two-hybrid assay by cloning a 900 bp fragment
35 of murine NF-ATp (McCaffrey, P.G. et al. ( 1993) Science 262:750-754), spanning
amino acids 228 to 520, into the BamHI site of vector pEG202 (Gyuris, J. et al. (1993)
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Cell 75:791-803). In frame fusion ofthe NF-AT(p) polypeptide sequences to the Gal4
sequences was confirmed by DNA sequence analysis. This bait was used to screen acDNA library prepared from the murine T cell line D10, constructed in the plasmid
pJG4-5, to select for clones encoding polypeptides that interacted with the bait, using
5 methodologies known in the art (see Gyuris, J. et al. (1993) Cell 75:791-803).One class of interactors encoding a fusion protein with apparently high affinityfor the NF-ATp(RHD)-Gal4 bait, as exhibited by high level of ,B-galactosidase activity
and ability to confer leucine prototrophy, was isolated and termed NIP45 (NF-AT
Interacting Protein 45). Figure 8 shows a photograph of yeast colonies (three
10 representatives for each plasmid combination), cotransformed with the NIP45 plasmid
and either the NF-ATp-RHD bait or control baits (Max-Gal4, CDK2-Gal4 and the
control vector pEG202, expressing only an epitope tagged Gal4 protein), together with
the LacZ reporter plasmid pSH18. The yeast colonies had been selected on app~,pl;ate
media and were spotted onto plates Cont:~ining Xgal and the nonrepressing carbon source
15 galactose. Yeast colonies cotransformed with the NIP45 plasmid and the NF-ATp-RHD
bait were blue in color, demonstrating expression of the LacZ reporter plasmid
(indicative of NIP-451NF-ATp-RHD interaction), whereas yeast colonies transformed
with the NIP45 plasmid and the control baits were white in color, indicating no
interaction of NIP45 with the control baits. Transformants were also tested on galactose
20 containing media lacking leucine, and only those con~ining the NIP45 plasmid and the
NF-ATp-RHD bait grew, further indicating the specific interaction of NIP45 with NF-
ATp-RHD. The NIP45 cDNA isolated by the two-hybrid assay was a 1.9 kb DNA
fragment.
25 EXAMPLE 9: Interaction of NIP45 and NF-ATp In vivo in Mammalian Cells
The ability of the NIP45 polypeptide to interact specifically with NF-ATp in vivo
was tested in m~mm~ n cells. The 1.9 kb NIP45 cDNA insert selected in the yeast
two-hybrid system (described in Example 8) was subcloned into a m~mm~ n
30 expression vector which fuses the coding region to an epitope tag from a influenza
hemagglutinin (HA) peptide. vector pCEP4-HA (Herrscher, R.F. et al. (1995) GenesDev. 2:3067-3082), to create the expression vector NIP45-HA. This tagged construct
was then cotransfected with an NF-ATp expression plasmid into HepG2 cells (whichexpress low levels of NF-ATp). As controls, HepG2 cells also were cotransfected with
35 NIP45-HA along with the parental eXpression vector for the NF-ATp construct (i.e., the
expression vector without the NF-ATp insert) or with the NF-ATp expression vector
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along with an out of frame fusion of NIP45 with the epitope tag. Lysates were prepared
from the transfected cells and immunoprecipitated with anti-NF-ATp antibody. Western
blot analysis was then performed on the immunprecipitated material using either anti-
NF-ATp or anti-HA antibodies.
The results of this experiment are shown in Figure 9. Western blot analysis of
these samples using an HA-specific monoclonal antibody (mAb) demonstrated that the
anti-NF-ATp antibody used for immunoprecipitation coimmunoprecipitated the HA-
tagged NIP45 polypeptide. The lane showing transfection with only NIP45-HA (middle
lane) reveals the low endogenous level of NF-ATp present in these cells. The amount of
HA-tagged NIP45 protein immunoprecipitated was further increased by cotransfection
with the NF-ATp expression plasmid demonstrating the specificity of this interaction
(right lane). Western blot analysis of untreated Iysates demonstrated that equivalent
levels of NIP45-HA polypeptide were expressed in the samples tested for
coimmunoprecipitation of NIP45-HA anti-NF-ATp antibodies. Furthermore, no
immunoreactive material for either NF-ATp or the HA tagged protein was detected
when performing immunoprecipitation using normal rabbit serum. These experimentsdemonstrate that NF-AT and NIP45 physically associate in vivo in mzlmm~ n cells.
EXAMPLE 10: Structural Analysis of NIP45 cDNAs
The 1.9 kb NIP45 cDNA insert from the clone isolated using the two-hybrid
assay (described in Example 1) was used to screen a DIO.G4 T cell lambda zap II cDNA
library (Stratagene) to identify full length clones. Screening of a library containing
approximately 8x 105 clones yielded 7 hybridizing clones most of which did not extend
as far towards the 5' end as the original isolate. Sequence analysis of the longest clone
(2.8 kb), however, demonstrated identity to the original clone at the 5' end. The
structures of the original 1.9 kb cDNA isolate and the longest 2.8 kb cDNA isolate are
compared in Figure 10. The 2.8 kb cDNA isolate contained an additional segment of
180 bp located 868 bp downstream from the 5' end of the original clone. Junctionsequences at the ends of this 180 nucleotide segment indicate it to be an unspliced intron
and conceptual translation of the nucleotide sequence within this region revealed an in-
frame stop codon. Much of the additional se~uence in this clone was at the 3' end and
represented an extensive 3' untranslated region followed by a poly-A+ tail (see Figure
10). Such extensive 3' untranslated regions have been observed in many genes.
Allowing for the splicing of the small intron and translation of the single large open
T
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reading frame, the 2.8 kb cDNA clone is predicted to encode an identical polypeptide to
that of the original 1.9 kb isolate.
The nucleotide and predicted amino acid sequences of the 1.9 kb cDNA isolate
are shown in Figure 11 (and in SEQ ID NOs: 5 and 6, respectively). The coding region
5 is shown from the first initiation codon through the first in frame stop codon. The
nucleotide and amino acid positions are indicated to the right of the primary sequence.
Conceptual translation of the 1.9 kb nucleotide sequence predicted a polypeptide of 412
amino acids with a molecular mass of 45 Kd, and hence the protein has been termed NF-
AT Interacting Protein 45 (NIP45). lnspection of the amino acid sequence of NIP45
10 revealed a highly basic domain at the N-terminus, in which 13 of 32 amino acid are
basic. This region is underlined in Figure 11. This basic region appears as a hydrophilic
stretch in the hydrophobicity plot shown in Figure 12.
EXAMPLE 11: Tissue Expression of NIP45 mRNA
Northern blot analysis of RNA from different murine tissues was perforrned to
investigate the tissue expression of NIP45 mRNA. 10 ~g of total RNA from varioustissues was separated on denaturing agarose gels, blotted and hybridized with a
radiolabelled 1.4 kb NIP45 cDNA fragment. Samples were controlled for equivalent20 loading of RNA by comparison of ethidium bromide fluorescence. The results of the
Northern blot analysis are shown in Figure 13. The hybridizations revealed a transcript
of approximately 3.1 kb, which is of comparable size to the longest cDNA clones. RNA
from testis contained an additional 1.4 Kb hybridizing species. The highest levels of
NIP45 transcripts were seen in spleen, thymus and testis. The preferential expression in
25 Iymphoid organs may indicate a specific function for NIP45 in the immune system. The
low intensity hybridization signal and the rare occurrence of NIP45 cDNA clones in the
T cell cDNA library indicate that the NIP45 RNA is a relatively rare message.
EXAMPLE 12: Subcellular Loc~ii7~tion of NIP45
Subcellular localization of epitope tagged NIP45 protein was determined by
indirect immunofluorescence. BHK cells were transfected with 1 ~lg of an expression
construct encoding an HA-epitope tagged NIP45 (pCEP4-HA), using methodologies
known in the art (see Heald, R. et al. (1993) Cell 74:463-474). Transfected cells were
35 incubated overnight, fixed, permeabilized as described (Heald, R. et al. (1993) supra)
and probed with an anti-HA mAb 12CA5 (Boehringer Mannheim) plus
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indocarbocyanine labelled donkey anti-mouse antibody (Jackson ImmunoResearch) and
then counterstained with the dye Hoechst 33258. The results are shown in Figures 14A-
B. Nuclear staining of NIP45 was observed with the indocarbocyanine labelled
secondary reagent (see Figure 14A) by comparison to the same cells counterstained with
5 the DNA staining dye Hoechst 33258 (see Figure 14B). The fluorescence pattern
indicates that NIP45 is evenly distributed throughout the nucleus. Furthermore, this
pattern matched that seen for cells transfected with NF-AT4 and stimulated with
ionomycin (Shibasaki, F. et al. (1996) Nature 382:370-373; see also below).
Stimulation with PMA and/or ionomycin did not affect the subcellular loc~li7~tion of
10 this NIP45.
Control experiments were also performed on BHK cells transfected with NF-
AT4. Cells were incubated overnight in culture media and either fixed directly or first
stimulated with I mM ionomycin for 10 minutes before fixation and then processed as
described above. The results are shown in Figures 14C-F. Unstimulated (Figs. 14C and
15 14D) or ionomycin treated (Figs. 14E and 14F) NF-AT4 transfectants were probed with
an anti-NF-AT4 specific antibody followed by a indocarbocyanine labelled secondary
reagent and Hoechst 33258. Indocarbocyanine fluorescence demonstrates the pattern of
staining for cytoplasmic localized NF-AT4 in unstimulated transfectants (Fig. 14C) and
nuclear localized NF-AT4 in stimulated cells (Fig.14E). Adjacent panels (Fig. 14D and
20 14F, respectively) show the same field exposed for detection of nuclei by staining with
Hoechst 33258.
The effect of NIP45 on the nuclear translocation of NF-AT4 also was
investigated. HepG2 cells were transfected with either NF-AT4 or NF-AT4 plus NIP45
and stimulated the following day with I ~M ionomycin for 0,2, 4, 8 or 15 minutes. For
25 one sample, the cells were stimulated for 15 minutes with ionomycin and then washed
with fresh media and allowed to rest for an additional 15 minutes (indicated as " 15 min.
+ 15 min. rest" in Table 1). This analysis is de~ignl~cl to examine the function of NIP45
as a nuclear retention factor. Fifteen minutes has been shown to be sufficient time for
NF-AT4 to be exported to the cytoplasm (Shibasaki, F. et al. (1996) Nature 382:370-
30 373). All samples were then fixed and analyzed by immunoflourescence fortranslocation of NF-AT4 as described above. The results are summarized below in
Table 2. Subcellular localization of NF-AT4 in the cytoplasm is indicated by a (-) and
nuclear translocation of NF-AT4 is indicated by (+).
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Table 2: Nuclear Translocation of NF-AT4
Time lonomycin Ionomycin + NIP45
0 min. - -
2min. +/- +/-
4 min. +t- +/-
8 min. + +
15 min. + +
15 min. + 15 min. rest
No difference in the rate of nuclear import or export of NF-AT4 was observed in
the presence of NIP45, indicating that nuclear trafficking of NF-AT4 in response to
5 changes in intracellular calcium levels was not affected by the overexpression of
exogenous NIP45.
EXAMPLE 13: Functional Activity of NIP45 in Regulating Gene Expression
To test for a functional role of NIP45 in NF-AT-driven transcription, NIP45 was
expressed at high levels in HepG2 cells. HepG2 cells were chosen because they have
low levels of endogenous NF-AT, and ectopic expression of NF-AT family member
proteins has been shown to transactivate NF-AT-driven transcription in this cell line in
the absence of exogenous stimulation (Hoey, T. e~ al. (1995) Immunity 2:461-472).
15 HepG2 cells were transfected with a 3X NF-AT-CAT reporter from the IL-2 gene
(Venkataraman, L. et al. ( I 994) Immunity I :189- 196) and control or expression plasmids
for a NIP45 and NF-AT family members (NF-ATp, NF-ATc, NF-AT3, NF-AT4).
HepG2 cells were transfected by the DEAE-Dextran method as described in Hoey, T. et
al. (1995) supra, and CAT assays were performed according to standard methodologies.
20 The results are shown in Figure 15. One representative assay for each combination is
shown adjacent to a bar graph representing relative CAT activity for each group. Fold
induction was calculated by normAIi7.ing the CAT activity of cells transfected with the
CAT reporter and each parental expression vector to one. Values represent the relative
level of CAT expression above this control transfection. All transfections were
25 performed at least three times with one representative autoradiograph shown.
Transfection of NIP45 alone into HepG2 cells with a 3X NF-AT-CAT reporter
did not lead to a significant increase in CAT expression demonstrating that NIP45
cannot act on its own to transactivate an NF-AT target sequence. Overexpression of
NF-ATp alone resulted in substantial (6-fold over vector control) transactivation of the
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NF-AT-CAT reporter, consistent with previous reports (Hoey, T. et al. (1995) supra).
Cotransfection of NIP45 plus NF-ATp resulted in a 4-5 fold increase in CAT activity
relative to transfection with NF-ATp alone and a 25-30 fold increase over that seen with
vector alone. This increase was not observed when a mutant 3X NF-AT-CAT reporter5 or a control MHC class Il promoter reporter was used thus demonstrating its target site
specificity. To confirm that the polypeptide product encoded by the NIP45 cDNA was
responsible for this enhanced transactivation, a frame shift mutation was introduced in
the coding region by creating a two base deletion at nucleotide 50. This alteration
results in the introduction of missense mutations at amino acid 13 and termination of the
10 polypeptide after an additional 22 residues. Assays using this NIP45~ construct
demonstrated its failure to transactivate the NF-AT reporter in the presence or absence
of NF-ATp thus conflrrning that the enhanced transactivation observed was due to the
polypeptide expressed from NIP45 cDNA. Transactivation experiments were also
performed in the B cell line M12 and the T cell clone D10 with similar although less
15 drarnatic results, which may be due to higher levels of endogenous NIP45 or NF-ATp in
these latter cell lines. These experiments demonstrate that NIP45 substantially and
specifically potentiates transcription induced by NF-ATp, an activity that re~uires
interaction with NF-ATp.
NF-AT proteins share approximately 70% identity within the RHD, raising the
20 possibility that NIP45 could also interact with other NF-AT family members. To test
this, NIP45 was cotransfected as above with expression constructs encoding either NF-
ATc, NF-AT3 or NF-AT4 plus the 3X NF-AT-CAT reporter plasmid. The results of
these experiments are also shown in Figure 8. It has previously been demonstrated that
all NF-AT family members can transactivate a reporter gene cont~ining 3 copies of an
25 NF-AT/AP 1 site when overexpressed in HepG2 cells, although to different levels (Hoey,
T. et al. ( l 99S) supra). In the absence of NIP45, NF-ATp was the most potent
transactivator of the NF-AT-CAT reporter followed by NF-ATc and NF-AT3 with onlyweak transactivation by NF-AT4, consistent with previous data (McCaffrey, P.G. et al.
(1993) Science 262:750-754). When NF-ATc, NF-AT3 or NF-AT4 were cotransfected
30 with NIP45, NIP45 substantially potentiated both NF-ATc and NF-AT3-driven
transactivation and weakly pot~nti~ted NF-AT4-mediated transactivation (Figure 15).
Cooperation with NF-ATc in HepG2 cells is consistent with the observation that NIP45
interacts with an NF-ATc RHD bait in yeast cells. Overall, NIP45 overexpression
resulted in a 4-fold increase in transactivation by NF-ATc, a 3-fold increase in NF-AT3-
35 driven transactivation and a 2-fold increase in NF-AT4-driven transcription. The ability
of NIP45 to potentiate the activity of all NF-AT family members is not surprising given
._ T
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the high degree of sequence conservation of the RHD of the NF-AT family members. A
sequence comparison of the NF-AT RHD domains reveals a higher level of sequence
identity in the amino terminal portion compared to that of the carboxyl terminus (Hoey,
- T. et al. (1995) supra). Thus it is likely that the NIP45/NF-AT interaction site is located
5 in the 5' portion of the RHD.
Although a reporter construct cont~ining multiple copies of the NF-AT binding
site provides a sensitive method for measuring transactivation by NF-AT and NIP45, we
sought to determine if NIP45 was functional in the context of a native NF-AT-dependent
promoter. IL-4 expression is highly tissue specific and restricted to the Th2 subset of T
10 cells and to mast cells. The IL-4 promoter contains multiple NF-AT binding sites which
have been shown to be critical for expression of IL-4 (Rooney, J.W. et al. (1995)
Immunity 2:473-483). Furthermore, the proto-oncogene c-Maf has been shown to direct
tissue specific expression of IL-4 (Examples 3 and 4). Thus, the IL-4 promoter is not
active in the HepG2 cell line but can be activated by the introduction of NF-ATp and c-
15 Maf. In cotransfection experiments carried out as described above, HepG2 cells weretransfected with an IL-4-CAT reporter construct (extending to -732 bp of the IL-4
promoter) and expression vectors or controls for NIP45, NF-ATp and c-Maf. The
controls for NIP45 was a frame shift mutant at amino acid 13. Controls for NF-ATp and
c-Maf were the empty expression vectors pREP4 and pMEX respectively (Ho, I.C. et al.
20 (1996) Cell 85 :973-983). The results of these experiments are shown in Figure 16
(representative CAT assays and bar graphs are depicted as in Figure 15). The data
indicate that introduction of NIP45 together with NF-ATp and c-Maf results in anadditional 9-fold increase in the activity of the IL-4 promoter relative to that seen for
NF-ATp and c-Maf alone. NIP45 also increased the activity of the IL-4 promoter in the
25 absence of transfected NF-ATp, an effect likely due to interaction with endogenous NF-
ATp.
EXAMPLE 14: Transient Overexpression of NIP45 with NF-ATp
and c-Maf Results in Endogenous IL-4 Production
To determine whether the combination of NIP45, NF-ATp and c-Maf was
sufficient to induce endogenous IL-4 expression by cells that do not normally produce
IL-4, M12 B lymphoma cells were transiently cotransfected with expression plasmids
for NF-ATp and
35 c-Maf together with NIP45 or pCI vector control. M12 cells were transiently transfected
by electroporation as previously described (Ho, I.C. et al. (1996) Cell 85:973-983) by
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incubating 3 x 1 o6 cells in 0.4 ml of PBS with 5 ~Lg of each plasmid for 10 minutes at
room temperature prior to electroporation at 975 ~F? 280 V. Levels of IL-4 in the
supernatants harvested 72 hours later were measured by a commercially available IL-4
ELISA (Pharmingen), performed according to the manufacturer's instructions except
5 with modification as described (Ho, I.C. et al. (1996) supra). Four independent sets of
transient transfections were done and assayed for secretion of IL-4 into the culture
supernatant. Results from a representative experiment from one of the four independent
transfections is shown in Figure 17. For each set of transfections, inclusion of NIP45
led to a dramatic increase in IL-4 production. Cells transfected with NIP45 produced
10 50-200 fold more endogenous IL-4 than cells that did not receive NIP45, in which IL-4
production was near the limit of detection.
EXAMPLE 15: Expression of plX mRNA is Downregulated During
T Helper Cell Differentiation In vitro
The Maf family protein p l 8 is a member of the "small mafs" that lack the aminoterrninal two thirds of c-Maf that contains the transactivating domain. To examine the
expression of p 18 transcripts during differentiation of normal T helper cells to the Th2
phenotype, in vitro differentiation experiments were perforrned as described above in
20 Example 2. Naive spleen cells (Th precursor (Thp) cells) were driven along a Thl or
Th2 pathway by treatment with anti-CD3 in the presence of cytokines and anti-cytokine
antibodies (IFN~ and anti-IL-4 for Thl, IL-4 and anti-IFN~ for Th2). Northern blot
analysis of differenti~ting cells harvested at various time points after stimulation (day 0,
1, 3, 5, or 7) were performed to analyze the expression of p 18 and c-maftranscripts. The
25 results for c-maf and pl8 expression in vitro differentiated Th2 cells are shown in Figure
18. Consistent with results described above, expression of the c-maftranscript was low
level or undetectable at day 0 but increased as the cells differentiated along the Th2
pathway. In contrast, expression of p 18 transcript was detectable at day 0 (i. e., in
undifferentiated T cells) but decreased to essentially undetectable levels as the cells
30 differentiated along the Th2 pathway. These results indicate that pl8 expression is
downregulated in norrnal T helper cells during differentiation to the Th2 phenotype.
EXAMPLE 16: p18 Represses IL-4 Promoter Activity
To examine whether pl 8 expression affects IL-4 promoter activity,
cotransfection experiments were perforrned in M12 B Iymphoma cells. Methodologies
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used for these experiments were as described above in Example 3. An IL-4
promoter/CAT reporter gene construct was transfected into M12 cells with either a c-
Maf expression vector, a pl 8 expression vector or both c-Maf and pl 8 expression
vectors. Representative results of CAT assays are shown in Figure 19. Expression of c-
5 Maf alone (5 ~g of plasmid) resulted in activation of the IL-4 promoter construct (see
lane 2 of Figure 19), evidenced by detectable CAT activity in the M12 cells.
Coexpression of the p l 8 expression vector (2.5, 5 or 10 ,ug) with c-Maf resulted in
decreased CAT activity (see lanes 3, 4, and 5 of Figure 19), with increasing amounts of
p 18 leading to greater decreases in the observed CAT activity. Expression of p 18 alone
in the M12 cells did not result in detectable CAT activity in the cells (see lane 6 of
Figure 19). These results demonstrate that pl 8 can repress IL-4 promoter activity that is
stimulated by c-Maf.
EXAMPLE 17: Transgenic Mice that Overexpress c-Maf
This example describes overexpression of c-Maf protein in T cells of transgenic
mice. A schematic diagram of the c-maf transgenic construct is shown in Figure 20.
This construct comprises the 4 kb mouse c-maf cDNA and the first intron of the mouse
c-maf gene. Expression of the c-maf cDNA is controlled by the CD4 promoter/enhancer
regulatory region, which is operatively linked to the 5' end of the c-maf first intron,
thereby conferring T cell-specific expression on the construct. An SV40
polyadenylation site is linked to the 3' end of the c-maf cDNA. This c-maf transgenic
construct was microinjected into fertilized mouse oocytes and transgenic mice were
prepared according to standard procedures.
The phenotype of the c-maf transgenic anim~l~ were compared to that of wild
type anim~ls using a number of different assays. First, total numbers of cells in
lymphoid organs (Iymph nodes, spleen and thymus) were quantitated, the results of
which are shown in Figure 21. This experiment demonstrated that the c-maf transgenic
mice exhibited decreased cell numbers in all three Iymphoid organs examined compared
to wild type mice. Next, the abundance of specific thymocyte populations was
compared in c-maf transgenic and wild type mice, using flow cytometry. Double
positive (CD4+CD8+), double negative (CD4-CD8-), and single positive thymocytes
(CD4+ or CD8+) were quantitated. The ratio of CD4 to CD8 cells (CD4/CD8) also was
determined. The results are summarized below in Table 3.
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Table 3: Abundance of Specific Thymocyte Populations in c-Maf Trans -enic Mice
CD4-CD8- CD4+CD8+ CD4+ CD8+ CD4/CD8*
Wildtype 4.2I0.6 71.6~9.5 8.5~1.6 2.8~0.6 3.2~0.8
Transgenic 2.8 + 0.6 11.8 + 8.7 3.8 ~ 0.8 2.0 ~ 0.6 2.0 ~ 0.2
These results demonstrate that c-maf transgenic mice have a dramatic decrease in the
number of double positive thymocytes compared to wild type mice. Moreover, the c-
5 maf transgenic mice have significantly decreased numbers of CD4+ single positivethymocytes. Finally, the basal level of IgE in the serum of c-maf transgenic and wild
type mice was q~l~nti~ted. The results are shown in Figure 22. This experiment
demonstrates that c-maf transgenic mice exhibit increased basal levels of serum IgE as
compared to the wild type mice.
The phenotype of the c-maf transgenic mice described above (namely small
spleen and thymus, decreased numbers of double positive thymocytes and single
positive CD4+ thymocytes, and increased basal levels of serum IgE) is very similar to
the phenotype described for IL-4 overexpressor transgenic mice (see Tepper et al. (1990)
Cell 62:457; and Lewis et al. (1991) ~ Exp. Med. 173:89).
EOUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific embodiments of the invention
described herein. Such e~uivalents are intended to be encompassed by the following
20 claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
s
(i) APPLICANT:
(A) NAME: President and Fellows of Harvard College
(B) STREET: 124 Mount Auburn Street
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02138
(ii) TITLE OF INVENTION: Methods and Compositions for Regulating
T Cell Subsets by Modulating Transcription
Factor Activity
(iii) NUM8ER OF SEQUENCES: 6
( iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD
(B) STREET: 60 State Street, suite 510
~C) CITY: Boston
(D) STATE: Massachusetts
~ E) COUNTRY: USA
(F) ZIP: 02109-1875
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/636,602
(B) FILING DATE: 23-APR-1996
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/755,592
(B) FILING DATE: 25-NOV-1996
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/755,584
(B) FILING DATE: 25-NOV-1996
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kara, Catherine J.
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(B) REGISTRATION NUMBER: P41,106
(C) REFERENCE/DOCKET NUMBER: HUI-021CPPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)227-7400
(B) TELEFAX: (617)227-5941
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CTCATTTTCC CTTGGTTTCA GCAACTTTAA CTC 33
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATAAAATTTT CCAATGTAAA 20
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGGTGTAATA AAATTTTCCA ATGTAAA 27
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(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGAATTGCTG ACTCAGCATT ACT 23
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1946 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..1248
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ACAGTGTGGG AG ATG GCG GAA CCA CTG AGG GGA CGT GGT CCG AGG TCC 48
Met Ala Glu Pro Leu Arg Gly Arg Gly Pro Arg Ser
1 5 10
CGC GGT GGC CGA GGC GCT CGG AGA GCC CGA GGC GCC CGT GGC CGG TGT 96
Arg Gly Gly Arg Gly Ala Arg Arg Ala Arg Gly Ala Arg Gly Arg Cys
15 20 25
CCT CGC GCC CGG CAG TCT CCG GCT AGG CTC ATT CCA GAC ACC GTG CTT 144
Pro Arg Ala Arg Gln Ser Pro Ala Arg Leu Ile Pro Asp Thr Val Leu
30 35 40
45 GTG GAC TTG GTC AGT GAC AGC GAC GAA GAG GTC TTG GAA GTC GCA GAC 192
Val Asp Leu Val Ser Asp Ser Asp Glu Glu Val Leu Glu Val Ala Asp
45 50 55 60
CCA GTA GAG GTG CCG GTC GCC CGC CTC CCC GCG CCG GCT AAA CCT GAG 240
50 Pro Val Glu Val Pro Val Ala Arg Leu Pro Ala Pro Ala Lys Pro Glu
65 70 75
CAG GAC AGC GAC AGT GAC AGT GAA GGG GCG GCC GAG GGG CCT GCG GGA 288
Gln Asp Ser Asp Ser Asp Ser Glu Gly Ala Ala Glu Gly Pro Ala Gly
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go
GCC CCG CGT ACA TTG GTG CGA CGG CGG CGG CGG CGG CTG CTG GAT CCC 336
Ala Pro Arg Thr Leu Val Arg Arg Arg Arg Arg Arg Leu Leu Asp Pro
95 100 105
GGA GAG GCG CCG GTG GTC CCA GTG TAC TCC GGG AAG GTA CAG AGC AGC 384
Gly Glu Ala Pro Val Val Pro Val Tyr Ser Gly Lys Val Gln Ser Ser
110 115 120
CTC AAC CTC ATT CCA GAT AAT TCA TCC CTC TTG AAA CTG TGC CCT TCA 432
Leu Asn Leu Ile Pro Asp Asn Ser Ser Leu Leu Lys Leu Cys Pro Ser
125 130 135 140
GAG CCT GAA GAT GAG GCA GAT CTG ACA AAT TCT GGC AGT TCT CCC TCT 480
Glu Pro Glu Asp Glu Ala Asp Leu Thr Asn Ser Gly Ser Ser Pro Ser
145 150 155
GAG GAT GAT GCC CTG CCT TCA GGT TCT CCC TGG AGA AAG AAG CTC AGA 528
Glu Asp Asp Ala Leu Pro Ser Gly Ser Pro Trp Arg Lys Lys Leu Arg
160 165 170
AAG AAG TGT GAG AAA GAA GAA AAG AAA ATG GAA GAG TTT CCG GAC CAG 576
Lys Lys Cys Glu Lys Glu Glu Lys Lys Met Glu Glu Phe Pro Asp Gln
175 180 185
GAC ATC TCT CCT TTG CCC CAA CCT TCG TCA AGG AAC AAA AGC AGA AAG 624
Asp Ile Ser Pro Leu Pro Gln Pro Ser Ser Arg Asn Lys Ser Arg Lys
190 195 200
CAT ACG GAG GCG CTC CAG AAG CTA AGG GAA GTG AAC AAG CGT CTC CAA 672
His Thr Glu Ala Leu Gln Lys Leu Arg Glu Val Asn Lys Arg Leu Gln
205 210 215 220
GAT CTC CGC TCC TGC CTG AGC CCC AAG CAG CAC CAG AGT CCA GCC CTT 720
Asp Leu Arg Ser Cys Leu Ser Pro Lys Gln His Gln Ser Pro Ala Leu
225 230 235
CAG AGC ACA GAT GAT GAG GTG GTC CTA GTG GAA GGG CCT GTC TTG CCA 768
Gln Ser Thr Asp Asp Glu Val Val Leu Val Glu Gly Pro Val Leu Pro
240 245 250
CAG AGC TCT CGA CTC TTT ACA CTC AAG ATC CGG TGC CGG GCT GAC CTA 816
Gln Ser Ser Arg Leu Phe Thr Leu Lys Ile Arg Cys Arg Ala Asp Leu
255 260 265
GTG AGA CTG CCT GTC AGG ATG TCG GAG CCC CTT CAG AAT GTG GTG GAT 864
Val Arg Leu Pro Val Arg Met Ser Glu Pro Leu Gln Asn Val Val Asp
270 275 280
CAC ATG GCC AAT CAT CTT GGG GTG TCT CCA AAC AGG ATT CTT TTG CTT 912
His Met Ala Asn His Leu Gly Val Ser Pro Asn Arg Ile Leu Leu Leu
285 290 295 300
TTT GGA GAG AGT GAA CTG TCT CCT ACT GCC ACC CCT AGT ACC CTA AAG 960
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Phe Gly Glu Ser Glu Leu Ser Pro Thr Ala Thr Pro Ser Thr Leu Lys
305 310 315
CTT GGA GTG GCT GAC ATC ATT GAT TGT GTG GTG CTA GCA AGC TCT TCA 1008
5 Leu Gly Val Ala Asp Ile Ile Asp Cys Val Val Leu Ala Ser Ser Ser
320 325 330
GAG GCC ACA GAG ACA TCC CAG GAG CTC CGG CTC CGG GTG CAG GGG AAG 1056
Glu Ala Thr Glu Thr Ser Gln Glu Leu Arg Leu Arg Val Gln Gly Lys
0 335 340 345
GAG AAA CAC CAG ATG TTG GAG ATC TCA CTG TCT CCT GAT TCT CCT CTT 1104
Glu Lys His Gln Met Leu Glu Ile Ser Leu Ser Pro Asp Ser Pro Leu
350 355 360
AAG GTT CTC ATG TCA CAC TAT GAG GAA GCC ATG GGA CTC TCT GGA CAC 1152
Lys Val Leu Met Ser His Tyr Glu Glu Ala Met Gly Leu Ser Gly His
365 370 375 380
20 AAG CTC TCC TTC TTC TTT GAT GGG ACA AAG CTT TCA GGC AAG GAG CTG 1200
Lys Leu Ser Phe Phe Phe Asp Gly Thr Lys Leu Ser Gly Lys Glu Leu
385 390 395
CCA GCT GAT CTG GGC CTG GAA TCC GGA GAT CTC ATC GAA GTC TGG GGC 1248
25 Pro Ala Asp Leu Gly Leu Glu Ser Gly Asp Leu Ile Glu Val Trp Gly
400 405 410
TGAAGCTCTC ACCCTGTTCG GACGCAAAGC CAAGACATGG AGACAATAGC TCCCAATTTT 1308
ATTATTGTGA 'l"l"l"l"l'C'~CCC CATAAGGGCT AACAGAAACT GAATTAGAAC ~ ACTT 1368
ATTTATTTCT GGTGCTGGGG ATTGAACCCC AGACTATGCA CATGCTAAGG ATGTATGAAG 1428
TGGAGGCA~A ACCAAGGCAT TACCTTTAGC CAGCCTCTAG TAGACTGTAG TGTCAAGCAA 1488
GTGGCTACTT GGTAGTTGTG TGGCTCTGTG TAl~lll~lG CTGTATTTGG CAGCCCCTGG 1548
GGCACATAGA AGGGACCTTG GCTTCCCTAC CATTTCACGT TCGCTGGTGC C~ C~llC 1608
ATCAGATGAC TTCTGTGAAG CTGCCTATGT TGA~l~l~l~ GAACTAAATG AGCTCTGCTT 1668
TGGGTGTCCA GGCCTGGGGT TTGTGCCGCA GTTGGAGCCA GCAGTGACTT CACTCTGACT 1728
TGGGACTGAG AATGCATTTC CTGGTGGAGA CACTCGGGTG CAGAAATATA ACAGAAGGTG 1788
ACATACATGC TGAAGCTGAG GACTAGGTCG AAAGTTAACG ACGTTGCATT TTCAGCCTTG 1848
GGTATCCTCT CTGCCTGCCA GGACTCTAGC CAGTGTCTGG TACACACTTC TTGGCATGGA 1908
CACCTAGGTC GACGCGGGCG CGATTCGGCC GACTCGAG 1946
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 412 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Glu Pro Leu Arg Gly Arg Gly Pro Arg Ser Arg Gly Gly Arg
0 l 5 10 15
Gly Ala Arg Arg Ala Arg Gly Ala Arg Gly Arg Cys Pro Arg Ala Arg
15 Gln Ser Pro Ala Arg Leu Ile Pro Asp Thr Val Leu Val Asp Leu Val
35 40 45
Ser Asp Ser Asp Glu Glu Val Leu Glu Val Ala Asp Pro Val Glu Val
50 55 60
Pro Val Ala Arg Leu Pro Ala Pro Ala Lys Pro Glu Gln Asp Ser Asp
65 70 75 80
Ser Asp Ser Glu Gly Ala Ala Glu Gly Pro Ala Gly Ala Pro Arg Thr
85 90 95
Leu Val Arg Arg Arg Arg Arg Arg Leu Leu Asp Pro Gly Glu Ala Pro
100 105 110
30 Val Val Pro Val Tyr Ser Gly Lys Val Gln Ser Ser Leu Asn Leu Ile
115 120 125
Pro Asp Asn Ser Ser Leu Leu Lys Leu Cys Pro Ser Glu Pro Glu Asp
130 135 140
Glu Ala Asp Leu Thr Asn Ser Gly Ser Ser Pro Ser Glu Asp Asp Ala
145 150 155 160
Leu Pro Ser Gly Ser Pro Trp Arg Lys Lys Leu Arg Lys Lys Cys Glu
165 170 175
Lys Glu Glu Lys Lys Met Glu Glu Phe Pro Asp Gln Asp Ile Ser Pro
180 185 190
~5 Leu Pro Gln Pro Ser Ser Arg Asn Lys Ser Arg Lys His Thr Glu Ala
195 200 205
Leu Gln Lys Leu Arg Glu Val Asn Lys Arg Leu Gln Asp Leu Arg Ser
210 215 220
Cys Leu Ser Pro Lys Gln His Gln Ser Pro Ala Leu Gln Ser Thr Asp
225 230 235 240
Asp Glu Val Val Leu Val Glu Gly Pro Val Leu Pro Gln Ser Ser Arg
245 250 255
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Leu Phe Thr Leu Lys Ile Arg Cys Arg Ala Asp Leu Val Arg Leu Pro
260 265 270
5 Val Arg Met Ser Glu Pro Leu Gln Asn Val Val Asp His Met Ala Asn
- 275 280 285
His Leu Gly Val Ser Pro Asn Arg Ile Leu Leu Leu Phe Gly Glu Ser
290 295 300
Glu Leu Ser Pro Thr Ala Thr Pro Ser Thr Leu Lys Leu Gly Val Ala
305 310 315 320
Asp Ile Ile Asp Cys Val Val Leu Ala Ser Ser Ser Glu Ala Thr Glu
325 330 335
Thr Ser Gln Glu Leu Arg Leu Arg Val Gln Gly Lys Glu Lys His Gln
340 345 350
20 Met Leu Glu Ile Ser Leu Ser Pro Asp Ser Pro Leu Lys Val Leu Met
355 360 365
Ser His Tyr Glu Glu Ala Met Gly Leu Ser Gly His Lys Leu Ser Phe
370 375 380
Phe Phe Asp Gly Thr Lys Leu Ser Gly Lys Glu Leu Pro Ala Asp Leu
385 390 395 400
Gly Leu Glu Ser Gly Asp Leu Ile Glu Val Trp Gly
405 410
... ,., .... ~ . ...
