U.S. patent application number 10/744379 was filed with the patent office on 2004-09-16 for novel receptor that causes cell death.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Degli-Esposti Rankin, Mariapia A., Goodwin, Raymond G..
Application Number | 20040180826 10/744379 |
Document ID | / |
Family ID | 21932492 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180826 |
Kind Code |
A1 |
Degli-Esposti Rankin, Mariapia A. ;
et al. |
September 16, 2004 |
Novel receptor that causes cell death
Abstract
Isolated apoptosis inducing receptors, DNAs encoding such
receptors, and pharmaceutical compositions made therefrom, are
disclosed. The isolated receptors can be used to regulate an immune
response. The receptors are also useful in screening for inhibitors
thereof.
Inventors: |
Degli-Esposti Rankin, Mariapia
A.; (Seattle, WA) ; Goodwin, Raymond G.;
(Seattle, WA) |
Correspondence
Address: |
Christine M. Bellas
Immunex Corporation
Law Department
51 University Street
Seattle
WA
98101
US
|
Assignee: |
Immunex Corporation
|
Family ID: |
21932492 |
Appl. No.: |
10/744379 |
Filed: |
December 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10744379 |
Dec 23, 2003 |
|
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|
08943776 |
Oct 3, 1997 |
|
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60044456 |
Oct 4, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/18.9; 514/44R; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/7151 20130101 |
Class at
Publication: |
514/012 ;
514/044; 435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
A61K 038/17; A61K
048/00; C07H 021/04; C07K 014/705 |
Claims
We claim:
1. An isolated DNA selected from the group consisting of: (a) a DNA
encoding a protein having an amino acid sequence of amino acids 1
through 417 of SEQ ID NO: 2; (b) a DNA encoding a protein having an
amino acid sequence of amino acids 1 through 411 of SEQ ID NO: 5;
(c) DNA molecules capable of hybridization to the DNA of (a) under
stringent conditions, and which encode biologically active AIR; and
(c) DNA molecules encoding biologically active fragments of
proteins encoded by the DNA of (a), (b) or (c).
2. The DNA according to claim 1, selected from the group consisting
of oligonucleotides of at least about 17 nucleotides in length,
oligonucleotides of at least about 25 nucleotides in length, and
oligonucleotides of at least about 30 nucleotides in length, having
a nucleotide sequence derived from the DNA of SEQ ID NO:1 that
encodes the cytoplasmic domain of AIR.
3. An isolated DNA according to claim 1, selected from the group
consisting of: (a) a DNA encoding a protein having an amino acid
sequence of amino acids 1 through 417 of SEQ ID NO: 2; (b) a DNA
encoding a protein having an amino acid sequence of amino acids 1
through 411 of SEQ ID NO: 5; (c) DNA molecules encoding
biologically active AIR polypeptides that are at least about 70%
identical in amino acid sequence to the protein of (a); and (d) DNA
molecules encoding fragments of proteins encoded by the DNA of (a),
(b) or (c), and which encode biologically active AIR.
4. An isolated DNA encoding an AIR polypeptide comprising an amino
acid sequence as set forth in SEQ ID NO:2, having an amino terminus
selected from the group consisting of an amino acid between amino
acid 1 and amino acid 29, inclusive, of SEQ ID NO:2, and a carboxy
terminus selected from the group consisting of an amino acid
between amino acid 190 and amino acid 200, inclusive.
5. An isolated DNA encoding an AIR polypeptide comprising an amino
acid sequence as set forth in SEQ ID NO:2, having an amino terminus
selected from the group consisting of an amino acid between amino
acid 225 and amino acid 335, inclusive, of SEQ ID NO:2, and a
carboxy terminus selected from the group consisting of an amino
acid between amino acid 410 and amino acid 417, inclusive.
6. A recombinant expression vector comprising a DNA sequence
according to claim 1.
7. A recombinant expression vector comprising a DNA sequence
according to claim 3.
8. A recombinant expression vector comprising a DNA sequence
according to claim 4.
9. A recombinant expression vector comprising a DNA sequence
according to claim 5.
10. A host cell transformed or transfected with an expression
vector according to claim 6.
11. A host cell transformed or transfected with an expression
vector according to claim 7.
12. A host cell transformed or transfected with an expression
vector according to claim 9.
13. A process for preparing an AIR protein, comprising culturing a
host cell according to claim 10 under conditions promoting
expression and recovering the AIR.
14. A process for preparing an AIR protein, comprising culturing a
host cell according to claim 11 under conditions promoting
expression and recovering the AIR.
15. A process for preparing an AIR protein, comprising culturing a
host cell according to claim 12 under conditions promoting
expression and recovering the AIR.
16. An isolated AIR polypeptide selected from the group consisting
of: (a) a polypeptide having an amino acid sequence of amino acids
1 through 417 of SEQ ID NO: 2; (b) a polypeptide having an amino
acid sequence of amino acids 1 through 411 of SEQ ID NO: 6; (c) an
AIR polypeptide encoded by a DNA capable of hybridization to a DNA
encoding the protein of (a) under stringent conditions, and which
is biologically active; and (d) biologically active fragments of
the polypeptides of (a), or (b).
17. An isolated AIR polypeptide according to claim 16, selected
from the group consisting of: (a) a polypeptide having an amino
acid sequence of amino acids 1 through 417 of SEQ ID NO: 2; (b) a
polypeptide having an amino acid sequence of amino acids 1 through
411 of SEQ ID NO: 6; (c) an AIR polypeptide that is at least about
70% identical in amino acid sequence to the polypeptide of (a), and
which is biologically active; and (d) biologically active fragments
of the polypeptides of (a), or (b).
18. An isolated and purified soluble AIR polypeptide selected from
the group consisting of a polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO:2, having an amino terminus
selected from the group consisting of an amino acid between amino
acid 225 and amino acid 335, inclusive, of SEQ ID NO:2, and a
carboxy terminus selected from the group consisting an amino acid
between amino acid 410 and amino acid 417, inclusive, and
biologically active fragments of the polypeptide.
19. A composition comprising an AIR polypeptide according to claim
18, and a suitable diluent or carrier.
20. An antibody immunoreactive with AIR.
21. The antibody of claim 20 which is a monoclonal antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
60/044,456, filed Oct. 4, 1996.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
cytokine receptors, and more specifically to cytokine receptor
proteins having immunoregulatory activity.
BACKGROUND OF THE INVENTION
[0003] Efficient functioning of the immune system requires a fine
balance between cell proliferation and differentiation and cell
death, to ensure that the immune system is capable of reacting to
foreign, but not self antigens. Central tolerance refers to the
mechanisms which lead to positive and negative selection of T cells
in the thymus where T cells are positively or negatively selected
depending on their capacity to interact with self MHC antigens
expressed in the thymus. In the periphery, mature T cells which
interact with self antigens expressed uniquely in the periphery are
deleted, as are T cells which have been activated by foreign
antigen. This is known as peripheral tolerance.
[0004] Deletion of inappropriately activated T cells is believed to
occur via programmed cell death known as apoptosis, which is
distinct from cell death due to necrosis. Two members of the TNF
family, Fas ligand (FasL) and TNF, have been reported to be
involved in some of the effector mechanisms which control immune
tolerance (reviewed in Cleveland and Ihle, Cell 81:479; 1995). FasL
and TNF mediate their biological effects by binding their
respective receptors, which are members of the TNFR superfamily
(Smith et al., Cell 76:959; 1994).
[0005] Fas (the receptor for FasL) and TNF receptor type I (TNFRI)
both contain a unique motif within their cytoplasmic regions, which
has been termed the death domain (Tartaglia et al., Cell 74:845,
1993; Itoh and Nagata, J. Biol. Chem. 268:10932, 1993).
Overexpression of the death domain in transient transfection
systems has been shown to result in apoptosis. The biological
effects of Fas/FasL and TNF/TNFRI interactions are thought to occur
through both distinct and similar signaling pathways
(Schultze-Osthoff et al., EMBO J. 13:4587, 1994; Wong and Goeddel,
J. Immunol. 152:1751, 1994).
[0006] The lpr and gld mouse models have implicated the Fas/FasL
system in peripheral tolerance; however, peripheral T cell deletion
does occur in lpr mice. This Fas-independent apoptosis of mature T
cells has been shown to be partly TNF mediated (Zheng et al.,
Nature 377:348, 1995) These data imply that multiple apoptotic
mechanisms, including unrecognized ones, may be involved in
peripheral tolerance. Moreover, the mechanisms mediating central
tolerance remain unknown. Investigation into the existence and
identity of other molecule(s) that play a role in apoptosis is
desirable. Identifying such molecules would provide an additional
means of regulating apoptosis, as well as providing further insight
into the development of self-tolerance by the immune system and the
etiology of autoimmune diseases.
SUMMARY OF THE INVENTION
[0007] The present invention identifies a novel receptor, referred
to as Apoptosis Inducing Receptor (AIR) that induces apoptosis of
certain cells on which it is expressed. The receptor is a Type I
transmembrane protein having 417 amino acid residues. Soluble forms
of the receptor can be prepared and used to regulate cell death in
a therapeutic setting; accordingly, pharmaceutical compositions
comprising soluble forms of the novel receptor are also provided.
Soluble forms of the receptor will also be useful in vitro to block
apoptosis of AIR-expressing cells, or to screen for agonists or
antagonists of AIR activity. The cytoplasmic domain of AIR will be
useful in developing assays for inhibitors of AIR-induced cell
death. Such inhibitors will have use in regulating cell death in a
therapeutic setting as well as in vitro. Deleted forms and fusion
proteins comprising the novel receptor are also disclosed. Agonists
of AIR activity can be used to kill tumor cells that express AIR,
or kill T cells expressing AIR in autoimmune diseases. These and
other aspects of the present invention w ill become evident upon
reference to the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 presents an alignment of AIR and TNFRI death domain
sequences. The 83 amino acid sequence of the TNFRI death domain is
compared to the homologous sequence from the AIR cytoplasmic
region. Identical and conserved amino acids (+) are shown in the
middle line.
DETAILED DESCRIPTION OF THE INVENTION
[0009] A new TNF receptor-like sequence was identified in the EST
database. Oligonucleotide primers were synthesized based on the EST
sequence, and a full-length cDNA was cloned from a peripheral blood
T cell library. The encoded protein was designated AIR, for
Apoptosis Inducing Receptor. The receptor is a Type I transmembrane
protein having 417 amino acid residues, with a predicted 24 amino
acid signal sequence, a 173 amino acid extracellular domain, a 27
amino acid transmembrane domain, and a 193 amino acid cytoplasmic
tail. The cytoplasmic region of AIR displayed significant amino
acid homology (48% identity, 64% similarity) to the 83 amino acid
sequence which encodes the Type I TNF receptor death domain. This
region, conserved between TNFRI and Fas, is necessary and
sufficient for transduction of an apoptotic signal to a cell
expressing AIR.
[0010] A search of the NCBI databank identified five expressed
sequence tags (ESTs) having regions of identity with AIR DNA. These
ESTs (NCBI accession numbers H41522, H46374, H46211, H46662, and
H46424) are all human cDNA fragments. The NCBI records do not
disclose any polypeptide encoded by the ESTs, and do not indicate
what the reading frame, if any, might be. However, even if the
knowledge of the reading frame revealed herein by disclosure of
complete AIR coding regions is used to express the ESTs, none of
the encoded polypeptides would have the biological properties of
the presently-claimed AIR polypeptides. In other words, if each of
the five ESTs were inserted into expression vectors downstream from
an initiator methionine codon, in the reading frame elucidated
herein, none of the resulting expressed polypeptides would contain
a sufficient portion of the AIR protein to induce apoptosis.
[0011] Apoptosis and the TNF/TNFR Superfamilies
[0012] Efficient functioning of the immune system requires a fine
balance between cell proliferation and differentiation and cell
death. Central tolerance refers to the mechanisms which lead to
positive and negative selection of T cells in the thymus. It is
believed that T cells are positively or negatively selected
depending on their capacity to interact with self MHC antigens
expressed in the thymus: autoreactive T cells are eliminated, while
those that recognize non-self antigens are selected for survival
and differentiation. In the periphery, mature T cells which
interact with self antigens expressed uniquely in the periphery are
deleted, as are T cells which have been activated by foreign
antigen. These mechanisms ensure that the immune system is capable
of reacting to foreign, but not self antigens and that activated
lymphocytes are removed after they have fulfilled their role
[0013] Two members of the TNFR superfamily, Fas and TNFRI, are
believed to play an important role in some of the effector
mechanisms which control peripheral tolerance. Fas has been
reported to mediate apoptosis and is believed to play a role in
clonal deletion of self-reactive T-cells (Itoh et al., Cell 66:233,
1991; Watanabe-Fukunage et al., Nature 356:314, 1992). DNAs
encoding Fas ligand have been isolated; binding of the Fas ligand
to cells expressing Fas antigen has been demonstrated to induce
apoptosis (Suda et al., Cell, 75:1169, 1993; Takahashi et al.,
International Immunology 6:1567, 1994). The lpr and gld mouse
models have implicated the Fas/FasL system in the processes which
lead to elimination of T cells after they have been activated by
self or foreign antigen in the periphery. However, some peripheral
T cell deletion still occurs in lpr mice. This Fas-independent
apoptosis of mature T cells has been shown to be partly TNF
mediated.
[0014] Elimination of these T cells occurs by apoptosis, a
morphologically defined type of cell death that can be
differentiated from necrosis. Fas and TNFRI both contain a death
domain, a unique motif present within their cytoplasmic regions
(Tartaglia et al., Cell 74:845, 1993; Itoh and Nagata, J. Biol.
Chem. 268:10932, 1993). This domain shows some similarity with a
Drosophila protein referred to as reaper which is required for
most, if not all programmed cell death in this species (White et
al., Science 264:677, 1994). Overexpression of the death domain in
transient transfection systems has been shown to result in
apoptosis.
[0015] The biological effects of Fas/FasL and TNF/TNFRI
interactions are thought to occur through both distinct and similar
signaling pathways (Schultze-Osthoff et al., EMBO J. 13:4587, 1994;
Won, and Goeddel, J. Immunol. 152:1751, 1994). Both receptors
couple ligand binding to tyrosine phosphorylation, and both
activate sphingomyelinases. Cysteine proteinases have also been
implicated in both FasL- and TNF-induced cell death; crmA, a
product of the cowpox virus that inhibits cysteine proteases,
inhibits both FasL- and TNF-induced apoptosis. Both FasL and TNF
induce cell death within a few hours of binding their respective
receptors, indicating that both signal pathways modulate latent
cytoplasmic effector molecules; however, death induced by TNF tends
to be slower than that induced by FasL.
[0016] The body of data currently available regarding apoptosis
imply that multiple apoptotic mechanisms, including those mediated
by Fas and TNFRI as well as additional, unrecognized
ligand/receptor interactions, may be involved in peripheral
tolerance within the immune system. In addition, the mechanism or
mechanisms mediating central tolerance remain unknown. FasL and TNF
are unlikely to be involved in the latter, since positive and
negative selection in the thymus appear normal in lpr and TNFR
knock-out mice. The novel receptor described herein shares certain
similarities with both Fas and TNFRI, and could be important in
regulating cell death during the development of self-tolerance
(either in the periphery or the thymus).
[0017] DNAs, Proteins and Analogs
[0018] The present invention provides isolated AIR polypeptides and
analogs (or muteins) thereof having an AIR activity (i.e., causing
apoptosis of cells expressing an AIR mutein or analog comprising
the death domain when triggered appropriately; or for soluble
forms, binding to AIR-specific antiboides or inhibition of
apoptosis induced by signalling through AIR). Such proteins are
substantially free of contaminating endogenous materials and,
optionally, without associated native-pattern glycosylation.
Derivatives of AIR within the scope of the invention also include
various structural forms of the primary protein which retain
biological activity. Due to the presence of ionizable amino and
carboxyl groups, for example, an AIR protein may be in the form of
acidic or basic salts, or may be in neutral form. Individual amino
acid residues may also be modified by oxidation or reduction. The
primary amino acid structure may be modified by forming covalent or
aggregative conjugates with other chemical moieties, such as
glycosyl groups, lipids, phosphate, acetyl groups and the like, or
by creating amino acid sequence mutants. Covalent derivatives are
prepared by linking particular functional groups to amino acid side
chains or at the N- or C-termini.
[0019] Derivatives of AIR may also be obtained by cross-linking
agents, such as M-maleimidobenzoyl succinimide ester and
N-hydroxysuccinimide, at cysteine and lysine residues. The
inventive proteins may also be covalently bound through reactive
side groups to various insoluble substrates, such as cyanogen
bromide-activated, bisoxirane-activated,
carbonyldiimidazole-activated or tosyl-activated agarose
structures, or by adsorbing to polyolefin surfaces (with or without
glutaraldehyde cross-linking). Once bound to a substrate, proteins
may be used to selectively bind (for purposes of assay or
purification) antibodies raised against the AIR or against other
proteins which are similar to the AIR, as well as other proteins
that bind AIR or its homologous proteins.
[0020] Soluble forms of AIR are also within the scope of the
invention. The nucleotide and predicted amino acid sequence of the
AIR is shown in SEQ ID NOs:1 and 2. Computer analysis indicated
that the protein has an N-terminal signal peptide with a cleavage
site between amino acid 24 and 25. Those skilled in the art will
recognize that the actual cleavage site may be different than that
predicted by computer analysis. Thus, the N-terminal amino acid of
the cleaved peptide is expected to be within about five amino acids
on either side of the predicted cleavage site. The signal peptide
is predicted to be followed by a 173 amino acid extracellular
domain, a 27 amino acid transmembrane domain, and a 193 amino acid
cytoplasmic tail.
[0021] Soluble AIR comprises the signal peptide and the
extracellular domain (residues 1 to 197 of SEQ ID NO:1) or a
fragment thereof. Alternatively, a different signal peptide can be
substituted for residues 1 through 24 of SEQ ID NO:1. Moreover,
fragments of the extracellular domain will also provide soluble
forms of AIR. Fragments can be prepared using known techniques to
isolate a desired portion of the extracellular region, and can be
prepared, for example, by comparing the extracellular region with
those of other members of the TNFR family and selecting forms
similar to those prepared for other family members. Alternatively,
unique restriction sites or PCR techniques that are known in the
art can be used to prepare numerous truncated forms which can be
expressed and analyzed for activity.
[0022] Other derivatives of the AIR protein within the scope of
this invention include covalent or aggregative conjugates of the
protein or its fragments with other proteins or polypeptides, such
as by synthesis in recombinant culture as N-terminal or C-terminal
fusions. For example, the conjugated peptide may be a signal (or
leader) polypeptide sequence at the N-terminal region of the
protein which co-translationally or post-translationally directs
transfer of the protein from its site of synthesis to its site of
function inside or outside of the cell membrane or wall (e.g., the
yeast .alpha.-factor leader).
[0023] Protein fusions can comprise peptides added to facilitate
purification or identification of AIR proteins and homologs (e.g.,
poly-His). The amino acid sequence of the inventive proteins can
also be linked to an identification peptide such as that described
by Hopp et al., BiolTechnology 6:1204 (1988). Such a highly
antigenic peptide provides an epitope reversibly bound by a
specific monoclonal antibody, enabling rapid assay and facile
purification of expressed recombinant protein. The sequence of Hopp
et al. is also specifically cleaved by bovine mucosal enterokinase,
allowing removal of the peptide from the purified protein. Fusion
proteins capped with such peptides may also be resistant to
intracellular degradation in E. coli.
[0024] Fusion proteins further comprise the amino acid sequence of
an AIR linked to an immunoglobulin Fc region. An exemplary Fc
region is a human IgG.sub.1 having a nucleotide and amino acid
sequence set forth in SEQ ID NO:4. Fragments of an Fc region may
also be used, as can Fc muteins. For example, certain residues
within the hinge region of an Fc region are critical for high
affinity binding to Fc.gamma.RI. Canfield and Morrison (J. Exp.
Med. 173:1483; 1991) reported that Leu.sub.(234) and Leu.sub.(235)
were critical to high affinity binding of IgG.sub.3 to Fc.gamma.RI
present on U937 cells. Similar results were obtained by Lund et al.
(J. Immunol. 147:2657, 1991; Molecular Immunol. 29:53, 1991). Such
mutations, alone or in combination, can be made in an IgG.sub.1 Fc
region to decrease the affinity of IgG.sub.1 for FcR. Depending on
the portion of the Fc region used, a fusion protein may be
expressed as a dimer, through formation of interchain disulfide
bonds. If the fusion proteins are made with both heavy and light
chains of an antibody, it is possible to form a protein oligomer
with as many as four AIR regions.
[0025] In another embodiment, AIR protein further comprises an
oligomerizing zipper domain. Zipper domains are described in U.S.
Ser. No. 08/107,353, filed Aug. 13, 1993, the relevant disclosure
of which is incorporated by reference herein. Examples of leucine
zipper domains are those found in the yeast transcription factor
GCN4 and a heat-stable DNA-binding protein found in rat liver
(C/EBP; Landschulz et al., Science 243:1681, 1989), the nuclear
transforming proteins, fos and jun, which preferentially form a
heterodimer (O'Shea et al., Science 245:646, 1989; Turner and
Tjian, Science 243:1689, 1989), and the gene product of the murine
proto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988).
The fusogenic proteins of several different viruses, including
paramyxovirus, coronavirus, measles virus and many retroviruses,
also possess leucine zipper domains (Buckland and Wild, Nature
338:547, 1989; Britton, Nature 353:394, 1991; Delwart and Mosialos,
AIDS Research and Human Retroviruses 6:703, 1990). Conservative
amino acids may be substituted for individual leucine residues in
dimer-forming zipper domains, with minimal decrease in the ability
to dimerize.
[0026] Also included within the scope of the invention are
fragments or derivatives of the intracellular domain of AIR. Such
fragments are prepared by any of the herein-mentioned techniques,
and include peptides that are identical to the cytoplasmic domain
of AIR as shown in SEQ ID NO:1, and those that comprise a portion
of the cytoplasmic region, for example, the death domain. All
techniques used in preparing soluble forms may also be used in
preparing fragments or analogs of the cytoplasmic domain (i.e.,
RT-PCR techniques or use of selected restriction enzymes to prepare
truncations).
[0027] The present invention also includes AIR with or without
associated native-pattern glycosylation. Proteins expressed in
yeast or mammalian expression systems, e.g., COS-7 cells, may be
similar or slightly different in molecular weight and glycosylation
pattern than the native molecules, depending upon the expression
system. Expression of DNAs encoding the inventive proteins in
bacteria such as E. coli provides non-glycosylated molecules.
Functional mutant analogs of AIR protein having inactivated
N-glycosylation sites can be produced by oligonucleotide synthesis
and ligation or by site-specific mutagenesis techniques. These
analog proteins can be produced in a homogeneous,
reduced-carbohydrate form in good yield using yeast expression
systems. N-glycosylation sites in eukaryotic proteins are
characterized by the amino acid triplet Asn-A.sub.1-Z, where
A.sub.1 is any amino acid except Pro, and Z is Ser or Thr. In this
sequence, asparagine provides a side chain amino group for covalent
attachment of carbohydrate. Such a site can be eliminated by
substituting another amino acid for Asn or for residue Z, deleting
Asn or Z. or inserting a non-Z amino acid between A.sub.1 and Z, or
an amino acid other than Asn between Asn and A.sub.1.
[0028] AIR protein derivatives may also be obtained by mutations of
the native AIR or its subunits. An AIR mutated protein, as referred
to herein, is a polypeptide homologous to an AIR protein but which
has an amino acid sequence different from the native AIR because of
one or a plurality of deletions, insertions or substitutions. The
effect of any mutation made in a DNA encoding an AIR peptide may be
easily determined by analyzing the ability of the mutated AIR
peptide to inhibit AIR-induced cell death, for example, by
AIR-specific antibodies. Moreover, activity of AIR analogs, muteins
or derivatives can be determined by any of the assays described
herein
[0029] Analogs of the inventive proteins may be constructed by, for
example, making various substitutions of residues or sequences or
deleting terminal or internal residues or sequences not needed for
biological activity. For example, cysteine residues can be deleted
or replaced with other amino acids to prevent formation of
incorrect intramolecular disulfide bridges upon renaturation. Other
approaches to mutagenesis involve modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which
KEX2 protease activity is present.
[0030] When a deletion or insertion strategy is adopted, the
potential effect of the deletion or insertion on biological
activity should be considered. Subunits of the inventive proteins
may be constructed by deleting terminal or internal residues or
sequences. Soluble forms of AIR can be readily prepared (for
example, by using restriction enzymes to delete portions of the DNA
encoding transmembrane and cytoplasmic regions) and tested for
their ability to inhibit AIR-induced cell death. Polypeptides
corresponding to the cytoplasmic regions, and fragments thereof
(for example, the death domain) can be prepared by similar
techniques. Additional guidance as to the types of mutations that
can be made is provided by a comparison of the sequence of AIR to
proteins that have similar structures, as well as by performing
structural analysis of the inventive AIR proteins.
[0031] Generally, substitutions should be made conservatively;
i.e., the most preferred substitute amino acids are those which do
not affect the biological activity of AIR (i.e., ability of the
inventive proteins to bind antibodies to AIR in a manner
substantially equivalent to that of native AIR for muteins of the
extracellular domain, or ability to induce apoptosis upon
overexpression in transient transfection systems, for example).
Examples of conservative substitutions include substitution of
amino acids outside of the binding domain(s) (either ligand or
antibody binding areas for the extracellular domain, or regions
that interact with other, intracellular proteins for the
cytoplasmic domain), and substitution of amino acids that do not
alter the secondary and/or tertiary structure of native AIR.
Additional examples include substituting one aliphatic residue for
another, such as Ile, Val, Leu, or Ala for one another, or
substitutions of one polar residue for another, such as between Lys
and Arg; Glu and Asp; or Gln and Asn. Other such conservative
substitutions, for example, substitutions of entire regions having
similar hydrophobicity characteristics, are well known
[0032] Mutations in nucleotide sequences constructed for expression
of analog AIR or fragments thereof must, of course, preserve the
reading frame phase of the coding sequences and preferably will not
create complementary regions that could hybridize to produce
secondary mRNA structures such as loops or hairpins which would
adversely affect translation of the receptor mRNA.
[0033] Not all mutations in the nucleotide sequence which encodes
an AIR protein or fragments thereof will be expressed in the final
product, for example, nucleotide substitutions may be made to
enhance expression, primarily to avoid secondary structure loops in
the transcribed mRNA (see EPA 75,444A, incorporated herein by
reference), or to provide codons that are more readily translated
by the selected host, e.g., the well-known E. coli preference
codons for E. coli expression.
[0034] Although a mutation site may be predetermined, it is not
necessary that the nature of the mutation per se be predetermined.
For example, in order to select for optimum characteristics of
mutants, random mutagenesis may be conducted and the expressed
mutated proteins screened for the desired activity. Mutations can
be introduced at particular loci by synthesizing oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes an analog having the
desired amino acid insertion, substitution, or deletion.
[0035] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
having particular codons altered according to the substitution,
deletion, or insertion required. Exemplary methods of making the
alterations set forth above are disclosed by Walder et al. (Gene
42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik
(BioTechniques, Jan. 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and U.S.
Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, and
are incorporated by reference herein.
[0036] Other embodiments of the inventive proteins include AIR
polypeptides encoded by DNAs capable of hybridizing under
moderately stringent conditions (prewashing solution of
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization
conditions of 50.degree. C., 5.times.SSC, overnight) to the DNA
sequences encoding AIR, and other sequences which are degenerate to
those which encode the AIR. In one embodiment, AIR polypeptides are
at least about 70% identical in amino acid sequence to the amino
acid sequence of native AIR protein as set forth in SEQ ID NO:1. In
a preferred embodiment, AIR polypeptides are at least about 80%
identical in amino acid sequence to the native form of AIR; most
preferred polypeptides are those that are at least about 90%
identical to native AIR.
[0037] Percent identity may be determined using a computer program,
for example, the GAP computer program described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG). For fragments derived
from the AIR protein, the identity is calculated based on that
portion of the AIR protein that is present in the fragment
[0038] The biological activity of AIR analogs of the extracellular
domain can be determined by testing the ability of the analogs or
muteins to inhibit AIR-induced cell death where death is triggered
by an agonistic antibody or other agonist of AIR. Alternatively,
suitable assays, for example, an enzyme immunoassay or a dot blot,
employing an antibody that binds native AIR, can be used to assess
the ability of AIR analogs or muteins to inhibit cell death. The
biological activity of AIR analogs or muteins of the extracellular
domain can be determined by testing the ability of the analogs to
cause apoptosis when over-expressed in transfected cells.
Alternatively, suitable assays, for example, signal transduction
assays and methods that evaluate the ability of the cytoplasmic
region of AIR to associate with other intracellular proteins
involved in signal transduction will also be useful to asses the
activity of AIR analogs or muteins. Such methods are well known in
the art.
[0039] The invention also includes AIR DNAs, including any DNAs
that encode the aforementioned AIR proteins and analogs. Due to
code degeneracy, there can be considerable variation in nucleotide
sequences encoding the same amino acid sequence. DNAs included
within the embodiments of the invention are those capable of
hybridizing under moderately stringent conditions (for example,
prewashing solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0)
and hybridization conditions of 50.degree. C., 5.times.SSC,
overnight), or, preferably, under stringent conditions (for
example, hybridization in 6.times.SSC at 63.degree. C. overnight;
washing in 3.times.SSC at 55.degree. C.), to the DNA of SEQ ID
NO:1.
[0040] Fragments of the AIR nucleotide sequences are also useful.
In one embodiment, such fragments comprise at least about 17
consecutive nucleotides, preferably at least about 25 nucleotides,
more preferably at least 30 consecutive nucleotides, of the AIR DNA
disclosed herein. DNA and RNA complements of such fragments are
provided herein, along with both single-stranded and
double-stranded forms of the AIR DNA of SEQ ID NO:1 or those
encoding the aforementioned polypeptides. A fragment of AIR DNA
generally comprises at least about 17 nucleotides, preferably from
about 17 to about 30 nucleotides. Such nucleic acid fragments (for
example, a probe corresponding to the extracellular domain of AIR)
are use as a probe or as primers in a polymerase chain reaction
(PCR).
[0041] The probes find use in detecting the presence of AIR nucleic
acids in in vitro assays and in such procedures as Northern and
Southern blots. Cell types expressing AIR can be identified as
well. Such procedures are well known, and the skilled artisan can
choose a probe of suitable length, depending on the particular
intended application. For PCR, 5' and 3' primers corresponding to
the termini of a desired AIR DNA sequence are employed to isolate
and amplify that sequence, using conventional techniques.
[0042] Other useful fragments of the AIR nucleic acids are
antisense or sense oligonucleotides comprising a single-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target AIR mRNA (sense) or AIR DNA (antisense) sequences. The
ability to create an antisense or a sense oligonucleotide, based
upon a cDNA sequence for a given protein is described in, for
example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der
Krol et al., BioTechniques 6:958, 1988.
[0043] Uses of DNAs, Proteins and Analogs
[0044] The AIR DNAs, proteins and analogs described herein will
have numerous uses, including the preparation of pharmaceutical
compositions. For example, soluble forms of AIR will be useful as
antagonists of AIR-mediated apoptosis. AIR compositions (both
protein and DNAs) will also be useful in development of both
agonistic and antagonistic antibodies to AIR. The former will be
useful in inducing cell death in, for example, tumor cells or
auto-reactive T cells that express AIR; the latter will be useful
in inhibiting apoptosis of AIR-expressing cells. The inventive DNAs
are useful for the expression of recombinant proteins, and as
probes for analysis (either quantitative or qualitative) of the
presence or distribution of AIR transcripts. Antagonists of AIR
will be useful in vitro to facilitate culture of cells (such as
PBT) that express AIR.
[0045] The inventive proteins will also be useful in preparing kits
that are used to detect soluble AIR or monitor AIR-related
apoptotic activity, for example, in patient specimens. AIR proteins
will also find uses in monitoring AIR-related apoptotic activity in
other samples or compositions, as is necessary when screening for
antagonists or mimetics of this activity (for example, peptides or
small molecules that inhibit or mimic, respectively, the
interaction). A variety of assay formats are useful in such kits,
including (but not limited to) ELISA, dot blot, solid phase binding
assays (such as those using a biosensor), rapid format assays and
bioassays.
[0046] The purified AIR according to the invention will facilitate
the discovery of inhibitors of AIR, and thus, inhibitors of
AIR-induced cell death. The use of a purified AIR polypeptide in
the screening for potential inhibitors is important and can
virtually eliminate the possibility of interfering reactions with
contaminants. Such a screening assay can utilize either the
extracellular domain of AIR, the intracellular domain, or a
fragment of either of these polypeptides. Detecting the AIR-induced
apoptotic inhibiting activity of a molecule would typically involve
use of a soluble form of AIR derived from the extracellular domain
in a screening assay to detect molecules capable of binding AIR and
inhibiting binding of, for example, an agonistic antibody, or using
a polypeptide derived from the extracellular domain in an assay to
detect inhibition of the interaction of AIR and other,
intracellular proteins involved in signal transduction.
[0047] Moreover, in vitro systems can be used to ascertain the
ability of molecules to antagonize or agonize AIR activity.
Included in such methods are uses of AIR chimeras, for example, a
chimera of the AIR intracellular domain and an extracellular domain
derived from a protein having a known ligand. The effects on signal
transduction of various molecule can then be monitored by utilizing
the known ligand to transduce an apoptotic signal.
[0048] In addition, AIR polypeptides can also be used for
structure-based design of AIR-inhibitors. Such structure-based
design is also known as "rational drug design." The AIR
polypeptides can be three-dimensionally analyzed by, for example,
X-ray crystallography, nuclear magnetic resonance or homology
modeling, all of which are well-known methods. The use of AIR
structural information in molecular modeling software systems to
assist in inhibitor design is also encompassed by the invention.
Such computer-assisted modeling and drug design may utilize
information such as chemical conformational analysis, electrostatic
potential of the molecules, protein folding, etc. A particular
method of the invention comprises analyzing the three dimensional
structure of AIR for likely binding sites of substrates,
synthesizing a new molecule that incorporates a predictive reactive
site, and assaying the new molecule as described above.
[0049] Expression of Recombinant AIR
[0050] The proteins of the present invention are preferably
produced by recombinant DNA methods by inserting a DNA sequence
encoding AIR protein or a analog thereof into a recombinant
expression vector and expressing the DNA sequence in a recombinant
microbial expression system under conditions promoting expression.
DNA sequences encoding the proteins provided by this invention can
be assembled from cDNA fragments and short oligonucleotide linkers,
or from a series of oligonucleotides, to provide a synthetic gene
which is capable of being inserted in a recombinant expression
vector and expressed in a recombinant transcriptional unit.
[0051] Recombinant expression vectors include synthetic or
cDNA-derived DNA fragments encoding AIR, homologs, or bioequivalent
analogs, operably linked to suitable transcriptional or
translational regulatory elements derived from mammalian,
microbial, viral or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences which control the termination of
transcription and translation, as described in detail below. The
ability to replicate in a host, usually conferred by an origin of
replication, and a selection gene to facilitate recognition of
transformants may additionally be incorporated.
[0052] DNA regions are operably linked when they are functionally
related to each other. For example, DNA for a signal peptide
(secretory leader) is operably linked to DNA for a polypeptide if
it is expressed as a precursor which participates in the secretion
of the polypeptide; a promoter is operably linked to a coding
sequence if it controls the transcription of the sequence; or a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to permit translation. Generally, operably
linked means contiguous and, in the case of secretory leaders,
contiguous and in reading frame. DNA sequences encoding AIR or
homologs which are to be expressed in a microorganism will
preferably contain no introns that could prematurely terminate
transcription of DNA into mRNA.
[0053] Useful expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. E. coli is
typically transformed using derivatives of pBR322, a plasmid
derived from an E. coli species (Bolivar et al., Gene 2:95, 1977).
pBR322 contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed
cells.
[0054] Promoters commonly used in recombinant microbial expression
vectors include the .beta.-lactamase (penicillinase) and lactose
promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et
al., Nature 281:544, 1979), the tryptophan (trp) promoter system
(Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and
tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful
bacterial expression system employs the phage .lambda.P.sub.L
promoter and cI857ts thermolabile repressor. Plasmid vectors
available from the American Type Culture Collection which
incorporate derivatives of the .lambda. P.sub.L promoter include
plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and
pPLc28, resident in E. coli RR1 (ATCC 53082).
[0055] Suitable promoter sequences in yeast vectors include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes
(Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al.,
Biochem. 17:4900, 1978), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Suitable
vectors and promoters for use in yeast expression are further
described in R. Hitzeman et al., EPA 73,657.
[0056] Preferred yeast vectors can be assembled using DNA sequences
from pBR322 for selection and replication in E. coli (Amp.sup.r
gene and origin of replication) and yeast DNA sequences including a
glucose-repressible ADH2 promoter and .alpha.-factor secretion
leader. The ADH2 promoter has been described by Russell et al. (J.
Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724,
1982). The yeast .alpha.-factor leader, which directs secretion of
heterologous proteins, can be inserted between the promoter and the
structural gene to be expressed. See, e.g., Kurjan et al., Cell
30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA
81:5330, 1984. The leader sequence may be modified to contain, near
its 3' end, one or more useful restriction sites to facilitate
fusion of the leader sequence to foreign genes.
[0057] The transcriptional and translational control sequences in
expression vectors to be used in transforming vertebrate cells may
be provided by viral sources. For example, commonly used promoters
and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus
40 (SV40), and human cytomegalovirus. DNA sequences derived from
the SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the other genetic elements required for expression of a
heterologous DNA sequence. The early and late promoters are
particularly useful because both are obtained easily from the virus
as a fragment which also contains the SV40 viral origin of
replication (Fiers et al., Nature 273:113, 1978). Smaller or larger
SV40 fragments may also be used, provided the approximately 250 bp
sequence extending from the Hind III site toward the BglI site
located in the viral origin of replication is included. Further,
viral genomic promoter, control and/or signal sequences may be
utilized, provided such control sequences are compatible with the
host cell chosen. Exemplary vectors can be constructed as disclosed
by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).
[0058] A useful system for stable high level expression of
mammalian receptor cDNAs in C127 murine mammary epithelial cells
can be constructed substantially as described by Cosman et al.
(Mol. Immunol. 23:935, 1986). A preferred eukaryotic vector for
expression of AIR DNA is referred to as pDC406 (McMahan et al.,
EMBO J. 10:2821, 1991), and includes regulatory sequences derived
from SV40, human immunodeficiency virus (HIV), and Epstein-Barr
virus (EBV). Other preferred vectors include pDC409 and pDC410,
which are derived from pDC406. pDC410 was derived from pDC406 by
substituting the EBV origin of replication with sequences encoding
the SV40 large T antigen. pDC409 differs from pDC406 in that a Bgl
II restriction site outside of the multiple cloning site has been
deleted, making the Bgl II site within the multiple cloning site
unique.
[0059] A useful cell line that allows for episomal replication of
expression vectors, such as pDC406 and pDC409, which contain the
EBV origin of replication, is CV-1/EBNA (ATCC CRL 10478). The
CV-1/EBNA cell line was derived by transfection of the CV-1 cell
line with a gene encoding Epstein-Barr virus nuclear antigen-1
(EBNA-1) and constitutively express EBNA-1 driven from human CMV
immediate-early enhancer/promoter.
[0060] Host Cells
[0061] Transformed host cells are cells which have been transformed
or transfected with expression vectors constructed using
recombinant DNA techniques and which contain sequences encoding the
proteins of the present invention. Transformed host cells may
express the desired protein (AIR or homologs thereof), but host
cells transformed for purposes of cloning or amplifying the
inventive DNA do not need to express the protein. Expressed
proteins will preferably be secreted into the culture supernatant,
depending on the DNA selected, but may be deposited in the cell
membrane.
[0062] Suitable host cells for expression of viral proteins include
prokaryotes, yeast or higher eukaryotic cells under the control of
appropriate promoters. Prokaryotes include gram negative or gram
positive organisms, for example E. coli or Bacillus spp. Higher
eukaryotic cells include established cell lines of mammalian origin
as described below. Cell-free translation systems could also be
employed to produce viral proteins using RNAs derived from the DNA
constructs disclosed herein. Appropriate cloning and expression
vectors for use with bacterial, fungal, yeast,.and mammalian
cellular hosts are described by Pouwels et al. (Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure
of which is hereby incorporated by reference.
[0063] Prokaryotic expression hosts may be used for expression of
AIR or homologs that do not require extensive proteolytic and
disulfide processing. Prokaryotic expression vectors generally
comprise one or more phenotypic selectable markers, for example a
gene encoding proteins conferring antibiotic resistance or
supplying an autotrophic requirement, and an origin of replication
recognized by the host to ensure amplification within the host.
Suitable prokaryotic hosts for transformation include E. coli,
Bacillus subtilis, Salmonella typhimurium, and various species
within the genera Pseudomonas, Streptomyces, and Staphylococcus,
although others may also be employed as a matter of choice.
[0064] Recombinant AIR may also be expressed in yeast hosts,
preferably from the Saccharomyces species, such as S. cerevisiae.
Yeast of other genera, such as Pichia or Kluyveromyces may also be
employed. Yeast vectors will generally contain an origin of
replication from the 2 .mu. yeast plasmid or an autonomously
replicating sequence (ARS), promoter, DNA encoding the viral
protein, sequences for polyadenylation and transcription
termination and a selection gene. Preferably, yeast vectors will
include an origin of replication and selectable marker permitting
transformation of both yeast and E. coli, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae trp1 gene, which
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, and a promoter derived from a
highly expressed yeast gene to induce transcription of a structural
sequence downstream. The presence of the trp1 lesion in the yeast
host cell genome then provides an effective environment for
detecting transformation by growth in the absence of
tryptophan.
[0065] Suitable yeast transformation protocols are known to those
of skill in the art; an exemplary technique is described by Hinnen
et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for
Trp.sup.+ transformants in a selective medium consisting of 0.67%
yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml
adenine and 20 .mu.g/ml uracil. Host strains transformed by vectors
comprising the ADH2 promoter may be grown for expression in a rich
medium consisting of 1% yeast extract, 2% peptone, and 1% glucose
supplemented with 80 .mu.g/ml adenine and 80 .mu.g/ml uracil.
Derepression of the ADH2 promoter occurs upon exhaustion of medium
glucose. Crude yeast supernatants are harvested by filtration and
held at 4.degree. C. prior to further purification.
[0066] Various mammalian or insect cell culture systems can be
employed to express recombinant protein. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, BiolTechnology 6:47 (1988). Examples of
suitable mammalian host cell lines include the COS-7 lines of
monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and
other cell lines capable of expressing an appropriate vector
including, for example, CV-1/EBNA (ATCC CRL 10478), L cells, C127,
3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.
Mammalian expression vectors may comprise nontranscribed elements
such as an origin of replication, a suitable promoter and enhancer
linked to the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a polyadenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences.
[0067] Purification of Recombinant AIR
[0068] Purified AIR, homologs, or analogs are prepared by culturing
suitable host/vector systems to express the recombinant translation
products of the DNAs of the present invention, which are then
purified from culture media or cell extracts. For example,
supernatants from systems which secrete recombinant protein into
culture media can be first concentrated using a commercially
available protein concentration filter, for example, an Amicon or
Millipore Pellicon ultrafiltration unit.
[0069] Following the concentration step, the concentrate can be
applied to a suitable purification matrix. For example, a suitable
affinity matrix can comprise a counter structure protein or lectin
or antibody molecule bound to a suitable support. Alternatively, an
anion exchange resin can be employed, for example, a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Alternatively, a
cation exchange step can be employed. Suitable cation exchangers
include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are preferred. Gel
filtration chromatography also provides a means of purifying the
inventive proteins.
[0070] Affinity chromatography is a particularly preferred method
of purifying AIR and homologs thereof. For example, an AIR
expressed as a fusion protein comprising an immunoglobulin Fc
region can be purified using Protein A or Protein G affinity
chromatography. Moreover, an AIR protein comprising an
oligomerizing zipper domain may be purified on a resin comprising
an antibody specific to the oligomerizing zipper domain. Monoclonal
antibodies against the AIR protein may also be useful in affinity
chromatography purification, by utilizing methods that are
well-known in the art. A ligand may also be used to prepare an
affinity matrix for affinity purification of AIR.
[0071] Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify an AIR composition. Some or all
of the foregoing purification steps, in various combinations, can
also be employed to provide a homogeneous recombinant protein.
[0072] Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of recombinant viral protein
can be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents.
[0073] Fermentation of yeast which express the inventive protein as
a secreted protein greatly simplifies purification. Secreted
recombinant protein resulting from a large-scale fermentation can
be purified by methods analogous to those disclosed by Urdal et al.
(J. Chromatog. 296:171, 1984). This reference describes two
sequential, reversed-phase HPLC steps for purification of
recombinant human GM-CSF on a preparative HPLC column.
[0074] Protein synthesized in recombinant culture is characterized
by the presence of cell components, including proteins, in amounts
and of a character which depend upon the purification steps taken
to recover the inventive protein from the culture. These components
ordinarily will be of yeast, prokaryotic or non-human higher
eukaryotic origin and preferably are present in innocuous
contaminant quantities, on the order of less than about 1 percent
by weight. Further, recombinant cell culture enables the production
of the inventive proteins free of other proteins which may be
normally associated with the proteins as they are found in nature
in the species of origin.
[0075] Uses and Administration of AIR Compositions
[0076] The present invention provides methods of using therapeutic
compositions comprising an effective amount of a protein and a
suitable diluent and carrier, and methods for regulating an immune
response. The use of AIR or homologs in conjunction with soluble
cytokine receptors or cytokines, or other immunoregulatory
molecules is also contemplated.
[0077] For therapeutic use, purified protein is administered to a
patient, preferably a human, for treatment in a manner appropriate
to the indication. Thus, for example, AIR protein compositions
administered to regulate immune function can be given by bolus
injection, continuous infusion, sustained release from implants, or
other suitable technique. Typically, a therapeutic agent will be
administered in the form of a composition comprising purified AIR,
in conjunction with physiologically acceptable carriers, excipients
or diluents. Such carriers will be nontoxic to recipients at the
dosages and concentrations employed.
[0078] Ordinarily, the preparation of such protein compositions
entails combining the inventive protein with buffers, antioxidants
such as ascorbic acid, low molecular weight (less than about 10
residues) polypeptides, proteins, amino acids, carbohydrates
including glucose, sucrose or dextrins, chelating agents such as
EDTA, glutathione and other stabilizers and excipients. Neutral
buffered saline or saline mixed with conspecific serum albumin are
exemplary appropriate diluents. Preferably, product is formulated
as a lyophilizate using appropriate excipient solutions (e.g.,
sucrose) as diluents. Appropriate dosages can be determined in
trials. The amount and frequency of administration will depend, of
course, on such factors as the nature and severity of the
indication being treated, the desired response, the condition of
the patient, and so forth.
[0079] Soluble forms of AIR and other AIR antagonists such as
antagonistic monoclonal antibodies can be administered for the
purpose of inhibiting AIR-induced cell death. AIR is expressed in
activated T cells, and may play a in role in apoptotic death of
these cells Soluble forms of AIR are thus likely to be useful in
preventing or treating diseases in which T cells are
inappropriately killed, for example, AIDS.
[0080] The following examples are offered by way of illustration,
and not by way of limitation. Those skilled in the art will
recognize that variations of the invention embodied in the examples
can be made, especially in light of the teachings of the various
references cited herein, the disclosures of which are incorporated
by reference.
EXAMPLE 1
[0081] This example describes identification and isolation of a new
tumor necrosis factor receptor-like protein. A cDNA having some
degree of homology to Tumor Necrosis Factor receptor type I (TNFRI;
p60, Schall, et al., Cell, 61:361, 1990); Loetscher, et al., Cell,
61:351, 1990), was identified in the NCBI EST (Expressed Sequence
Tag) database (accession # H41522). Additional homologous ESTs were
then identified (accession numbers H46374, H46424, H46211, H46662),
and oligonucleotide primers were synthesized based on portions of
the sequences that were conserved in the overlapping ESTs, had
sequence of only about 50% GC, and contained no ambiguous
nucleotides. The primers were used to determine the distribution of
a potential corresponding RNA transcript in a variety of tissues
using RT-PCR (reverse transcriptase-polymerase chain reaction) on a
panel of first strand cDNAs substantially as described in U.S. Ser.
No. 08/496,632, filed Jun. 29, 1995. Fetal brain was identified as
a tissue in which mRNA corresponding to the identified sequence was
transcribed; a human fetal brain cDNA library (Clontech, Palo Alto,
Calif.)was screened using the PCR product described previously,
which was labeled with .sup.32P-dCTP by random priming or by
additional PCR reaction. This process allowed the isolation of a
number of cDNA clones which contained the sequences identified in
the ESTs.
[0082] Sequence analysis confirmed the homology of the isolated
cDNA to the TNF receptor family and showed conservation of the
cysteine-rich pseudo-repeats which characterize the extracellular
domain of members of this family. However, all the clones isolated
lacked a leader peptide and a transmembrane region. Accordingly,
additional clones were isolated form other libraries. Analysis of
clones derived from a human peripheral blood T-cell (PBT) library
(Park et al., Blood 74:56, 1989) allowed the isolation of a full
length cDNA transcript, and also indicated that the clones obtained
from fetal brain contained unspliced introns. After the full-length
clone was isolated, it became clear that the ESTs contained
nucleotide sequence derived from introns such as those present in
the clones obtained from fetal brain.
[0083] The novel transcript isolated from the PBT library, referred
to as apoptosis inducing receptor (AIR), encoded a 417 amino acids
(aa) type I membrane protein with a 27 aa signal sequence, a 170 aa
extracellular domain characterized by four cysteine-rich
pseudo-repeats, a 27 aa transmembrane region and a 193 aa
cytoplasmic region. The cytoplasmic region AIR contains a sequence
that has the characteristics of a death domain, a region, conserved
between TNFRI and Fas, which is necessary and sufficient for
transduction of an apoptotic signal when signaling occurs via TNFRI
or Fas (Tartaglia et al., Cell 74:845, 1993; Itoh and Nagata, J.
Biol. Chem. 268:10932, 1993). The death domain of AIR exhibits
significant homology (48% identity, 64% similarity) to the 80 aa
sequence which encodes the TNFRI death domain, and is also similar
to the death domain of Fas (19.5% identity, 47% similarity). The
nucleotide and amino acid sequence of AIR is shown in SEQ ID NO:1.
The predicted signal peptide spans amino acids 1 through 24 of SEQ
ID NO:1. Amino acids 198 through 224 are predicted to form a
transmembrane region. Within the cytoplasmic region (amino acids
225 through 417), the putative death domain spans amino acids 335
through 413.
EXAMPLE 2
[0084] This example describes the cellular and tissue distribution
of AIR mRNA. In order to determine the cellular and tissue
distribution of AIR mRNA, poly (A).sup.+ RNA derived from various
cell lines or tissues was examined by Northern blot analysis using
the AIR cDNA as a probe. Filters containing poly(A).sup.+ RNA (2
.mu.g per lane) from various tissues were purchased from Clontech
(Palo Alto, Calif.). Polyadenylated RNA from various cells or cell
lines were isolated, fractionated (2 .mu.g per lane) on a 1%
agarose formaldehyde gel, blotted onto Hybond nylon membrane
(Amersham). Filters were probed with an anti-sense RNA riboprobe
corresponding to the coding region of AIR cDNA. Hybridization was
performed at 63.degree. C. followed by three washings in
0.2%.times.SSC, 0.1% SDS at 68.degree. C. Blots were exposed for 8
to 48 hr at -70.degree. C.
[0085] Northern analysis showed strong expression of AIR in spleen,
thymus and peripheral blood lymphocytes, with a predominant
transcript .about.3.0 kb together with a smaller .about.1.5 kb
transcript. Low level expression was observed in colon and small
intestine. No transcripts were detected in ovary, testis, and
prostate. The K299 cell line (established from peripheral blood of
a patient diagnosed with high grade large cell anaplastic lymphoma)
and the T cell line termed clone 22 also express AIR. Expression of
AIR was also observed by RT-PCR in dendritic cells. Expression of
the receptor can be regulated: PMA plus ionomycin stimulation of
fresh PBTs leads to up-regulation of AIR message, whereas treatment
of K299 cells with PMA plus ionomycin down-regulates AIR
expression.
[0086] Chromosomal localization of AIR using a panel of radiation
hybrids maps the gene to the telomeric end of human chromosome 1p,
in close proximity to the region which encodes the genes for
TNFRII, CD30 and OX40.
EXAMPLE 3
[0087] This example describes construction of a construct to
express a soluble AIR/Flag.RTM. protein referred to as AIR/Flag.
AIR/Flag.RTM. contains a leader sequence, and the region of AIR
from amino acid 25 to amino acid 197 (SEQ ID NO:1), and the
octapeptide referred to as Flag.RTM. (SEQ ID NO:3). The construct
is prepared essentially as described for other soluble constructs,
by ligating a DNA fragment encoding amino acids 25 through 197 of
SEQ ID NO:1 (obtained by PCR or another suitable method) into an
appropriate expression vector which contains a suitable leader
sequence. The resultant DNA construct is transfected into a
suitable cell line such as the monkey kidney cell line CV-1/EBNA
(ATCC CRL 10478). AIR/Flag.RTM. may be purified using a Flag.RTM.
antibody affinity column, and analyzed for biological activity
using any of the methods described herein.
EXAMPLE 4
[0088] This example describes construction of an AIR DNA construct
to express an AIR/Fc fusion protein. A soluble form of AIR fused to
the Fc region of human IgG1 was constructed in the mammalian
expression vector pDC409 (U.S. Ser. No. 08/571,579). This
expression vector encompasses the leader sequence of the
Cytomegalovirus open reading frame R27080, followed by amino acids
25-199 of AIR, followed by a mutated form of the constant domain of
human IgG.sub.1 (SEQ ID NO:4).
[0089] The AIR/Fc expression plasmid was transfected into CV-1/EBNA
cells, and supernatants were collected for one week. The AIR/Fc
fusion protein was purified on a protein A sepharose column
(Pharmacia, Uppsala, Sweden) substantially as described in Example
5. Protein concentration was determined by an enzyme-linked
immunoadsorbent assay specific for the constant domain of human
IgG1 and by BCA analysis (Pharmacia), and purity was confirmed by
SDS-polyacrylamide gel electrophoresis analysis followed by silver
stain of the gel. SDS-PAGE (in the presence of a reducing agent)
analysis of the purified AIR-Fc showed the protein migrated with a
molecular weight of .about.52 kDa Interestingly, disulfide linked
homodimers were found to only represent .about.30% of the material,
with higher order aggregates representing the remaining 70%.
EXAMPLE 5
[0090] This example describes purification of AIR fusion proteins.
AIR/Fc fusion protein is purified by conventional methods using
Protein A or Protein G chromatography. Approximately one liter of
culture supernatant containing AIR/Fc fusion protein is purified by
filtering mammalian cell supernatants (e.g., in a 0.45 m filter)
and applying filtrate to a protein A/G antibody affinity column
(Schleicher and Schuell, Keene, N.H.) at 4.degree. C. at a flow
rate of 80 ml/hr for a 1.5 cm.times.12.0 cm column. The column is
washed with 0.5 M NaCl in PBS until free protein is not detected in
the wash buffer. Finally, the column is washed with PBS. Bound
fusion protein is eluted from the column with 25 mM citrate buffer,
pH 2.8, and brought to pH 7 with 500 mM Hepes buffer, pH 9.1.
[0091] An AIR fusion protein comprising Flag.RTM. may also be
detected and/or purified using an antibody that binds Flag.RTM.,
substantially as described in Hopp et al., Bio/Technology 6:1204
(1988). Biological activity is measured by inhibition of
AIR-induced cell death, for example, as described in the Examples
herein.
EXAMPLE 6
[0092] This example illustrates the preparation of monoclonal
antibodies against AIR. Preparations of purified recombinant AIR,
for example, or transfected cells expressing high levels of AIR,
are employed to generate monoclonal antibodies against AIR using
conventional techniques, such as those disclosed in U.S. Pat. No.
4,411,993. DNA encoding AIR can also be used as an immunogen, for
example, as reviewed by Pardoll and Beckerleg in Immunity 3:165,
1995. Such antibodies are likely to be useful in interfering with
AIR-induced cell death (antagonistic antibodies) or in inducing
apoptosis by cross-linking AIR (agonistic antibodies), as
components of diagnostic or research assays for AIR or AIR
activity, or in affinity purification of AIR.
[0093] To immunize rodents, AIR immunogen is emulsified in an
adjuvant (such as complete or incomplete Freund's adjuvant, alum,
or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton,
Mont.), and injected in amounts ranging from 10-100 .mu.g
subcutaneously into a selected rodent, for example, BALB/c mice or
Lewis rats. DNA may be given intradermally (Raz et al., Proc. Natl.
Acad. Sci. USA 91:9519, 1994) or intamuscularly (Wang et al., Proc.
Natl. Acad. Sci. USA 90:4156, 1993); saline has been found to be a
suitable diluent for DNA-based antigens. Ten days to three weeks
days later, the immunized animals are boosted with additional
immunogen and periodically boosted thereafter on a weekly, biweekly
or every third week immunization schedule.
[0094] Serum samples are periodically taken by retro-orbital
bleeding or tail-tip excision for testing by dot-blot assay
(antibody sandwich), ELISA (enzyme-linked immunosorbent assay),
immunoprecipitation, or other suitable assays, including FACS
analysis. Following detection of an appropriate antibody titer,
positive animals are given an intravenous injection of antigen in
saline. Three to four days later, the animals are sacrificed,
splenocytes harvested, and fused to a murine myeloma cell line
(e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridoma cell
lines generated by this procedure are plated in multiple microtiter
plates in a selective medium (for example, one containing
hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit
proliferation of non-fused cells, myeloma-myeloma hybrids, and
splenocyte-splenocyte hybrids.
[0095] Hybridoma clones thus generated can be screened by ELISA for
reactivity with AIR, for example, by adaptations of the techniques
disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S.
Pat. No. 4,703,004. A preferred screening technique is the antibody
capture technique described by Beckman et al., J. Immunol. 144:4212
(1990). Positive clones are then injected into the peritoneal
cavities of syngeneic rodents to produce ascites containing high
concentrations (>1 mg/ml) of anti-AIR monoclonal antibody. The
resulting monoclonal antibody can be purified by ammonium sulfate
precipitation followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can also be used, as can
affinity chromatography based upon binding to AIR protein.
[0096] Monoclonal antibodies were generated using the AIR-Fc fusion
protein as the immunogen. These reagents are screened to confirm
reactivity against the AIR protein. Using the methods described
herein to monitor the activity of the mAbs, both agonistic (i.e.,
antibodies that bind AIR and transduce an apoptotic signal) and
antagonistic (i.e., antibodies that bind AIR and do not transduce
an apoptotic signal, and in fact can inhibit apoptosis) are
isolated.
EXAMPLE 7
[0097] This example illustrates the ability of AIR to cause
apoptosis of transfected cells. To determine the apoptotic activity
of AIR in the absence of a ligand, the full length transcript was
over-expressed in a transient transfection system. This approach
has been used successfully to demonstrate Fas and TNFRI mediated
apoptosis and relies on the fact that self aggregation of death
domain sequences induced by over-expression mimics the ligand
mediated juxta-position of the cytoplasmic regions required for
productive signaling.
[0098] CVI/EBNA cells (1.65.times.10.sup.5 cells per slide) were
transiently co-transfected a with total of 2 .mu.g of DNA per
slide, by the DEAE-dextran method. Each DNA mixture contained 0.5
.mu.g of test transcript (pDC409-AIR; pDC302-TNFRI, or pDC302-FAS;
pDC302 is described in Mosley et al., Cell 59:335, 1989), 0.25
.mu.g of pDC409-.beta.-glactosidase and 0.25 .mu.g of pSV3neo plus
1.0 .mu.g of pDC303-crmA (pDC303 is described in U.S. Pat. No.
5,350,683; crmA is a cowpox open reading frame which encodes an
inhibitor of Interleukin-1.beta.-converting enzyme that is capable
of blocking apoptosis through Fas or TNFRI; see Cell 69:597, 1992)
or 1.0 .mu.g of empty pDC302 vector DNA. crmA, pDC302 and pDC409
controls were included in the experiment. All samples were
supplemented with an appropriate amount of empty pDC302 vector DNA
to keep the total amount of DNA per transfection constant. After 48
hours cells were washed with PBS, lysed with 1.0 ml of PBS+1% NP-40
for 30 min and the .beta.-galactosidase activity determined using
o-nitrophenyl-b-D-galactopyranoside as a substrate. The amount of
.beta.-galactosidase in the lysates (X-Gal u/ml) was determined by
measuring the absorbance of the resulting colored product and
comparing it to a known .beta.-galactosidase standard. A decrease
in the absorbance obtained in this assay indicates loss of
.beta.-galactosidase expression and correlates with death of cells
that express the protein(s) co-transfected with
.beta.-galactosidase.
[0099] Over-expression of AIR in CVI/EBNA cells lead to cell death.
Morphological and confocal microscope analyses suggested that the
observed cell death was mediated by an apoptotic mechanism. Further
evidence that AIR mediated apoptosis was obtained by Hoechst
staining of the transfected cells. Like Fas-induced apoptosis, the
AIR-mediated cell death was partially blocked by co-expression of
crmA, the cowpox gene product which specifically inhibits the
Interleukin-1.beta.-converting enzyme. The cytoplasmic domain of
AIR carrying the 80 aa death domain sequence is essential for
transducing the apoptotic signal. This was demonstrated using a
chimera containing the extracellular domain of CD40 fused to the
trans membrane and cytoplasmic domains of AIR. Over-expression of
the CD40-AIR chimera led to the same apoptosis observed when full
length AIR was over-expressed.
EXAMPLE 7
[0100] This example demonstrates that a soluble AIR/Fc fusion
protein can enhance the allostimulatory capacity of
CD8.alpha..sup.+ dendritic cells (DC). CD8.alpha..sup.+ DC are a
recently identified subset of antigen presenting cells which have
been described as lymphoid-related DC (i.e. derived from a lymphoid
committed BM progenitor which can give rise to T cells B cells and
NK cells (Wu et al., J. Exp. Med. 184: 903, 1996). CD8.alpha..sup.+
DC express high levels of FasL and have been shown to kill
activated, Fas expressing CD4.sup.+ T cells in vitro (Suss and
Shortman, J. Exp. Med. 183: 1789, 1996). In vitro studies have
shown that CD8.alpha..sup.+ splenic DC are less efficient at
stimulating the proliferation of allo-reactive and
hemagglutinin-specific CD4.sup.+ T cells (Sss and
Shortman,supra).
[0101] Maraskovsky et al. (J. Exp. Med. 184:1953, 1996) found that
the predominantly CD8.alpha..sup.+ lymphoid-related DC were
2-3-fold less efficient at stimulating the proliferation of
allo-reactive or KLH-specific CD4.sup.+ T cells as compared to
other DC. Furthermore, the apparent deficit in T cell-stimulating
capacity of CD8.alpha..sup.+ DC was actually due to their ability
to directly induce the apoptotic death of the CD4.sup.+ T cells
during in vitro stimulation; this effect was obviated when using
either DC from gld (FasL-/-) mice or CD4.sup.+ T cells from lpr
(Fas-/-) mice, implicating a direct role for FasL expressed on the
CD8.alpha..sup.+ (Suss and Shortman, supra). This suggests that
FasL expressing CD8.alpha..sup.+ DC can negatively regulate T cell
activation via a programmed cell death pathway. However, the role
of Fas-mediated killing has been shown to be only partially
responsible for apoptosis of the target T cells, especially when
the targets are CD8.sup.+ T cells (Kronin et al, J. Immunol. 157:
3819, 1996).
[0102] In the case of CD8.sup.+ T cells, the CD8.alpha..sup.+ DC
were initially efficient at stimulating CD8.sup.+ T cells to
proliferate, but T cell proliferation was significantly reduced
later in culture (Kronin et al, supra). Studies performed with lpr
or gld mice indicated that Fas-induced apoptosis was not involved
suggesting that the mechanism was distinct from that observed for
CD4.sup.+ T cells. The reduced proliferation was rescued only when
high levels of IL-2 were added to the cultures, correlating with
the significantly reduced IL-2 production observed for the
CD8.sup.+ T cells (IL-3, GM-CSF and IFN-.gamma. production was also
reduced, but IL-2 appeared to be the most limiting cytokine for the
CD8.sup.+ T cells). This indicated that CD8.alpha..sup.+ DC were
actually not deficient in their ability to induce T cell
proliferation but did not induce adequate cytokine production in
the responding CD8.sup.+ T cells. Thus, CD8.sup.+ DC can kill
activated CD4.sup.+ T cells via a Fas-mediated pathway but also
regulate CD8.sup.+ T cell proliferation by controlling T cell
cytokine production via an unknown pathway.
[0103] The regulatory nature of CD8.alpha..sup.+ DC is further
demonstrated by Inaba, et al., J. Exp, Med. 186, 665 (1997), who
show that the CD8.alpha..sup.+ DC which are localized in the T-cell
areas of secondary lymphoid tissue, predominantly express MHC
molecules occupied by self-peptides. Furthermore, these
CD8.alpha..sup.+ DC induced the initial proliferation and then
death of self-peptide reactive T cell clones in vitro. These
findings indicate that CD8.alpha..sup.+ DC are functionally
specialized to regulate the course of T cell activation and may
play a critical role in the maintenance of peripheral tolerance to
self-reactive T cells as well as regulate the development of the T
cell cytokine repertoire.
[0104] Functionally mature CD8.alpha..sup.+ DC were isolated from
the spleens of mice treated for nine consecutive days with
CHO-derived human Flt3 ligand (FL) as previously reported
(Maraskovsky et al., supra, and Pulendran et al., J. Immunol.
159:2222, 1997). FL has been shown to dramatically increase the
numbers of DC in vivo (U.S. Ser. No. 08/539,142, filed Oct. 4,
1995). Briefly, spleen cells from FL-treated mice were incubated
with the monoclonal antibodies (mAb), anti-B220 (for B cells),
anti-Thy 1 (for T cells), anti-Gr-1 (for myeloid- cells), anti-NK
1.1 (for NK cells) and anti-Ter 119 (for erythroid cells) to label
mature lineage cells.
[0105] The mAb coated cells were then incubated with magnetic beads
coated with goat-anti-mouse and goat-anti-rat Ig (Dyna beads, Dynal
Oslo, Norway). The subsequent mAb and magnetic bead coated cells
(mature lineage cells) were removed from the cell suspension using
a magnet leaving behind an enriched population of DC. The depleted
spleen cells were incubated with anti-CD11c (to identify DC) and
anti- CD8.alpha..sup.+ to identify the CD8.alpha..sup.+
lymphoid-related DC. CD11c.sup.+ CD8.alpha..sup.+ DC were then
isolated at high purity (>95%) by fluorescence activated cell
sorting (FACS) using a FACStar Plus.TM. (Becton Dickinson, San
Jose, Calif.), and used for biological evaluation of AIR/Fc.
[0106] The mouse CD8.alpha..sup.+ DC expressed a counterstructure
that bound the human AIR/Fc protein (prepared substantially as
described in Example 4) at the cell surface, as assessed by flow
cytometric analysis. Accordingly, the effect of AIR/Fc on the
biological activity of the CD8.alpha..sup.+ DC was assessed. The
addition of AIR/Fc to CD8.alpha..sup.+ DC enhanced their
allo-stimulatory capacity in a mixed lymphocyte reaction (MLR).
Allogeneic T cells (1.times.10.sup.5) were incubated with varying
numbers of irradiated (2000 rad) DC cultured as indicated above in
96-well round bottomed culture plates in 0.2 ml culture medium for
four days. The cultures were pulsed with 0.5 mCi
[.sup.3H]-thymidine for eight hours and the cells harvested onto
glass fiber sheets for counting on a gas phase .beta. counter.
[0107] The potent apoptotic cell death induced by AIR suggests that
in vivo the AIR/AIR ligand system may modulate cell death via the
induction of apoptosis in a manner similar to that used by the
Fas/FasL system. Given the high expression of AIR on T cells, and
the of presence of an AIR counterstructure on the CD8.alpha..sup.+
DCs, it is possible that an interaction between AIR and the
DC-expressed countersturcture may also contribute to the poor
allostimulatory capacity of the CD8.alpha..sup.+ DC.
EXAMPLE 8
[0108] This example illustrates a cross-species hybridization
technique which was used to isolate a murine AIR homolog using a
probe designed from the sequence of human AIR. A human AIR probe
was produced by .sup.32P-labeling a 451 nucleotide fragment
(327-778), encoding the extracellular domain of the human AIR
protein, with random primers using the Primer It Kit (Stratagene,
San Diego, Calif.). The murine 7B9 T cell (Mosley et al., Cell
59:335; 1989) derived cDNA library was constructed in a .lambda.
ZAP phage vector and packaged in vitro using a commercially
available kit (Gigapak.RTM. Stratagene, San Diego, Calif.)
according to the manufacturer's instructions, substantially as
described in U.S. Pat. No. 5,599,905, issued Feb. 4, 1997).
[0109] The human AIR probe was hybridized to the cDNA library in a
buffer containing 10.times. Denhardt's solution, 5 mM Tris pH7.5,
1M NaCl, 0.1% sodium pyrophosphate, 1.times.SDS and 200 mg/ml
denatured salmon sperm DNA at 63.degree. C. overnight.
Hybridization was followed by washing 30 min in 6.times.SSC, 30 min
in 2.times.SSC, 60 min in 1.times.SSC at 63.degree. C. and 30 min
in 0.5.times.SSC at room temperature. Hybridizing clones were
visualized by autoradiography.
[0110] A clone was isolated carrying a sequence containing a
complete coding region with homology to human AIR. This clone,
however, also contained what appeared to be two intronic sequences.
A fragment encoding the second and third cysteine-rich
pseudo-repeats in the extracellular domain of the murine AIR cDNA
clone was used to generate a single-stranded PCR probe which was
used to re-screen the 7B9 cDNA library. The murine probe was
hybridized to phage cDNA in Starks' buffer containing 50% formamide
(Wahl et al, 1979, PNAS 76: 3683-3687) at 37.degree. C. overnight.
Hybridization was followed by 2.times.30 min washes in 2.times.SSC
and 2.times.30 min washes in 0.5.times.SSC at 42.degree. C.
Hybridizing clones were visualized by autoradiography.
[0111] A clone was isolated containing a coding region identical to
that of the previously isolated clone, but without intronic
sequences. This clone, however, lacked the first 78 amino acids of
murine AIR from the start methionine. A full length coding sequence
cDNA construct was derived by PCR by joining the sequences from the
two original cDNA clones without the introns. The nucleotide
sequence and predicted amino acid sequence of this clone are
illustrated SEQ ID NO:5 and 6. Similarly to human AIR, murine AIR
is a Type I transmembrane protein having 411 amino acid residues,
with a predicted 30 amino acid signal sequence, a 159 amino
acid-extracellular domain, a 21 amino acid transmembrane domain,
and a 195 amino acid cytoplasmic tail. The cytoplasmic region of
murine AIR displays significant amino acid homology to cytoplasmic
region of human AIR and encodes a death domain motif.
Sequence CWU 1
1
* * * * *