U.S. patent application number 10/923520 was filed with the patent office on 2005-01-06 for human c-maf compositions and methods of use therefor.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Douhan, John III, Glimcher, Laurie H..
Application Number | 20050004034 10/923520 |
Document ID | / |
Family ID | 26706199 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050004034 |
Kind Code |
A1 |
Glimcher, Laurie H. ; et
al. |
January 6, 2005 |
Human C-Maf compositions and methods of use therefor
Abstract
Isolated nucleic acid molecules encoding human c-Maf, and
isolated c-Maf proteins, are provided. The invention further
provides antisense nucleic acid molecules, recombinant expression
vectors containing a nucleic acid molecule of the invention, host
cells into which the expression vectors have been introduced and
non-human transgenic animals carrying a human c-Maf transgene. The
invention further provides human c-Maf fusion proteins and
anti-human c-Maf antibodies. Methods of using the human c-maf
compositions of the invention are also disclosed, including methods
for detecting human c-Maf activity in a biological sample, methods
of modulating human c-Maf activity in a cell, and methods for
identifying agents that modulate the activity of human c-Maf.
Inventors: |
Glimcher, Laurie H.; (West
Newton, MA) ; Douhan, John III; (Boston, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
26706199 |
Appl. No.: |
10/923520 |
Filed: |
August 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10923520 |
Aug 20, 2004 |
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09888370 |
Jun 22, 2001 |
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09888370 |
Jun 22, 2001 |
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09879312 |
Jun 12, 2001 |
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09879312 |
Jun 12, 2001 |
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09086010 |
May 27, 1998 |
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6274338 |
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09086010 |
May 27, 1998 |
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09030579 |
Feb 24, 1998 |
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Current U.S.
Class: |
424/185.1 ;
514/1.7; 514/16.6; 514/17.9; 514/18.7; 514/2.3; 514/4.3; 514/44R;
514/7.3; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; C07K 14/82 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
514/012 ;
514/044; 536/023.2 |
International
Class: |
A61K 038/17; A61K
048/00; C12Q 001/68; C07H 021/04 |
Goverment Interests
[0002] Work described herein was supported, at least in part, under
grant AI/AG 37833 awarded by the National Institutes of Health. The
U.S. government therefore may have certain rights in this
invention.
Claims
We claim:
1. A method for modulating human c-Maf activity in a cell
comprising contacting the cell with an agent that modulates human
c-Maf activity such that human c-Maf activity in the cell is
modulated.
2. The method of claim 1, wherein said agent stimulates human c-Maf
activity.
3. The method of claim 1, wherein said agent modulates human c-Maf
activity by modulating the activity of the c-Maf polypeptide.
4. The method of claim 1, wherein said agent modulates human c-Maf
activity by modulating the transcription of the c-Maf gene.
5. The method of claim 1, wherein said agent modulates human c-Maf
activity by modulating the transcription of the c-Maf mRNA.
6. The method of claim 1, wherein said agent that modulates c-Maf
activity is a nucleic acid molecule encoding human c-maf.
7. A method for stimulating human c-Maf activity in a cell
comprising introducing into the cell a nucleic acid molecule
encoding human c-Maf such that human c-Maf activity in the cell is
stimulated.
8. The method of claim 7, wherein said nucleic acid molecule
comprises a nucleotide sequence that encodes the polypeptide of SEQ
ID NO:2.
9. The method of claim 7, wherein said nucleic acid has at least
98% nucleotide identity with the nucleotide sequence of SEQ ID NO:
1.
10. The method of claim 7, wherein said nucleic acid has at least
99% nucleotide identity with the nucleotide sequence of SEQ ID NO:
1.
11. The method of claim 7, wherein said nucleic acid has at least
99.5% nucleotide identity with the nucleotide sequence of SEQ ID
NO: 1.
12. The method of claim 7, wherein said nucleic acid comprises the
nucleotide sequence of the coding region of the NheI/XbaI insert of
plasmid pHu-c-Maf (ATCC Accession No. 98671).
13. The method of claim 7, wherein said nucleic acid molecule is
introduced into the cell by contacting the cell with the nucleic
acid molecule.
14. The method of claim 7, wherein said nucleic acid molecule is
introduced into the cell by contacting the cell with a composition
comprising the nucleic acid molecule and a lipophillic carrier.
15. The method of claim 7, wherein said nucleic acid molecule is
introduced into the cell by transfecting or infecting the cell with
a vector comprising nucleic acid sequences capable of producing the
nucleic acid molecule when transcribed in situ.
16. The method of claim 7, wherein said nucleic acid molecule is
introduced into the cell by injecting into the cell a vector
comprising nucleic acid sequences capable of producing the nucleic
acid molecule when transcribed in situ.
17. The method of claim 7, wherein said nucleic acid molecule is
introduced into the cell by contacting the cell with a recombinant
virus having the nucleic acid molecule incorporated into the
retroviral genome.
Description
RELATED APPLICATION
[0001] This application is a divisional application of U.S. Ser.
No. 09/888,370, filed Jun. 22, 2001; which is a divisional
application of pending U.S. Ser. No. 09/879,312, filed Jun. 12,
2001; which is a divisional application of U.S. Ser. No.09/086,010,
filed May 27, 1998 (now U.S. Pat. No 6,274,338, granted Aug. 14,
2001); which is a continuation-in-part of abandoned U.S. Ser. No.
09/030,579, filed Feb. 24, 1998. The entire contents of each of
these applications is hereby incorporated herein by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0003] The Maf family of proteins are a sub-family of AP-1/CREB/ATF
proteins. The first member of the family to be identified, the
v-maf oncogene, was originally isolated from a spontaneous
musculoaponeurotic 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, which has been isolated from murine cells, has only
two structural changes in the 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 p18. The latter four, mafK, mafF, mafG and p18, each encode
proteins that lack the amino terminal two thirds of c-Maf that
contains the transactivating domain ("small maf proteins")
(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).
[0004] 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 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. 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 QR1
retina-specific gene (Swaroop, A. et al. (1992) Proc. Natl. Acad.
Sci. USA 89:266-270). Additionally, the small mafs have been shown
to function as repressors of .alpha. and .beta.-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 (Igarashi, K. et al. (1995)
Proc. Natl. Acad Sci. USA 92:7445-7449). Moreover, c-Maf has been
shown to control the tissue-specific expression of the cytokine
interleukin-4 in T helper 2 (Th2) cells (Ho, I-C. et al. (1996)
Cell 85:973-983).
[0005] The nucleotide sequence of the mouse c-maf proto-oncogene,
and predicted amino acid sequence for the mouse c-Maf protein, have
been described (Kurscher, C. and Morgan, J. I. (1995) Mol. Cell.
Biol. 15:246-254; and Genbank Accession number S74567). The
nucleotide sequence of the chicken c-maf proto-oncogene, and
predicted amino acid sequence for the chicken c-Maf protein, also
have been described (Kataoka et al., Genbank Accession number
D28596). However, these non-human c-Maf compositions may not
function optimally in human cells and, moreover, use of these
compositions in humans is likely to stimulate an immune response,
since the chicken or mouse c-Maf would be recognized as "foreign"
by the human immune system. Accordingly, there is still a need for
human c-Maf compositions that are suitable for use in humans.
SUMMARY OF THE INVENTION
[0006] This invention provides human c-Maf compositions. In
particular, this invention provides isolated nucleic acid molecules
encoding human c-Maf and isolated human c-Maf protein. Since the
c-Maf compositions of the invention are human-derived, they
function optimally in human cells (compared with non-human c-Maf
compositions) and do not stimulate an immune response in
humans.
[0007] One aspect of the invention pertains to an isolated nucleic
acid molecule comprising a nucleotide sequence encoding human
c-Maf. In a preferred embodiment, the nucleic acid molecule
comprises the nucleotide sequence of the coding region of the
NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).
In another preferred embodiment, the nucleic acid molecule
comprises the nucleotide sequence of SEQ ID NO: 1. In other
embodiments, the nucleic acid molecule has at least 98% nucleotide
identity, more preferably 99% nucleotide identity, and even more
preferably 99.5% nucleotide identity with the nucleotide sequence
of SEQ ID NO: 1 or the nucleotide sequence of the NheI/XbaI insert
of plasmid pHu-c-Maf (ATCC Accession No. 98671). In yet another
embodiment, the nucleic acid molecule comprises the nucleotide
sequence of the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC
Accession No. 98671).
[0008] The isolated nucleic acid molecules of the invention
encoding human c-Maf can be incorporated into a vector, such as an
expression vector, and this vector can be introduced into a host
cell. The invention also provides a method for producing a human
c-Maf protein by culturing a host cell of the invention (carrying a
hu-c-Maf expression vector) in a suitable medium until a human
c-Maf protein is produced. The method can further involve isolating
the human c-Maf protein from the medium or the host cell.
[0009] Another aspect of the invention pertains to an isolated
human c-Maf protein. Preferably, the human c-Maf protein comprises
the amino acid sequence encoded by the coding region of the
NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No. 98671).
In another preferred embodiment, the protein comprises the amino
acid sequence of SEQ ID NO: 2. In other embodiments, the protein
has at least 98% amino acid identity, more preferably 99% amino
identity, and even more preferably 99.5% amino acid identity with
SEQ ID NO: 2 or the protein encoded by the coding region of the
NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No.
98671).
[0010] Fusion proteins, comprising a human c-Maf protein
operatively linked to a polypeptide other than human c-Maf, are
also encompassed by the invention, as well as antibodies that
specifically bind a human c-Maf protein. The antibodies can be, for
example, polyclonal antibodies or monoclonal antibodies. In one
embodiment, the antibodies are coupled to a detectable
substance.
[0011] Another aspect of the invention pertains to a nonhuman
transgenic animal that contains cells carrying a transgene encoding
a human c-Maf protein.
[0012] Yet another aspect of the invention pertains to a method for
detecting the presence of human c-Maf in a biological sample. The
method involves contacting the biological sample with an agent
capable of detecting an indicator of human c-Maf activity such that
the presence of human c-Maf is detected in the biological sample.
The invention also provides a method for modulating human c-Maf
activity in a cell comprising, involving contacting the cell with
an agent that modulates human c-Maf activity such that human c-Maf
activity in the cell is modulated.
[0013] Still another aspect of the invention pertains to methods
for identifying a compound that modulates the activity of a human
c-Maf protein. These methods generally involve:
[0014] providing an indicator composition that comprises a human
c-Maf protein;
[0015] contacting the indicator composition with a test compound;
and
[0016] determining the effect of the test compound on the activity
of the human c-Maf protein in the indicator composition to thereby
identify a compound that modulates the activity of a human c-Maf
protein. In a preferred embodiment, the indicator composition
comprises a human c-Maf protein and a DNA molecule to which the
human c-Maf protein binds and the effect of the test compound on
the activity of the human c-Maf protein is determined by evaluating
the binding of the human c-Maf protein to the DNA molecule in the
presence and absence of the test compound. In another preferred
embodiment, the indicator composition is a cell comprising a human
c-Maf protein and a reporter gene responsive to the human c-Maf
protein and the effect of the test compound on the activity of the
human c-Maf protein is determined by evaluating the expression of
the reporter gene in the presence and absence of the test compound.
In yet another embodiment, the method further involves the step of
determining the effect of the test compound on an immune response
to thereby identify a compound that modulates an immune
response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A-1B is an alignment of the nucleotide sequence of the
human c-maf coding region with the mouse c-maf coding region.
Nucleotide differences between the two sequences are boxed.
[0018] FIG. 2 is an alignment of the amino acid sequence of the
human c-Maf protein with the mouse c-Maf protein. Amino acid
differences between the two sequences are boxed.
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention pertains to human c-Maf compositions, such as
isolated nucleic acid molecules encoding human c-Maf and isolated
human c-Maf proteins, as well as methods of use therefore. The
human compositions of the invention have the advantages that they
function optimally in human cells (compared with non-human c-Maf
compositions) and do not stimulate an immune response in
humans.
[0020] So that the invention may be more readily understood,
certain terms are first defined.
[0021] As used herein, the term "human c-Maf" is intended to
encompass proteins that share the distinguishing structural and
functional features (described further herein) of the human c-Maf
protein encoded by the NheI/XbaI insert of plasmid pHu-c-Maf, which
was deposited under the provisions of the Budapest Treaty with the
American Type Culture Collection, Rockville, Md. on Feb. 24, 1998
and assigned ATCC Accession No. 98671, and having the amino acid
sequence of SEQ ID NO: 2, including the amino acid residues unique
to human c-Maf (as compared to mouse c-Maf), which are boxed in
FIG. 2.
[0022] 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.
[0023] 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., genetic sequences
that are located adjacent to the gene for the isolated nucleic
molecule in the genomic DNA of the organism from which the nucleic
acid is derived). For example, in various embodiments, an isolated
human c-Maf nucleic acid molecule typically contains less than
about 10 kb of nucleotide sequences which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived, and more preferably contains less than
about 5, kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of naturally
flanking nucleotide sequences. An "isolated" human c-Maf nucleic
acid molecule may, however, be linked to other nucleotide sequences
that do not normally flank the human c-Maf sequences in genomic DNA
(e.g., the human c-Maf nucleotide sequences may be linked to vector
sequences). In certain preferred embodiments, an "isolated" nucleic
acid molecule, such as a cDNA molecule, also may be free of other
cellular material. However, it is not necessary for the human c-Maf
nucleic acid molecule to be free of other cellular material to be
considered "isolated" (e.g., a human c-Maf DNA molecule separated
from other mammalian DNA and inserted into a bacterial cell would
still be considered to be "isolated").
[0024] As used herein, the term "hybridizes under high stringency
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences having substantial
homology (e.g., typically greater than 70% homology) to each other
remain stably hybridized to each other. A preferred, non-limiting
example of high stringency conditions are hybridization in a
hybridization buffer that contains 6.times. sodium chloride/ sodium
citrate (SSC) at a temperature of about 45.degree. C. for several
hours to overnight, followed by one or more washes in a washing
buffer containing 0.2.times.SSC, 0.1% SDS at a temperature of about
50-65.degree. C.
[0025] The term "% identity" as used in the context of nucleotide
and amino acid sequences (e.g., when one amino acid sequence is
said to be X % identical to another amino acid sequence) refers to
the percentage of identical residues shared between the two
sequences, when optimally aligned. To determine the percent
identity of two nucleotide or amino acid sequences, the sequences
are aligned for optimal comparison purposes (e.g., gaps may be
introduced in one sequence for optimal alignment with the other
sequence). The residues at corresponding positions are then
compared and when a position in one sequence is occupied by the
same residue as the corresponding position in the other sequence,
then the molecules are identical at that position. The percent
identity between two sequences, therefore, is a function of the
number of identical positions shared by two sequences (i e., %
identity=# of identical positions/total# of
positions.times.100).
[0026] Computer algorithms known in the art can be used to
optimally align and compare two nucleotide or amino acid sequences
to define the percent identity between the two sequences. A
preferred, non-limiting example of a mathematical algorithim
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68,
modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA 90:5873-77. Such an algorithm is incorporated into the NBLAST
and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Research 25(17):3389-3402. When utilizing
BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting
example of a mathematical algorithim utilized for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS (1989). Such
an algorithm is incorporated into the ALIGN program (version 2.0)
which is part of the GCG sequence alignment software package. When
utilizing the ALIGN program for comparing amino acid sequences, a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4 can be used. If multiple programs are used to compare
sequences, the program that provides optimal alignment (i.e., the
highest percent identity between the two sequences) is used for
comparison purposes.
[0027] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0028] 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.
[0029] As used herein, the term "coding region" refers to regions
of a nucleotide sequence comprising codons which are translated
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).
[0030] 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 mammalian
vectors). Other vectors (e.g., non-episomal mammalian 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 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
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.
[0031] As used herein, the term "host cell" is intended to refer to
a cell into which a nucleic 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.
[0032] As used herein, a "transgenic animal" refers to a non-human
animal, preferably a mammal, more preferably a mouse, in which one
or more of the cells of the animal includes 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.
[0033] As used herein, a "homologous recombinant animal" refers to
a type of transgenic non-human animal, preferably a mammal, 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.
[0034] As used herein, an "isolated protein" refers to a protein
that is substantially free of other proteins, cellular material and
culture medium when isolated from cells or produced by recombinant
DNA techniques, or chemical precursors or other chemicals when
chemically synthesized.
[0035] As used herein, the term "antibody" is intended to include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen, such as Fab and F(ab').sub.2 fragments. The terms
"monoclonal antibodies" 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, whereas the
term "polyclonal antibodies" and "polyclonal antibody composition"
refer to a population of antibody molecules that contain multiple
species of antigen binding sites capable of interacing with a
particular antigen. A monoclonal antibody compositions thus
typically display a single binding affinity for a particular
antigen with which it immunoreacts.
[0036] There is a known and definite correspondence between the
amino acid sequence of a particular protein and the nucleotide
sequences that can code for the protein, as defined by the genetic
code (shown below). Likewise, there is a known and definite
correspondence between the nucleotide sequence of a particular
nucleic acid molecule and the amino acid sequence encoded by that
nucleic acid molecule, as defined by the genetic code.
1 GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg,
R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT
Aspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic
acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G)
GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I)
ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine
(Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F)
TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC,
AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT
Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine (Val, V)
GTA, GTC, GTG, GTT Termination signal (end) TAA, TAG, TGA
[0037] An important and well known feature of the genetic code is
its redundancy, whereby, for most of the amino acids used to make
proteins, more than one coding nucleotide triplet may be employed
(illustrated above). Therefore, a number of different nucleotide
sequences may code for a given amino acid sequence. Such nucleotide
sequences are considered functionally equivalent since they result
in the production of the same amino acid sequence in all organisms
(although certain organisms may translate some sequences more
efficiently than they do others). Moreover, occasionally, a
methylated variant of a purine or pyrimidine may be found in a
given nucleotide sequence. Such methylations do not affect the
coding relationship between the trinucleotide codon and the
corresponding amino acid.
[0038] In view of the foregoing, the nucleotide sequence of a DNA
or RNA molecule coding for a human c-Maf protein of the invention
(or any portion thereof) can be use to derive the human c-Maf amino
acid sequence, using the genetic code to translate the DNA or RNA
molecule into an amino acid sequence. Likewise, for any human
c-Maf-amino acid sequence, corresponding nucleotide sequences that
can encode the human c-Maf protein can be deduced from the genetic
code (which, because of its redundancy, will produce multiple
nucleic acid sequences for any given amino acid sequence). Thus,
description and/or disclosure herein of a human c-Maf nucleotide
sequence should be considered to also include description and/or
disclosure of the amino acid sequence encoded by the nucleotide
sequence. Similarly, description and/or disclosure of a human c-Maf
amino acid sequence herein should be considered to also include
description and/or disclosure of all possible nucleotide sequences
that can encode the amino acid sequence.
[0039] Various aspects of the invention are described in further
detail in the following subsections:
[0040] I. Isolated Nucleic Acid Molecules
[0041] One aspect of the invention pertains to isolated nucleic
acid molecules that encode human c-Maf. An approximately 4.2
kilobase fragment of DNA encoding human c-Maf has been isolated
from a genomic DNA library and subcloned into the plasmid
pBluescriptKS/II. E. coli bacteria carrying this plasmid, referred
to as phu-c-Maf, have been deposited under the provisions of the
Budapest Treaty with the American Type Culture Collection,
Rockville, Md., on Feb. 24, 1998 and assigned ATCC Accession No.
98671. This plasmid was constructed by insertion of a .about.4.2 kb
NheI fragment encompassing the human c-Maf coding region into the
compatible XbaI site of the plasmid vector, to thereby create a
.about.4.2 kb NheI/XbaI insert that encodes human c-Maf. It should
be noted that upon ligation of the NheI fragment into the XbaI
site, these restriction sites are not regenerated and, thus, to
excise the fragment from the plasmid, it is necessary to use
adjacent restriction sites within the pBluescript polylinker. The
nucleotide sequence of the human c-Maf coding region, and
corresponding predicted amino acid sequence, are shown in SEQ ID
NOs: 1 and 2, respectively. This nucleotide sequence, and predicted
amino acid sequence, of human c-Maf were obtained by sequencing of
the NheI/XbaI insert of the pHu-c-Maf plasmid using standard
sequencing methods. Primers for sequencing are designed based on
the nucleotide sequence shown in SEQ ID NO: 1. Isolation and
characterization of the human c-Maf-encoding DNA is described
further in the Example.
[0042] In a preferred embodiment, the nucleic acid molecule of the
invention comprises the nucleotide sequence of the coding region of
the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC Accession No.
98671). In another preferred embodiment, the nucleic acid
moleculecomprises the nucleotide sequence of SEQ ID NO: 1. In other
embodiments, the nucleic acid molecule has at least 98% nucleotide
identity, more preferably 99% nucleotide identity, and even more
preferably 99.5% nucleotide identity with the nucleotide sequence
of SEQ ID NO: 1 or the nucleotide sequence of the NheI/XbaI insert
of plasmid pHu-c-Maf (ATCC Accession No. 98671). In yet another
embodiment, the nucleic acid molecule comprising the nucleotide
sequence of the NheI/XbaI insert of plasmid pHu-c-Maf (ATCC
Accession No. 98671).
[0043] Nucleic acid molecules that differ from SEQ ID NO: 1 (and
nucleotide sequence of the NheI/XbaI insert of p-Hu-c-Maf) due to
degeneracy of the genetic code, and thus encode the same human
c-Maf protein as that encoded by SEQ ID NO: 1 and pHu-c-Maf, are
encompassed by the invention. 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: 2 or having the amino acid sequence encoded by the
coding region of the NheI/XbaI insert of p-Hu-c-Maf.
[0044] A nucleic acid molecule having the nucleotide sequence of
human c-Maf can be obtained from plasmid pHu-c-Maf or can be
isolated using standard molecular biology techniques and the
sequence information provided herein. For example, a human c-Maf
DNA can be isolated from a human genomic DNA library using all or
portion of SEQ ID NO: 1 as a hybridization probe and standard
hybridization techniques (e.g., as described in Sambrook, J., et
al. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Moreover, a
nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1
can be isolated by the polymerase chain reaction using
oligonucleotide primers designed based upon the sequence of SEQ ID
NO: 1. 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 America, Inc., St.
Petersburg, Fla.). Synthetic oligonucleotide primers for PCR
amplification can be designed based upon the nucleotide sequence
shown in SEQ ID NO: 1. 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 into an appropriate vector and characterized by DNA sequence
analysis. Furthermore, oligonucleotides corresponding to a human
c-Maf nucleotide sequence can be prepared by standard synthetic
techniques, e.g., using an automated DNA synthesizer.
[0045] In addition to the human c-Maf nucleotide sequence shown in
SEQ ID NO: 1 and carried by plasmid pHu-c-Maf, it will be
appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to minor changes in the nucleotide or amino
acid sequences of human c-Maf may exist within a population. Such
genetic polymorphism in the human c-Maf gene may exist among
individuals within a population due to natural allelic variation.
Such natural allelic variations can typically result in 1-2%
variance in the nucleotide sequence of the a gene. Any and all such
nucleotide variations and resulting amino acid polymorphisms in
human c-Maf that are the result of natural allelic variation and
that do not alter the functional activity of human c-Maf are
intended to be within the scope of the invention.
[0046] Nucleic acid molecules corresponding to natural allelic
variants of the human c-Maf DNAs of the invention can be isolated
based on their homology to the human c-Maf nucleic acid molecules
disclosed herein using the human DNA, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under high stringency hybridization conditions. Accordingly, in
another embodiment, an isolated nucleic acid molecule of the
invention hybridizes under high stringency conditions to a second
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 1. In certain embodiment, the isolated nucleic acid molecule
comprises at least 30, 50, 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 2000 or 3000 contiguous nucleotides of SEQ ID NO: 1.
Preferably, an isolated nucleic acid molecule of the invention that
hybridizes under high stringency conditions to the sequence of SEQ
ID NO: 1 corresponds to a naturally-occurring allelic variant of a
human c-Maf nucleic acid molecule.
[0047] In addition to naturally-occurring allelic variants of the
human c-Maf sequence that may exist in the population, the skilled
artisan will further appreciate that minor changes may be
introduced by mutation into the nucleotide sequence of SEQ ID NO:
1, thereby leading to changes in the amino acid sequence of the
encoded protein, without altering the functional activity of the
human c-Maf 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: 1. A "non-essential"
amino acid residue is a residue that can be altered from the
wild-type sequence of human c-Maf (e.g., the sequence of SEQ ID NO:
2) without altering the functional activity of c-Maf, such as its
ability to interact with DNA or its ability to enhance
transcription from an IL-4 promoters whereas an "essential" amino
acid residue is required for functional activity.
[0048] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding human c-Maf proteins that contain
changes in amino acid residues that are not essential for human
c-Maf activity. Such human c-Maf proteins differ in amino acid
sequence from SEQ ID NO: 2 (or the amino acid sequence encoded by
pHu-c-Maf) yet retain human c-Maf activity. These non-natural
variants of human c-Maf also differ from non-human c-Maf proteins
(e.g., chicken or mouse c-Maf) in that they encode at least one
amino acid residue that is unique to human c-Maf (i.e., at least
one residue that is not present in chicken or mouse c-Maf).
Preferably, these non-natural variants of human c-Maf encode at
least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues that are
unique to human c-Maf (i.e., that are not present in chicken or
mouse c-Maf).
[0049] An isolated nucleic acid molecule encoding a non-natural
variant of a human c-Maf protein can be created by introducing one
or more nucleotide substitutions, additions or deletions into the
nucleotide sequence of SEQ ID NO: 1 (or plasmid pHu-c-Maf) such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into SEQ ID NO: 1 by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
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 side chain. Families of
amino acid residues having similar side chains have been defined in
the art, including basic side chains (e.g., lysine, 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., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
nonessential amino acid residue in human c-Maf 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 the human c-Maf coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for their ability to bind to DNA and/or
activate transcription, to identify mutants that retain functional
activity. Following mutagenesis, the encoded human c-Maf mutant
protein can be expressed recombinantly in a host cell and the
functional activity of the mutant protein can be determined using
assays available in the art for assessing c-Maf activity (e.g.,
assays such as those described in detail in PCT Publication WO
97/39721.
[0050] Another aspect of the invention pertains to isolated nucleic
acid molecules that are antisense to the coding strand of a human
c-Maf mRNA or gene. An antisense nucleic acid of the invention can
be complementary to an entire human c-Maf 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 human c-Maf that is unique to human
c-Maf (as compared to non-human c-Mafs, such as chicken or mouse
c-Maf). In another embodiment, the antisense nucleic acid molecule
is antisense to a noncoding region of the coding strand of a
nucleotide sequence encoding human c-Maf that is unique to human
c-Maf (as compared to non-human c-Mafs, such as chicken or mouse
c-Maf). In preferred embodiments, an antisense of the invention
comprises at least 30 contiguous nucleotides of the noncoding
strand of SEQ ID NO: 1, more preferably at least 50, 100, 200, 300,
400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the
noncoding strand of SEQ ID NO: 1.
[0051] Given the coding strand sequences encoding human c-Maf
disclosed herein (e.g., SEQ ID NO: 1 and plasmid pHu-c-Maf),
antisense nucleic acids of the invention can be designed according
to the rules of Watson and Crick base pairing. The antisense
nucleic acid molecule may be complementary to the entire coding
region of human c-Maf mRNA, or alternatively can be an
oligonucleotide which is antisense to only a portion of the coding
or noncoding region of human c-Maf mRNA. For example, the antisense
oligonucleotide may be complementary to the region surrounding the
translation start site of human c-Maf mRNA. An antisense
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 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 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).
[0052] 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 cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. A ribozyme having specificity for a human
c-Maf-encoding nucleic acid can be designed based upon the
nucleotide sequence of a human c-Maf gene disclosed herein. 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 human
c-Maf-encoding mRNA. See for example Cech et al. U.S. Pat. No.
4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,
human c-Maf 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.
[0053] Yet another aspect of the invention pertains to isolated
nucleic acid molecules encoding human c-Maf fusion proteins. Such
nucleic acid molecules, comprising at least a first nucleotide
sequence encoding a human c-Maf protein, polypeptide or peptide
operatively linked to a second nucleotide sequence encoding a
non-human c-Maf protein, polypeptide or peptide, can be prepared by
standard recombinant DNA techniques. Human c-Maf fusion proteins
are described in further detail below in subsection III.
[0054] II. Recombinant Expression Vectors and Host Cells
[0055] Another aspect of the invention pertains to vectors,
preferably recombinant expression vectors, containing a nucleic
acid encoding human c-Maf (or a portion thereof). The expression
vectors of the invention comprise a nucleic acid of the invention
in a form 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 expression, 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
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, Calif. (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
the expression vector may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of 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., human c-Maf proteins, mutant forms of human
c-Maf proteins, human c-Maf fusion proteins and the like).
[0056] The recombinant expression vectors of the invention can be
designed for expression of human c-Maf protein in prokaryotic or
eukaryotic cells. For example, human c-Maf can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector may be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0057] 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
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors can serve one or more purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification; 4) to provide an epitope tag to aid in
detection and/or purification of the protein; and/or 5) to provide
a marker to aid in detection of the protein (e.g., a color marker
using .beta.-galactosidase fusions). Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation 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, Mass.) and
pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein. Recombinant
proteins also can be expressed in eukaryotic cells as fusion
proteins for the same purposes discussed above.
[0058] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al. (1 988) Gene 69:301-315) and pET
11 d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (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 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident
.lambda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0059] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (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 Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0060] In another embodiment, the human c-Maf expression vector is
a yeast expression vector. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (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, Calif.).
[0061] Alternatively, human c-Maf can be expressed in insect cells
using baculovirus expression vectors. Baculovirus vectors available
for expression of proteins in cultured insect cells (e.g., Sf9
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) Virology 170:31-39).
[0062] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pMex-NeoI,
pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987), EMBO J. 6:187-195). When used in mammalian 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.
[0063] In another embodiment, the recombinant mammalian 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 known 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 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. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0064] Moreover, inducible regulatory systems for use in mammalian
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, Fla., ppl67-220), hormones (see e.g., Lee et al.
(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 & Kaufman (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 268:1766-1769; PCT Publication No. WO
94/29442; and PCT Publication No. WO 96/01313). Accordingly, in
another embodiment, the invention provides a recombinant expression
vector in which human c-Maf DNA is operatively linked to an
inducible eukaryotic promoter, thereby allowing for inducible
expression of human c-Maf protein in eukaryotic cells.
[0065] 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
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to human c-Maf 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 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 antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0066] 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, human c-Maf
protein may be expressed in bacterial cells such as E. coli, insect
cells, yeast or mammalian 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 to refer to a variety of
art-recognized techniques 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 Laboratory press (1989)), and other laboratory
manuals.
[0067] For stable transfection of mammalian 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 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 human c-Maf or may be introduced on a separate
vector. Cells stably transfected with the introduced 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).
[0068] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) human c-Maf protein. Accordingly, the invention further
provides methods for producing human c-Maf protein using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant
expression vector encoding human c-Maf has been introduced) in a
suitable medium until human c-Maf is produced. In another
embodiment, the method further comprises isolating human c-Maf from
the medium or the host cell. In its native form the human c-Maf
protein is an intracellular protein and, accordingly, recombinant
human c-Maf protein can be expressed intracellularly in a
recombinant host cell and then isolated from the host cell, e.g.,
by lysing the host cell and recovering the recombinant human c-Maf
protein from the lysate. Alternatively, recombinant human c-Maf
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. In
this case, recombinant human c-Maf protein can be recovered from
the culture medium in which the cells are cultured.
[0069] Certain host cells of the invention can also be used to
produce nonhuman transgenic animals. For example, in one
embodiment, a host cell of the invention is a fertilized oocyte or
an embryonic stem cell into which human c-Maf-coding sequences have
been introduced. Such host cells can then be used to create
non-human transgenic animals in which exogenous human c-Maf
sequences have been introduced into their genome or homologous
recombinant animals in which endogenous c-Maf sequences have been
altered. Such animals are useful for studying the function and/or
activity of human c-Maf and for identifying and/or evaluating
modulators of human c-Maf activity. Accordingly, another aspect of
the invention pertains to nonhuman transgenic animals which contain
cells carrying a transgene encoding a human c-Maf protein or a
portion of a human c-Maf protein. In a subembodiment, of the
transgenic animals of the invention, the transgene alters an
endogenous gene encoding an endogenous c-Maf protein (e.g.,
homologous recombinant animals in which the endogenous c-Maf gene
has been functionally disrupted or "knocked out", or the nucleotide
sequence of the endogenous c-Maf gene has been mutated or the
transcriptional regulatory region of the endogenous c-Maf gene has
been altered).
[0070] A transgenic animal of the invention can be created by
introducing human c-Maf-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
animal. The human c-Maf nucleotide sequence of SEQ ID NO: 1 (and
plasmid pHu-c-Maf) can be introduced as a transgene into the genome
of a non-human animal. Intronic sequences and polyadenylation
signals can also be included in the transgene to increase the
efficiency of expression of the transgene. A tissue-specific
regulatory sequence(s) can be operably linked to the human c-Maf
transgene to direct expression of human c-Maf protein to particular
cells. Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. 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 animals. A transgenic founder
animal can be identified based upon the presence of the human c-Maf
transgene in its genome and/or expression of human c-Maf mRNA in
tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene encoding human
c-Maf can further be bred to other transgenic animals carrying
other transgenes.
[0071] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a human c-Maf gene
into which a deletion, addition or substitution has been introduced
to thereby alter, e.g., functionally disrupt, the endogenous c-Maf
gene. In one embodiment, a homologous recombination vector is
designed such that, upon homologous recombination, the endogenous
c-Maf 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 c-Maf gene replaced by the
human c-Maf gene. In the homologous recombination vector, the
altered portion of the c-Maf gene is flanked at its 5' and 3' ends
by additional nucleic acid of the c-Maf gene to allow for
homologous recombination to occur between the exogenous human c-Maf
gene carried by the vector and an endogenous c-Maf gene in an
embryonic stem cell. The additional flanking c-Maf nucleic acid is
of sufficient length for successful homologous recombination with
the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a
description of homologous recombination vectors). The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced human c-Maf gene
has homologously recombined with the endogenous c-Maf 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
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, 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 animals in which all cells of the animal contain the
homologously recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals 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 et al.; WO 92/0968 by Zijlstra et
al.; and WO 93/04169 by Berns et al.
[0072] In addition to the foregoing, the skilled artisan will
appreciate that other approaches known in the art for homologous
recombination can be applied to the instant invention.
Enzyme-assisted site-specific integration systems are known in the
art and can be applied to integrate a DNA molecule at a
predetermined location in a second target DNA molecule. Examples of
such enzyme-assisted integration systems include the Cre
recombinase-lox target system (e.g., as described in Baubonis, W.
and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige,
S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909)
and the FLP recombinase-FRT target system (e.g., as described in
Dang, D. T. and Perrimon, N. (1992) Dev. Genet. 13:367-375; and
Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA 90:8469-8473).
Tetracycline-regulated inducible homologous recombination systems,
such as described in PCT Publication No. WO 94/29442 and PCT
Publication No. WO 96/01313, also can be used.
[0073] III. Isolated Human c-Maf Proteins and Anti-Human c-Maf
Antibodies
[0074] Another aspect of the invention pertains to isolated human
c-Maf proteins. Preferably, the human c-Maf protein comprises the
amino acid sequence encoded by the coding region of the NheI/XbaI
insert of plasmid pHu-c-Maf (ATCC Accession No. 98671). In another
preferred embodiment, the protein comprises the amino acid sequence
of SEQ ID NO: 2. In other embodiments, the protein has at least 98%
amino acid identity, more preferably 99% amino identity, and even
more preferably 99.5% amino acid identity with SEQ ID NO: 2 or the
protein encoded by the coding region of the NheI/XbaI insert of
plasmid pHu-c-Maf (ATCC Accession No. 98671).
[0075] In other embodiments, the invention provides isolated
portions of the human c-Maf protein. For example, the invention
further encompasses an amino-terminal portion of human c-Maf that
includes a transcriptional activation domain. In various
embodiments, this amino terminal portion encompasses at least amino
acids 1-122, at least amino acids 1-187, or at least amino acids
1-257. Another isolated portion of human c-Maf provided by the
invention is a portion encompassing a carboxy-terminal leucine
zipper domain. This portion encompasses at least amino acids
313-348.
[0076] Human c-Maf proteins of the invention 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 described above) and the human c-Maf protein is
expressed in the host cell. The human c-Maf protein can then be
isolated from the cells by an appropriate purification scheme using
standard protein purification techniques. Alternative to
recombinant expression, a human c-Maf polypeptide can be
synthesized chemically using standard peptide synthesis techniques.
Moreover, native human c-Maf protein can be isolated from cells
(e.g., from T cells), for example by immunoprecipitation using an
anti-human c-Maf antibody.
[0077] The invention also provides human c-Maf fusion proteins. As
used herein, a human c-Maf "fusion protein" comprises a human c-Maf
polypeptide operatively linked to a polypeptide other than human
c-Maf. A "human c-Maf polypeptide" refers to a polypeptide having
an amino acid sequence corresponding to human c-Maf protein, or a
peptide fragment thereof which is unique to human c-Maf protein (as
compared to non-human c-Maf proteins, such as mouse or chicken
c-Maf", whereas a "polypeptide other than human c-Maf" refers to a
polypeptide having an amino acid sequence corresponding to another
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the human c-Maf polypeptide and the
other polypeptide are fused in-frame to each other. The other
polypeptide may be fused to the N-terminus or C-terminus of the
human c-Maf polypeptide. For example, in one embodiment, the fusion
protein is a GST-human c-Maf fusion protein in which the human
c-Maf sequences are fused to the C-terminus of the GST sequences.
In another embodiment, the fusion protein is a human c-Maf-HA
fusion protein in which the human c-Maf nucleotide sequence is
inserted in a vector such as pCEP4-HA vector (Herrscher, R. F. et
al. (1995) Genes Dev. 9:3067-3082) such that the human c-Maf
sequences are fused in frame to an influenza hemagglutinin epitope
tag. Such fusion proteins can facilitate the purification of
recombinant human c-Maf.
[0078] Preferably, a human c-Maf 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 appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification 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 human c-Maf-encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the human c-Maf protein.
[0079] An isolated human c-Maf protein, or fragment thereof, can be
used as an immunogen to generate antibodies that bind specifically
to human c-Maf using standard techniques for polyclonal and
monoclonal antibody preparation. The human c-Maf protein can be
used to generate antibodies or, alternatively, an antigenic peptide
fragment of human c-Maf can be used as the immunogen. An antigenic
peptide fragment of human c-Maf typically comprises at least 8
amino acid residues of the amino acid sequence shown in SEQ ID NO:
2 and encompasses an epitope of human c-Maf such that an antibody
raised against the peptide forms a specific immune complex with
human c-Maf. 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 human c-Maf
that are located on the surface of the protein, e.g., hydrophilic
regions, and that are unique to human c-Maf, as compared to c-Maf
proteins from other species, such as chicken or mouse (i.e., an
antigenic peptide that spans a region of human c-Maf that is not
conserved across species is used as immunogen; such non-conserved
regions/residues are boxed in FIG. 2). A standard hydrophobicity
analysis of the human c-Maf protein can be performed to identify
hydrophilic regions.
[0080] A human c-Maf immunogen typically is used to prepare
antibodies by immunizing a suitable subject, (e.g., rabbit, goat,
mouse or other mammal) with the immunogen. An appropriate
immunogenic preparation can contain, for examples, recombinantly
expressed human c-Maf protein or a chemically synthesized human
c-Maf 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
immunogenic human c-Maf preparation induces a polyclonal anti-human
c-Maf antibody response.
[0081] Accordingly, another aspect of the invention pertains to
anti-human c-Maf antibodies. Polyclonal anti-human c-Maf antibodies
can be prepared as described above by immunizing a suitable subject
with a human c-Maf immunogen. The anti-human c-Maf antibody titer
in the immunized subject can be monitored over time by standard
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized human c-Maf. If desired, the antibody
molecules directed against human c-Maf can be isolated from the
mammal (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-human c-Maf antibody titers are highest, 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) J Biol Chem 255:4980-83;Yehetal.
(1976)PNAS76:2927-31; and Yehetal. (1982) Int. J. Cancer
29:269-75), the more recent human B cell 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., New
York, N.Y. (1980); E. A. Lemer (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 lymphocytes (typically splenocytes) from a mammal
immunized with a human c-Maf 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 human c-Maf.
[0082] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-human c-Maf monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; 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 mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes 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
containing 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-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 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
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind human c-Maf, e.g., using a
standard ELISA assay.
[0083] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-human c-Maf antibody can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with human c-Maf to thereby isolate immunoglobulin library members
that bind human c-Maf. 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 SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. 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.
International Publication WO 93/01288; McCafferty et al.
International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Ladner et al.
International 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) J
Mol 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 Acid Res
19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and
McCafferty et al. Nature (1990) 348:552-554.
[0084] Additionally, recombinant anti-human c-Maf antibodies, such
as chimeric and humanized 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 humanized 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.
Pat. 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. Res. 47:999-1005; Wood et 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. Pat. No. 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.
[0085] An anti-human c-Maf antibody (e.g., monoclonal antibody) can
be used to isolate human c-Maf by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-human c-Maf
antibody can facilitate the purification of natural human c-Maf
from cells and of recombinantly produced human c-Maf expressed in
host cells. Moreover, an anti-human c-Maf antibody can be used to
detect human c-Maf protein (e.g., in a cellular lysate 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-human c-Maf 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, alkaline phosphatase, .beta.-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, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0086] Yet another aspect of the invention pertains to anti-human
c-Maf antibodies that are obtainable by a process comprising:
[0087] (a) immunizing an animal with an immunogenic human c-Maf
protein, or an immunogenic portion thereof unique to human c-Maf
protein; and
[0088] (b) isolating from the animal antibodies that specifically
bind to a human c-Maf protein.
[0089] Methods for immunization and recovery of the specific
anti-human c-Maf antibodies are described further above.
[0090] IV. Pharmaceutical Compositions
[0091] Human c-Maf modulators of the invention (e.g., human c-Maf
inhibitory or stimulatory agents, including human c-Maf proteins
and antibodies) can be incorporated into pharmaceutical
compositions suitable for administration. Such compositions
typically comprise the modulatory agent 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 administration. 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.
[0092] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. 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 ethylenediaminetetraacetic
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 be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0093] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) 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
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, 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
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms 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 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.
[0094] Sterile injectable solutions can be prepared by
incorporating the active compound 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 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.
[0095] 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
administration, 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 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 tragacanth 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.
[0096] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
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. Methods for preparation of such formulations will
be apparent 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
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0097] V. Methods of the Invention
[0098] Another aspect of the invention pertains to methods of using
the various human c-Maf compositions of the invention. For example,
the invention provides a method for detecting the presence of human
c-Maf activity in a biological sample. The method involves
contacting the biological sample with an agent capable of detecting
human c-Maf activity, such as human c-Maf protein or human c-Maf
mRNA, such that the presence of human c-Maf activity is detected in
the biological sample.
[0099] A preferred agent for detecting human c-Maf mRNA is a
labeled nucleic acid probe capable of specifically hybridizing to
human c-Maf mRNA. The nucleic acid probe can be, for example, the
human c-Maf DNA of SEQ ID NO: 1 (or plasmid pHu-c-Maf), or a
portion thereof unique to human c-Maf (as compared to c-Maf from
other species, such as chicken or mouse), 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 conditions to human c-Maf
mRNA.
[0100] A preferred agent for detecting human c-Maf protein is a
labeled antibody capable of binding to human c-Maf protein.
Antibodies can be polyclonal, or more preferably, monoclonal. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2)
can be used. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) 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 intended to include tissues, cells and
biological fluids. For example, techniques for detection of human
c-Maf mRNA include Northern hybridizations and in situ
hybridizations. Techniques for detection of human c-Maf protein
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence.
[0101] The invention further provides methods for identifying
compounds that modulate the activity of a human c-Maf protein. For
example, the invention provides a method for identifying a compound
that modulates the activity of a human c-Maf protein,
comprising
[0102] providing an indicator composition that comprises a human
c-Maf protein;
[0103] contacting the indicator composition with a test compound;
and
[0104] determining the effect of the test compound on the activity
of the human c-Maf protein in the indicator composition to thereby
identify a compound that modulates the activity of a human c-Maf
protein.
[0105] Specific embodiments of the screening methods of the
invention exploit the ability of c-Maf proteins to bind to DNA
(e.g., the ability to bind to a Maf Response Element (MARE)) and/or
to regulate gene expression (e.g., regulate expression of a
Th2-associated cytokine gene). For further description of these
activities of Maf proteins, in general, see for example PCT
Publication WO 97/39721, Kurschner and Morgan (1995) Mol. Cell.
Biol., 15:246-254; Kataoka et al. (1993) J. Virol. 67:2133-2141;
Kataoka et al. (1996) Oncogene 12:53-62; Kataoka et al. (1994) Mol.
Cell. Biol. 14:700-712; and Ho, I-C. et al. (1996) Cell 85:973-983;
the contents of each of which are expressly incorporated herein by
reference.
[0106] In a preferred embodiment of the screening assays of the
invention, the indicator composition comprises an indicator cell,
wherein said indicator cell comprises: (i) the a human c-Maf
protein and (ii) a reporter gene responsive to the human c-Maf
protein. Preferably, the indicator cell contains:
[0107] i) a recombinant expression vector encoding the human c-Maf;
and
[0108] ii) a vector comprising regulatory sequences of a
Th2-associated cytokine gene operatively linked a reporter gene;
and
[0109] said method comprises:
[0110] a) contacting the indicator cell with a test compound;
[0111] b) determining the level of expression of the reporter gene
in the indicator cell in the presence of the test compound; and
[0112] 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 human c-Maf.
[0113] In another preferred embodiment, the indicator composition
comprises a preparation of: (i) a human c-Maf protein and (ii) a
DNA molecule to which the human c-Maf binds, and
[0114] said method comprises:
[0115] a) contacting the indicator composition with a test
compound;
[0116] b) determining the degree of interaction of the human c-Maf
protein and the DNA molecule in the presence of the test compound;
and
[0117] c) comparing the degree of interaction of the human c-Maf
and the DNA molecule in the presence of the test compound with the
degree of interaction of the human c-Maf protein and the DNA
molecule in the absence of the test compound to thereby identify a
compound that modulates the activity of human c-Maf.
[0118] Preferably, the DNA molecule to which human c-Maf binds
comprises a maf response element (MARE).
[0119] In another preferred embodiment, the method identifies
proteins that interact with human c-Maf. In this embodiment,
[0120] the indicator composition is an indicator cell, which
indicator cell comprises:
[0121] i) a reporter gene operably linked to a transcriptional
regulatory sequence; and
[0122] ii) a first chimeric gene which encodes a first fusion
protein, said first fusion protein including human c-Maf;
[0123] the test compound comprises a library of second chimeric
genes, which library encodes second fusion proteins;
[0124] expression of the reporter gene being sensitive to
interactions between the first fusion protein, the second fusion
protein and the transcriptional regulatory sequence; and
[0125] wherein the effect of the test compound on human c-Maf 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 that interacts with
human c-Maf.
[0126] In a preferred embodiment, the library of second chimeric
genes is prepared from cDNA library from Th2 cells.
[0127] In a preferred embodiment of the screening assays of the
invention, once a test compound is identified as modulating the
activity of human c-Maf, 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
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. 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 Th1 cells. Examples of Th2-associated cytokines include IL-4,
IL-5, IL-6 and IL-13. In another embodiment, the effect of the
compound of interest on an immune response is determined by
determining the effect of the compound on development of T helper
type 1 (Th1) or T helper type 2 (Th2) cells.
[0128] Recombinant expression vectors that can be used for
expression of human c-Maf in the indicator cell are known in the
art (see discussions above). In one embodiment, within the
expression vector the human c-Maf-coding sequences are operatively
linked to regulatory sequences that allow for constitutive
expression of human c-Maf 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 human c-Maf in the indicator cell is
preferred for identification of compounds that enhance or inhibit
the activity of human c-Maf. In an alternative embodiment, within
the expression vector the human c-Maf-coding sequences are
operatively linked to regulatory sequences of the endogenous human
c-Maf gene (i.e., the promoter regulatory region derived from the
endogenous human c-Maf gene). Use of a recombinant expression
vector in which human c-Maf expression is controlled by the
endogenous regulatory sequences is preferred for identification of
compounds that enhance or inhibit the transcriptional expression of
human c-Maf.
[0129] In methods in which a Th2-associated cytokine gene is
utilized (e.g., as a reporter gene), 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. 154:6397-6405). 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. 177:1663-1674.
See also PCT Publication WO 97/39721.
[0130] A variety of reporter genes are known in the art and are
suitable for use in the screening 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.
[0131] 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 human c-Maf, such as a B cell (e.g., the
M12 B lymphoma cell line) or a Th1 cell clone (e.g., AE7 cells).
Nonlymphoid cell lines can also be used as indicator cells, such as
the HepG2 hepatoma cell line. Yeast cells also can be used as
indicator cells.
[0132] In one embodiment, the level of expression of the reporter
gene in the indicator cell 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 human c-Maf. 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 and the test compound is identified as a compound
that inhibits the expression or activity of human c-Maf.
[0133] Alternative to the use of a reporter gene construct,
compounds that modulate the expression or activity of human c-Maf
can be identified by using other "read-outs." For example, an
indicator cell can be transfected with a human c-Maf expression
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 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.
[0134] As described above, the invention provides a screening assay
for identifying proteins (e.g., proteins in Th2 cells) that
interact with human c-Maf. 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. Pat. No.
5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al.
(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)
Biotechniques 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-binding
domain and a transcriptional activation domain, wherein both
domains are required 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) 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
functional GAL4 transactivator).
[0135] This general two-hybrid system can be applied to the
identification of proteins in cells (e.g., Th2 cells) that interact
with human c-Maf by construction of a target human c-Maf fusion
protein (e.g., a human c-Maf/GAL4 binding domain fusion as the
"bait") and a cDNA library of "fish" fusion proteins (e.g., a
cDNA/GAL4 activation domain library), wherein the cDNA library is
prepared from mRNA of a cell type of interest (e.g., Th2 cells),
and introducing these constructs into a host cell that also
contains a reporter gene construct linked to a regulatory sequence
responsive to human c-Maf (e.g., a MARE sequence, for example a
region of the IL-4 promoter, as discussed above). cDNAs encoding
proteins that interact with human c-Maf can be identified based
upon transactivation of the reporter gene construct.
[0136] 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 that interact with human 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 been removed (e.g., human 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 human c-Maf
(e.g., a MARE sequence, for example a region of the IL-4 promoter,
responsive to human c-Maf). cDNAs encoding proteins that interact
with human c-Maf can be identified based upon transactivation of
the reporter gene construct.
[0137] As described above, the invention provides a screening assay
for identifying compounds that modulate the interaction of human
c-Maf and a MARE (e.g., a MARE in an IL-4 gene regulatory region).
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).
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.
[0138] In one embodiment, the amount of binding of human c-Maf to
the DNA fragment in the presence of the test compound is greater
than the amount of binding of human c-Maf 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 human c-Maf.
In another embodiment, the amount of binding of human c-Maf to the
DNA fragment in the presence of the test compound is less than the
amount of binding of human c-Maf to the DNA fragment in the absence
of the test compound, in which case the test compound is identified
as a compound that inhibits binding of human c-Maf.
[0139] Yet another aspect of the invention pertains to methods of
modulating human c-Maf activity in a cell. The modulatory methods
of the invention involve contacting the cell with an agent that
modulates human c-Maf activity such that human c-Maf activity in
the cell is modulated. The agent may act by modulating the activity
of human c-Maf protein in the cell or by modulating transcription
of the human c-Maf gene or translation of the human c-Maf mRNA. As
used herein, the term "modulating" is intended to include
inhibiting or decreasing human c-Maf activity and stimulating or
increasing human c-Maf activity. Accordingly, in one embodiment,
the agent inhibits human c-Maf activity. In another embodiment, the
agent stimulates human c-Maf activity.
[0140] A. Inhibitory Agents
[0141] According to a modulatory method of the invention, human
c-Maf activity is inhibited in a cell by contacting the cell with
an inhibitory agent. Inhibitory agents of the invention can be, for
example, intracellular binding molecules that act to inhibit the
expression or activity of human c-Maf. 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 itself, to a nucleic acid (e.g.,
an mRNA molecule) that encodes the protein or to a target with
which the protein normally interacts (e.g., to a DNA target
sequence to which c-Maf binds). Examples of intracellular binding
molecules, described in further detail below, include antisense
human c-Maf nucleic acid molecules (e.g., to inhibit translation of
human c-Maf mRNA), intracellular anti-human c-Maf antibodies (e.g.,
to inhibit the activity of human c-Maf protein) and dominant
negative mutants of the human c-Maf protein.
[0142] In one embodiment, an inhibitory agent of the invention is
an antisense nucleic acid molecule that is complementary to a gene
encoding human c-Maf or to a portion of said gene, or a recombinant
expression vector encoding said antisense 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. et al., Antisense RNA as a molecular tool for
genetic analysis, Reviews--Trends in Genetics, Vol. 1(1) 1986;
Askari, F. K. and McDonnell, W. M. (1996) N. Eng J. 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 Ther.
2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R.
W. (1994) Nature 372:333-335). An antisense 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 be complementary to a
sequence found in the coding region of the mRNA, the 5' or 3'
untranslated region of the mRNA or a region bridging the coding
region and an untranslated 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 to be
complementary to a region preceding or spanning the initiation
codon on the coding strand or in the 3' untranslated region of an
mRNA. An antisense nucleic acid for inhibiting the expression of
human c-Maf protein in a cell can be designed based upon the
nucleotide sequence encoding the human c-Maf protein (e.g., SEQ ID
NO: 1 and plasmid pHu-c-Maf), constructed according to the rules of
Watson and Crick base pairing.
[0143] An antisense nucleic acid can exist in a variety of
different forms. For example, the antisense nucleic acid can be an
oligonucleotide that is complementary to only a portion of a human
c-Maf gene. An antisense oligonucleotides can be constructed using
chemical synthesis procedures known in the art. An antisense
oligonucleotide can be 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 human
c-Maf expression in cells in culture, one or more antisense
oligonucleotides can be added to cells in culture media, typically
at about 200 .mu.g oligonucleotide/ml.
[0144] 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 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 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. For example, for inducible expression
of antisense RNA, an inducible eukaryotic regulatory system, such
as the Tet system (e.g., as described in Gossen, M. and Bujard, H.
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.
(1995) Science 268:1766-1769; PCT Publication No. WO 94/29442; and
PCT Publication No. WO 96/01313) can be used. The antisense
expression vector is prepared as described above for recombinant
expression vectors, except that the cDNA (or portion thereof) is
cloned into the vector in the antisense orientation. The antisense
expression vector can be in the form 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.
[0145] 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 human
c-Maf mRNA can be designed based upon the nucleotide sequence of
the human c-Maf cDNA. 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 human c-Maf mRNA. See for example U.S. Pat. Nos. 4,987,071 and
5,116,742, both by Cech et al. Alternatively, human c-Maf 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.
[0146] Another type of inhibitory agent that can be used to inhibit
the expression and/or activity of human c-Maf in a cell is an
intracellular antibody specific for the human c-Maf protein. 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) FEBS Letters 274:193-198;
Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428;
Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA
90:7889-7893; Biocca, S. et 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.
et al. (1994) J. Biol. Chem. 269:23931-23936; Beerli, R. R. et al.
(1994) Biochem. Biophys. Res. Commun. 204:666-672; Mhashilkar, A.
M. et al. (1995) EMBO J. 14:1542-1551; Richardson, J. H. et al.
(1995) Proc. Natl. Acad. Sci. USA 92:3137-3141; PCT Publication No.
WO 94/02610 by Marasco et al.; and PCT Publication No. WO 95/03832
by Duan et al.).
[0147] 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 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 human c-Maf activity according to the inhibitory
methods of the invention, an intracellular antibody that
specifically binds the human c-Maf protein is expressed in the
cytoplasm of the cell. To prepare an intracellular antibody
expression vector, antibody light and heavy chain cDNAs encoding
antibody chains specific for the target protein of interest, e.g.,
human c-Maf, are isolated, typically from a hybridoma that secretes
a monoclonal antibody specific for the human c-Maf protein.
Hybridomas secreting anti- human c-Maf monoclonal antibodies, or
recombinant anti-human c-Maf monoclonal antibodies, can be prepared
as described above. Once a monoclonal antibody specific for human
c-Maf protein has been 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 which PCR 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 and Human Services, NIH Publication No. 91-3242 and in
the "Vbase" human germline sequence database.
[0148] Once obtained, the antibody light and heavy chain sequences
are cloned into a recombinant expression vector using standard
methods. To allow for cytoplasmic expression of the light and heavy
chains, the nucleotide sequences encoding the hydrophobic leaders
of the light and heavy chains are removed. An intracellular
antibody expression vector can encode an intracellular antibody in
one of several different forms. For example, in one embodiment, the
vector encodes 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/CH1 region of the heavy chain such that a Fab
fragment is expressed intracellularly. In the most preferred
embodiment, the vector encodes a single chain antibody (scFv)
wherein the variable regions of the light and heavy chains are
linked by a flexible peptide linker (e.g., (Gly.sub.4Ser).sub.3)
and expressed as a single chain molecule. To inhibit human c-Maf
activity in a cell, the expression vector encoding the anti-human
c-Maf intracellular antibody is introduced into the cell by
standard transfection methods, as discussed hereinbefore.
[0149] Yet another form of an inhibitory agent of the invention is
an inhibitory form of human c-Maf, 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. et al. (1994) 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). By dimerizing with functional
transcription factors, such dominant negative inhibitors can
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 mafK, mafF, mafG and p18,
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. (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 12:53-62). Additionally, MafB has been identified as an
interaction partner of Ets-1 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).
[0150] Accordingly, an inhibitory agent of the invention can be a
form of a human c-Maf protein that has the ability to dimerize with
other proteins but that lacks the ability to activate
transcription. This dominant negative form of a human c-Maf protein
may be, for example, a mutated form of human c-Maf in which the
transactivation domain has been removed. Such dominant negative
human c-Maf proteins can be expressed in cells using a recombinant
expression vector encoding the human c-Maf protein, which is
introduced into the cell by standard transfection methods. To
express a mutant form of human c-Maf lacking a transactivation
domain, nucleotide sequences encoding the amino terminal
transactivation domain of human c-Maf are removed from the c-maf
coding sequences 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 1-257, are
removed. Nucleotide sequences encoding the basic-leucine zipper
region are maintained. The truncated DNA is inserted into a
recombinant expression vector, which is then introduced into a cell
to allow for expression of the truncated human c-Maf, lacking a
transactivation domain, in the cell.
[0151] Other inhibitory agents that can be used to inhibit the
activity of a human c-Maf protein are chemical compounds that
directly inhibit human c-Maf activity or inhibit the interaction
between human c-Maf and target DNA or another protein. Such
compounds can be identified using screening assays that select for
such compounds, as described in detail above.
[0152] B. Stimulatory Agents
[0153] According to a modulatory method of the invention, human
c-Maf activity is stimulated in a cell by contacting the cell with
a stimulatory agent. Examples of such stimulatory agents include
active human c-Maf protein and nucleic acid molecules encoding
human c-Maf that are introduced into the cell to increase human
c-Maf activity in the cell. A preferred stimulatory agent is a
nucleic acid molecule encoding a human c-Maf protein, wherein the
nucleic acid molecule is introduced into the cell in a form
suitable for expression of the active human c-Maf protein in the
cell. To express a human c-Maf protein in a cell, typically a human
c-Maf-encoding DNA is first introduced into a recombinant
expression vector using standard molecular biology techniques, as
described herein. A human c-Maf-encoding DNA can be obtained, for
example, from plasmid pHu-c-Maf or by amplification using the
polymerase chain reaction (PCR), using primers based on the human
c-Maf nucleotide sequence. Following isolation or amplification of
human c-Maf-encoding DNA, the DNA fragment is introduced into an
expression vector and transfected into target cells by standard
methods, as described herein.
[0154] Other stimulatory agents that can be used to stimulate the
activity of a human c-Maf protein are chemical compounds that
stimulate human c-Maf activity in cells, such as compounds that
directly stimulate human c-Maf protein and compounds that promote
the interaction between human c-Maf and target DNA or other
proteins. Such compounds can be identified using screening assays
that select for such compounds, as described in detail above.
[0155] The modulatory methods of the invention can be performed in
vitro (e.g., by culturing the cell with the agent or by introducing
the agent into cells in culture) or, alternatively, in vivo (e.g.,
by administering the agent to a subject or by introducing the agent
into cells of a subject, such as by gene therapy). For practicing
the modulatory method in vitro, cells can be obtained from a
subject by standard methods and incubated (i.e., cultured) in vitro
with a modulatory agent of the invention to modulate human c-Maf
activity in the cells. For example, peripheral blood mononuclear
cells (PBMCs) can be obtained from a subject and isolated by
density gradient 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. B cells can be enriched for
example, by positive selection using antibodies to B cell surface
markers, for example by incubating cells with a specific primary
monoclonal antibody (mAb), followed by isolation of cells that bind
the mAb using magnetic beads coated with a secondary antibody that
binds the primary mAb. Specific cell populations can also be
isolated by fluorescence activated cell sorting according to
standard methods. If desired, cells treated in vitro with a
modulatory agent of the invention can be readministered to the
subject. For administration to a subject, it may be preferable to
first remove residual agents in the culture from the cells before
administering 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
readministration to a subject, see also U.S. Pat. No. 5,399,346 by
W. F. Anderson et al.
[0156] For practicing the modulatory method in vivo in a subject,
the modulatory agent can be administered to the subject such that
human c-Maf activity in cells of the subject is modulated. The term
"subject" is intended to include living organisms in which an
immune response can be elicited. Preferred subjects are mammals.
Examples of subjects include humans, monkeys, dogs, cats, mice,
rats, cows, horses, goats and sheep.
[0157] For stimulatory or inhibitory agents that comprise nucleic
acids (including recombinant expression vectors encoding human
c-Maf protein, antisense RNA, intracellular antibodies or dominant
negative inhibitors), the agents can be introduced 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
encompass both non-viral and viral methods, including:
[0158] 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; 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).
[0159] Cationic Lipids: Naked DNA can be introduced into cells in
vivo by complexing the DNA with cationic lipids or encapsulating
the DNA in cationic liposomes. Examples of suitable cationic lipid
formulations include
N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride
(DOTMA) and a 1:1 molar ratio of
1,2-dimyristyloxy-propyl-3-dimethylhydro- xyethylammonium bromide
(DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) (see e.g.,
Logan, J. J. et al. (1995) Gene Therapy 2:38-49; San, H. et al.
(1993) Human Gene Therapy 4:781-788).
[0160] 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 ligand for a cell-surface
receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol.
Chem. 263:14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967;
and U.S. Pat. No. 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 cytoplasm can be used to avoid degradation of the complex by
intracellular lysosomes (see for example Curiel et al. (1991) Proc.
Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl.
Acad. Sci. USA 90:2122-2126).
[0161] 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). 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 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 .psi. Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, endothelial cells, lymphocytes,
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 85:3014-3018;
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. 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 et al. (1992) Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwuetal. (1993) J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. 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 replication of the target cell.
[0162] 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
lytic 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. (1992) Cell 68:143-155. Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 d1324 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
supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)
Proc. Natl. 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 avoiding 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 vectors currently in use are deleted for all or parts of
the viral E1 and E3 genes but retain as much as 80% of the
adenoviral genetic material.
[0163] Adeno-Associated Viruses: 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
see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)
158:97-129). It is also one of the few viruses that may integrate
its DNA into non-dividing cells, and exhibits a high frequency of
stable integration (see for example Flotte et al. (1992) Am. J.
Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J.
Virol. 63:3822-3828; and McLaughlin 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
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. 2:32-39; Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte etal. (1993) J. Biol. Chem.
268:3781-3790).
[0164] 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
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 appropriate 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 of the gene product.
[0165] In a preferred embodiment, a retroviral expression vector
encoding human c-Maf is used to express human c-Maf protein in
cells in vivo, to thereby stimulate c-Maf protein activity in vivo.
Such retroviral vectors can be prepared according to standard
methods known in the art (discussed further above).
[0166] A modulatory agent, such as a chemical compound, can be
administered to a subject as a pharmaceutical composition. Such
compositions typically comprise the modulatory agent 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 administration. 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. Pharmaceutical
compositions can be prepared as described above in subsection
IV.
[0167] The identification of c-Maf as a key regulator of the
production of IL-4 (see e.g., PCT Publication WO 97/39721 and Ho,
I-C. et al. (1996) Cell 85:973-983), and hence 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. The stimulatory methods of the invention (i.e.,
methods that use a stimulatory agent to enhance human c-Maf
activity) result in production of Th2-associated cytokines, with
concomitant promotion of a Th2 response and downregulation of a Th1
response. In contrast, the inhibitory methods of the invention
(i.e., methods that use an inhibitory agent to downmodulate human
c-Maf activity) inhibit the production of Th2-associated cytokines,
with concomitant downregulation of a Th2 response and promotion of
a Th1 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
Th1 responses. Alternatively, to treat a disease condition wherein
a Th1 response is beneficial, an inhibitory method of the invention
is selected such that Th2 responses are downregulated while
promoting Th1 responses. 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.
[0168] Numerous disease conditions associated with a predominant
Th1 or Th2-type 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.
[0169] A. Allergies
[0170] 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 activation of cells that mediate allergic reactions, i.e., mast
cells and basophils. IL-4 also plays an important role in
eosinophil mediated inflammatory 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 may be directly
administered 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 readministered to the subject. Moreover, in
certain situations it may be beneficial to coadminister 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 administering other Th1-promoting agents, such as the
cytokine IL-12 or antibodies to Th2-associated cytokines (e.g.,
anti-IL-4 antibodies), to the allergic subject in amounts
sufficient to further stimulate a Th1-type response.
[0171] B. Cancer
[0172] The expression of Th2-promoting cytokines has been reported
to be elevated in cancer patients (see e.g., Yamamura, M., et al.
(1993) J. Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc.
Natl. Acad. Sci. USA 89:7708-7712) and malignant disease is often
associated with a shift from Th1 type responses to Th2 type
responses along with a worsening of the course of the disease.
Accordingly, the inhibitory methods of the invention can be used to
inhibit the production of Th2-associated cytokines in cancer
patients, as a means to counteract the Th1 to Th2 shift and thereby
promote an ongoing Th1 response in the patients to ameliorate the
course of the disease. The inhibitory method can involve either
direct administration 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) with an inhibitory agent followed by
readministration of the cells to the subject. The treatment may be
further enhanced by administering other Th1-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 Th1-type response.
[0173] C. Infectious Diseases
[0174] The expression of Th2-promoting cytokines also has been
reported to increase during a variety of infectious diseases,
including HIV infection, tuberculosis, leishmaniasis,
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 14:107-1 11; Fauci, A. S. (1988) Science
239:617-623; Locksley, R. M. and Scott, P. (1992)
Immunoparasitology Today 1:A58-A61; Pearce, E. J., etal. (1991) J.
Exp. Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol.
141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.
148:3264-3270; Bancroft, A. J., etal. (1993) J. Immunol.
150:1395-1402; Pearlman, E., et al. (1993) Infect. Immun.
61:1105-1112; Else, K. J., etal. (1994) J. Exp. Med. 179:347-351)
and such infectious diseases are also associated with a Th1 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 diseases, as a
means to counteract the Th1 to Th2 shift and thereby promote an
ongoing Th1 response in the patients to ameliorate the course of
the infection. The inhibitory method can involve either direct
administration 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 readministration of the cells to the subject. The treatment may
be further enhanced by administering other Th1-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 Th1-type response.
[0175] D. Autoimmune Diseases
[0176] 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 inappropriate activation of T cells that are
reactive against self tissue and that promote the production of
cytokines and autoantibodies 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 example, in
experimental allergic encephalomyelitis (EAE), stimulation of a
Th2-type response by administration of IL-4 at the time of the
induction of the disease diminishes the intensity of the autoimmune
disease (Paul, W. E., et al. (1994) Cell 76:241-251). Furthermore,
recovery of the animals from the disease has been shown to be
associated with an increase in a Th2-type response as evidenced by
an increase of Th2-specific cytokines (Koury, S. J., et al. (1992)
J. Exp. Med. 176:1355-1364). Moreover, T cells that can suppress
EAE secrete Th2-specific cytokines (Chen, C., et al. (1994)
Immunity 1:147-154). 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.
[0177] 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 promotes a Th2
response) prevents or delays onset of type I diabetes that normally
develops in these mice (Rapoport, M. J., et al. (1993) J. Exp. Med.
178:87-99). Thus, stimulation of a Th2 response in a subject
suffering from or susceptible to diabetes may ameliorate the
effects of the disease or inhibit the onset of the disease.
[0178] 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 Th1 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 Th1 response can
be concomitantly downmodulated to thereby ameliorate the effects of
the disease.
[0179] 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
administration of a stimulatory agent to the subject or ex vivo
treatment of cells obtained from the subject (e.g., Thp, Th1 cells,
B cells, non-lymphoid cells) with a stimulatory agent followed by
readministration of the cells to the subject. The treatment may be
further enhanced by administering other Th2-promoting agents, such
as IL-4 itself or antibodies to Th1-associated cytokines, to the
subject in amounts sufficient to further stimulate a Th2-type
response.
[0180] In contrast to the autoimmune diseases described above in
which a Th2 response is desirable, other autoimmune diseases may be
ameliorated by a Th1-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 administrating a Th1-promoting cytokine (e.g.,
IFN-.gamma.) to the subject in amounts sufficient to further
stimulate a Th1-type response.
[0181] 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 multiple sclerosis and the NOD mice as a
model for diabetes) or other well characterized animal models of
human autoimmune diseases. 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., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856). A modulatory
(i.e., stimulatory or inhibitory) agent of the invention is
administered to test animals and the course of the disease in the
test animals 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 animals
treated with the agent as compared to untreated animals (or animals
treated with a control agent).
[0182] 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 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 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, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis.
[0183] E. Transplantation
[0184] While graft rejection or graft acceptance may not be
attributable exclusively to the action of a particular T cell
subset (i.e., Th1 or Th2 cells) in the graft 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 graft survival or a predominant Th2 response
in graft rejection. For example, graft acceptance has been
associated with production of a Th2 cytokine pattern and/or graft
rejection has been associated with production of a Th1 cytokine
pattern (see e.g., Takeuchi, T. et al. (1992) Transplantation
53:1281-1291; Tzakis, A. G. et al. (1994) J. Pediatr. Surg.
29:754-756; Thai, N. L. et al. (1995) Transplantation 59:274-281).
Additionally, adoptive transfer of cells having a Th2 cytokine
phenotype prolongs skin graft 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,
administration of IL-4, which promotes Th2 differentiation,
prolongs cardiac allograft survival (Levy, A. E. and Alexander, J.
W. (1995) Transplantation 60:405-406), whereas administration of
IL-12 in combination with anti-IL-10 antibodies, which promotes Th1
differentiation, enhances skin allograft rejection (Gorczynski, R.
M. et al. (1995) Transplantation 60:1337-1341).
[0185] 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 graft. 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 administration of a stimulatory agent to the
transplant recipient or ex vivo treatment of cells obtained from
the subject (e.g., Thp, Th1 cells, B cells, non-lymphoid cells)
with a stimulatory agent followed by readministration of the cells
to the subject. The treatment may be further enhanced by
administering other Th2-promoting agents, such as IL-4 itself or
antibodies to Th1-associated cytokines, to the recipient in amounts
sufficient to further stimulate a Th2-type response.
[0186] 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 stimulate
production 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 stimulate IL-4
production and the IL-4 can be recovered from the culture
supernatant, further purified if necessary, and packaged for
commercial use).
[0187] Furthermore, the modulatory methods of the invention can be
applied to vaccinations to promote either a Th1 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 response to a
vaccine either to a Th1 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 coadministered 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 immune response to an antigen of interest,
the antigen and an inhibitory agent of the invention can be
coadministered to a subject to promote a Th1 response to the
antigen in a subject, since Th1 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 or in
separate compositions. In a preferred embodiment, the antigen of
interest and the modulatory agent are administered simultaneously
to the subject. Alternatively, in certain situations it may be
desirable to administer the antigen first and then the modulatory
agent or vice versa (for example, in the case of an antigen that
naturally evokes a Th1 response, it may be beneficial to first
administer the antigen alone to stimulate a Th1 response and then
administer a stimulatory agent, alone or together with a boost of
antigen, to shift the immune response to a Th2 response).
[0188] This invention is further illustrated by the following
example, which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by reference.
Additionally, all nucleotide and amino acid sequences deposited in
public databases referred to herein are also hereby incorporated by
reference.
EXAMPLE
Isolation and Characterization of a Human c-Maf Nucleic Acid
[0189] To isolate a nucleic acid molecule encoding human c-maf, a
human genomic DNA library in lambda phage (commercially available
from Stratagene) was screened with a radiolabeled DNA probe derived
from the 3' untranslated region of the mouse c-Maf gene. Following
hybridization under standard conditions, filters were washed under
stringent conditions in 0.2.times.SSC, 0.1% SDS wash buffer at
approximately 62.degree. C. Phage clones that remained hybridized
to the probe under these conditions were selected and isolated to
purity. The genomic inserts of the isolated phage were subcloned
into the plasmid vector pBluescript KS/II, by restriction digestion
of the phage DNA with NheI and insertion into the Xbal site of the
plasmid. E. coli bacterial cells carrying a pBluescript plasmid
containing the human c-Maf coding region, referred to herein as
pHu-c-Maf, has been deposited under the provisions of the Budapest
Treaty with the American Type Culture Collection, Rockville, Md.,
on Feb. 24, 1998 and assigned ATCC Accession No. 98671. This
plasmid contains an NheI fragment of approximately 4.2 kb (derived
from the isolated phage), cloned into the compatible XbaI site of
the plasmid vector, to thereby create a .about.4.2 kb NheI/XbaI
insert that encodes human c-Maf. It should be noted that upon
ligation of the NheI fragment into the Xbal site, these restriction
sites are not regenerated and, thus, to excise the fragment from
the plasmid, it is necessary to use adjacent restriction sites
within the pBluescript polylinker.
[0190] The coding region of human c-Maf, contained in the pHu-c-Maf
plasmid, was sequenced by standard dideoxy sequencing methods. The
nucleotide and predicted amino acid sequences are shown in SEQ ID
NOs: 1 and 2, respectively.
[0191] The coding region encompasses approximately 1.2 kb of DNA
and thus, the remainder of the 4.2 kb insert of pHu-c-Maf
represents 5' and 3' untranslated sequences. FIG. 1 shows a
comparison of the nucleotide sequence of hu-c-Maf shown of SEQ ID
NO: 1 with the mouse c-Maf coding region. A number of nucleotide
differences between the two coding regions are evident, which
differences are boxed in FIG. 1. FIG. 2 shows a comparison of the
amino acid sequence of hu-c-Maf shown of SEQ ID NO: 2 with the
mouse c-Maf amino acid sequence. Again, a number of amino acid
differences between the two proteins are evident, which differences
are boxed in FIG. 2. The overall structure of the human c-Maf
protein, however, is conserved with the mouse c-Maf protein,
including the presence of a leucine zipper domain at amino acid
positions 313-348 of SEQ ID NO: 2.
[0192] Equivalents
[0193] 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 equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *
References