U.S. patent application number 11/299182 was filed with the patent office on 2006-04-13 for family of immunoregulators designated leukocyte immunoglobulin-like receptors (lir).
This patent application is currently assigned to Immunex Corporation. Invention is credited to David J. Cosman.
Application Number | 20060078564 11/299182 |
Document ID | / |
Family ID | 36145617 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060078564 |
Kind Code |
A1 |
Cosman; David J. |
April 13, 2006 |
Family of immunoregulators designated leukocyte immunoglobulin-like
receptors (LIR)
Abstract
A new family of immunoreceptor molecules of the immunoglobulin
superfamily, (LIR) polypeptides is described. Disclosed are
sequences encoding LIR family members and their deduced amino acid
sequences, polypeptides encoded by DNA that hybridize to
oligonucleotide probes having defined sequences, processes for
producing polypeptides of the LIR family, and antagonistic
antibodies to LIR family members. LIR family members can be used to
treat autoimmune diseases and disease states associated with
suppressed immune function.
Inventors: |
Cosman; David J.;
(Bainbridge Island, WA) |
Correspondence
Address: |
AMGEN INC.
MAIL STOP 28-2-C
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Immunex Corporation
Seattle
WA
|
Family ID: |
36145617 |
Appl. No.: |
11/299182 |
Filed: |
December 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10143618 |
May 8, 2002 |
|
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11299182 |
Dec 9, 2005 |
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Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 14/70503 20130101 |
Class at
Publication: |
424/185.1 ;
530/350; 536/023.5; 435/069.1; 435/320.1; 435/325 |
International
Class: |
C07K 14/715 20060101
C07K014/715; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 39/00 20060101 A61K039/00 |
Claims
1. An isolated DNA encoding an LIR polypeptide, wherein said LIR
polypeptide comprises amino acid sequences selected from the group
consisting of: a) amino acids 5 to 50 of SEQ ID NO:2; and b) an
amino acid sequence that is at least 77% identical to the sequence
of a).
2. DNA encoding an LIR polypeptide, wherein said LIR polypeptide
comprises the amino acid sequence: Leu Xaa.sub.a Leu Ser Xaa.sub.b
Xaa.sub.c Pro Arg Thr Xaa.sub.d Xaa.sub.e Gln Xaa.sub.f Gly
Xaa.sub.g Xaa.sub.h Pro Xaa.sub.i Pro Thr Leu Trp Ala Glu Pro
Xaa.sub.j Ser Phe Ile Xaa.sub.j Xaa.sub.70 Ser Asp Pro Lys Leu
Xaa.sub.k Leu Val Xaa.sub.m Thr Gly where Xaa.sub.a is Gly or Arg;
Xaa.sub.b is Leu or Val; Xaa.sub.c is Gly or Asp; Xaa.sub.d is His
Arg or Cys; Xaa.sub.e is Val or Met; Xaa.sub.f is Ala or Thr;
Xaa.sub.g is His Pro or Thr; Xaa.sub.h Leu Ile or Phe; Xaa.sub.i is
Gly Asp or Ala; Xaa.sub.j is Thr Ile Ser or Ala; Xaa.sub.k is Gly
or Val; Xaa.sub.m is Met or Ala; and Xaa.sub.70 is a sequence of 70
amino acids.
3. DNA encoding an LIR polypeptide, wherein said LIR polypeptide is
encoded by DNA selected from the group consisting of: a) DNA
capable of hybridizing under highly stringent conditions to a probe
consisting essentially of nucleotides 310 to 1684 of SEQ ID NO: 1;
and b) DNA capable of hybridizing under highly stringent conditions
to DNA complementary to the probe of a), wherein the highly
stringent conditions comprise a hybridizing temperature of at least
63.degree. C.
4. An isolated DNA encoding an LIR polypeptide, wherein said LIR
polypeptide comprises an amino acid sequence that is at least 90%
identical to an amino acid sequence selected from the group
consisting of: a) SEQ ID NO:2; b) SEQ ID NO:4; c) SEQ ID NO:8; d)
SEQ ID NO:10; e) SEQ ID NO:12; f) SEQ ID NO:14; g) SEQ ID NO:16; h.
SEQ ID NO:18; i) SEQ ID NO:20; and j ) SEQ ID NO:22.
5. An isolated DNA of claim 4, wherein said LIR polypeptide
comprises an amino acid sequence selected from the group consisting
of: a) SEQ ID NO:2; b) SEQ ID NO:4; c) SEQ ID NO:8; d) SEQ ID NO:
10; e) SEQ ID NO:12; f) SEQ ID NO:14; g) SEQ ID NO:16; h. SEQ ID
NO: 18; i) SEQ ID NO:20; and j ) SEQ ID NO:22.
6. An isolated DNA encoding a soluble LIR polypeptide, wherein said
soluble LIR polypeptide comprises an amino acid sequence that is at
least 90% identical to a sequence selected from the group
consisting of: a) the extracellular domain of LIR family members
comprising sequences selected from the group consisting of: amino
acids x.sub.1 to 458 of SEQ ID NO:2, wherein x.sub.1 is amino acid
1 or 17; amino acids x.sub.2 to 459 of SEQ ID NO:4, wherein x.sub.2
is amino acid 1 or 17; amino acids x.sub.3 to 439 of SEQ ID NO:8,
wherein x.sub.3 is amino acids 1 or 17; amino acids x.sub.4 to 458
of SEQ ID NO:10, wherein x.sub.4 is amino acid 1 or 17; amino acids
x.sub.5 to 241 of SEQ ID NO:12, wherein x.sub.5 is amino acid 1 or
17; amino acids x.sub.6 to 461 of SEQ ID NO:14, wherein x.sub.6 is
amino acid 1 or 17; amino acids x.sub.7 to 449 of SEQ ID NO:16,
wherein x.sub.7 is amino acid 1 or 17; amino acids x.sub.8 to 259
of SEQ ID NO:18, wherein x.sub.8 is amino acid 1 or 17; amino acids
x.sub.9 to 443 of SEQ ID NO:20, wherein x.sub.9 is amino acid 1 or
17; and, amino acids x.sub.10 to 456 of SEQ ID NO:22, wherein
x.sub.10 is amino acid 1 or 17; b) a fragment of any of the LIR
extracellular domains of a), wherein said soluble LIR polypeptide
binds an MHC molecule.
7. A DNA of claim 6, wherein said soluble LIR polypeptide comprises
an amino acid sequence selected from the group consisting of: a)
the extracellular domain of LIR family members comprising sequences
selected from the group consisting of: amino acids x.sub.1 to 458
of SEQ ID NO:2, wherein x.sub.1 is amino acid 1 or 17; amino acids
x.sub.2 to 459 of SEQ ID NO:4, wherein x.sub.2 is amino acid 1 or
17; amino acids x.sub.3 to 439 of SEQ ID NO:8, wherein x.sub.3 is
amino acids 1 or 17; amino acids x.sub.4 to 458 of SEQ ID NO:10,
wherein x.sub.4 is amino acid 1 or 17; amino acids x.sub.5 to 241
of SEQ ID NO:12, wherein x.sub.5 is amino acid 1 or 17; amino acids
x.sub.6 to 461 of SEQ ID NO:14, wherein x.sub.6 is amino acid 1 or
17; amino acids x.sub.7 to 449 of SEQ ID NO:16, wherein x.sub.7 is
amino acid 1 or 17; amino acids x.sub.8 to 259 of SEQ ID NO:18,
wherein x.sub.8 is amino acid 1 or 17; amino acids x.sub.9 to 443
of SEQ ID NO:20, wherein x.sub.9 is amino acid 1 or 17; and, amino
acids x.sub.10 to 456 of SEQ ID NO:22, wherein x10 is amino acid 1
or 17; b) a fragment of any of the LIR extracellular domains of
a).
8. An LIR polypeptide comprising an amino acid sequence selected
from the group consisting of: a) amino acids 5 to 50 of SEQ ID
NO:2; and b) an amino acid sequence that is at least 77% identical
to the sequence of a).
9. An LIR polypeptide comprising the amino acid sequence: Leu
Xaa.sub.a Leu Ser Xaa.sub.b Xaa.sub.c Pro Arg Thr Xaa.sub.d
Xaa.sub.e Gln Xaa.sub.f Gly Xaa.sub.g Xaa.sub.h Pro Xaa.sub.i Pro
Thr Leu Trp Ala Glu Pro Xaa.sub.j Ser Phe Ile Xaa.sub.j Xaa.sub.70
Ser Asp Pro Lys Leu Xaa.sub.k Leu Val Xaa.sub.m Thr Gly where
Xaa.sub.a is Gly or Arg; Xaa.sub.b is Leu or Val; Xaa.sub.c is Gly
or Asp; Xaa.sub.d is His Arg or Cys; Xaa.sub.e is Val or Met;
Xaa.sub.f is Ala or Thr; Xaa.sub.g is His Pro or Thr; Xaa.sub.h Leu
Ile or Phe; Xaa.sub.i is Gly Asp or Ala; Xaa.sub.j is Thr Ile Ser
or Ala; Xaa.sub.k is Gly or Val; Xaa.sub.m is Met or Ala; and
Xaa.sub.70 is a sequence of 70 amino acids.
10. An isolated polypeptide encoded by DNA selected from the group
consisting of: a) DNA capable of hybridizing under highly stringent
conditions to a probe consisting essentially of nucleotides 310 to
1684 of SEQ ID NO: 1; and b) DNA capable of hybridizing under
highly stringent conditions to DNA complementary to the probe of
a), wherein the highly stringent conditions comprise a hybridizing
temperature of at least 63.degree. C.
11. A soluble LIR polypeptide comprising an amino acid sequence
that is at least 90% identical to a sequence selected from the
group consisting of: a) the extracellular domain of LIR family
members, the extracellular domains selected from the group
consisting of amino acids x.sub.1 to 458 of SEQ ID NO:2, wherein
x.sub.1 is amino acid 1 or 17; amino acids x.sub.2 to 458 of SEQ ID
NO:4, wherein x.sub.2 is amino acid 1 or 17; amino acids x.sub.3 to
439 of SEQ ID NO:8, wherein x.sub.3 is amino acid 1 or 17; amino
acids x.sub.4 to 458 of SEQ ID NO:10, wherein x.sub.4 is amino acid
1 or 17; amino acids x.sub.5 to 242 of SEQ ID NO:12, wherein
x.sub.5 is amino acid 1 or 17; amino acids x.sub.6 to 461 of SEQ ID
NO:14, wherein x.sub.6 is amino acid 1 or 17; amino acids x.sub.7
to 449 of SEQ ID NO:16, wherein x.sub.7 is amino acid 1 or 17;
amino acids x.sub.8 to 259 of SEQ ID NO:18, wherein x.sub.8 is
amino acid 1 or 17; amino acids x.sub.9 to 443 of SEQ ID NO:20,
wherein x.sub.9 is amino acid 1 or 17; and, amino acids x.sub.10 to
456 of SEQ ID NO:22, wherein x.sub.10 is amino acid 1 or 17; b) a
fragment of any of the LIR extracellular domains of a), wherein the
fragment is capable of binding a ligand.
12. A soluble polypeptide of claim 11 comprising an amino acid
sequence selected from the group consisting of: a) the
extracellular domain of LIR family members, the extracellular
domains selected from the group consisting of amino acids x.sub.1
to 458 of SEQ ID NO:2, wherein x.sub.1 is amino acid 1 or 17; amino
acids x.sub.2 to 458 of SEQ ID NO:4, wherein x.sub.2 is amino acid
1 or 17; amino acids x.sub.3 to 439 of SEQ ID NO:8, wherein x.sub.3
is amino acid 1 or 17; amino acids x.sub.4 to 458 of SEQ ID NO:10,
wherein x.sub.4 is amino acid 1 or 17; amino acids x.sub.5 to 242
of SEQ ID NO:12, wherein x.sub.5 is amino acid 1 or 17; amino acids
x.sub.6 to 461 of SEQ ID NO:14, wherein x.sub.6 is amino acid 1 or
17; amino acids x.sub.7 to 449 of SEQ ID NO:16, wherein x.sub.7 is
amino acid 1 or 17; amino acids x.sub.8 to 259 of SEQ ID NO:18,
wherein x.sub.8 is amino acid 1 or 17; amino acids x.sub.9 to 443
of SEQ ID NO:20, wherein x.sub.9 is amino acid 1 or 17; and, amino
acids x.sub.10 to 456 of SEQ ID NO:22, wherein x10 is amino acid 1
or 17; b) a fragment of any of the human P3G2 extracellular domains
of a).
13. An isolated polypeptide encoded by a DNA selected from the
group consisting of: a) DNA capable of hybridizing under highly
stringent conditions to DNA selected from the group consisting of
SEQ ID NO:5 and SEQ ID NO:6, the highly stringent conditions
including a hybridizing temperature of at least 68.degree. C.
14. A fusion protein comprising amino acids 17 to 458 of SEQ ID
NO:2 and the Fc region of Ig.
15. A fusion DNA construct comprising DNA encoding amino acids 17
to 458 of SEQ ID NO:2 and DNA encoding the Fc region of Ig.
16. A recombinant expression vector comprising DNA of claim 1.
17. A process for preparing an LIR polypeptide, the process
comprising culturing a host cell transformed with an expression
vector of claim 16 under conditions that promote expression of said
polypeptide, and recovering said polypeptide.
18. A composition comprising a suitable diluent carrier and a
polypeptide of claim 8.
19. A host cell transformed or transfected with an expression
vector according to claim 16.
20. An antibody that is immunoreactive with a polypeptide of claim
8.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/143,618 filed on May 8, 2002, which is incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0002] Immune system cellular activity is controlled by a complex
network of cell surface interactions and associated signaling
processes. When a cell surface receptor is activated by its ligand
a signal is sent to the cell, and, depending upon the signal
transduction pathway that is engaged, the signal can be inhibitory
or activatory. For many receptor systems cellular activity is
regulated by a balance between activatory signals and inhibitory
signals. In some of these it is known that positive signals
associated with the engagement of a cell surface receptor by its
ligand are downmodulated or inhibited by negative signals sent by
the engagement of a different cell surface receptor by its
ligand.
[0003] The biochemical mechanisms of these positive and negative
signaling pathways have been studied for a number of known immune
system receptor and ligand interactions. Many receptors that
mediate positive signaling have cytoplasmic tails containing sites
of tyrosine phosphatase phosphorylation known as immunoreceptor
tyrosine-based activation motifs (ITAM). A common mechanistic
pathway for positive signaling involves the activation of tyrosine
kinases which phosphorylate sites on the cytoplasmic domains of the
receptors and on other signaling molecules. Once the receptors are
phosphorylated, binding sites for signal transduction molecules are
created which initiate the signaling pathways and activate the
cell. The inhibitory pathways involve receptors having
immunoreceptor tyrosine based inhibitory motifs (ITIM) which, like
the ITAMs, are phosphorylated by tyrosine kinases. Receptors having
these motifs are involved in inhibitory signaling because these
motifs provide binding sites for tyrosine phosphatases which block
signaling by removing tyrosine from activated receptors or signal
transduction molecules. While many of the details of the activation
and inhibitory mechanisms are unknown, it is clear that functional
balance in the immune system depends upon opposing activatory and
inhibitory signals.
[0004] One example of immune system activity that is regulated by a
balance of positive and negative signaling is B cell proliferation.
The B cell antigen receptor is a B cell surface immunoglobulin
which, when bound to antigen, mediates a positive signal leading to
B cell proliferation. However, B cells also express Fc.gamma. RIIb1
, a low affinity IgG receptor. When an antigen is part of an immune
complex with soluble immunoglobulin, the immune complex can bind B
cells by engaging both the B cell antigen receptor via the antigen
and Fc.gamma. RIIb1 via the soluble immunoglobulin. Co-engagement
of the Fc.gamma. RIIb1 with the B cell receptor complex
downmodulates the activation signal and prevents B cell
proliferation. Fc.gamma. RIIb1 receptors contain ITIM motifs which
are thought to deliver inhibitory signals to B cells via
interaction of the ITIMs with tyrosine phosphatases upon
co-engagement with B cell receptors.
[0005] The cytolytic activity of Natural Killer (NK) cells is
another example of immune system activity which is regulated by a
balance between positive signals that initiate cell function and
inhibitory signals which prevent the activity. The receptors that
activate NK cytotoxic activity are not fully understood. However,
if the target cells express cell-surface MHC class I antigens for
which the NK cell has a specific receptor, the target cell is
protected from NK killing. These specific receptors, known as
Killer Inhibitory Receptors (KIRs) send a negative signal when
engaged by their MHC ligand, downregulating NK cell cytotoxic
activity.
[0006] KIRs belong to the immunoglobulin superfamily or the C-type
lectin family (see Lanier et al., Immunology Today 17:86-91,1996).
Known human NK KIRs are members of the immunoglobulin superfamily
and display differences and similarities in their extracellular,
transmembrane and cytoplasmic regions. A cytoplasmic domain amino
acid sequence common to many of the KIRs is an ITIM motif having
the sequence YxxL/V. In some cases, it has been shown that
phosphorylated ITIMs recruit tyrosine phosphatases which
dephosphorylate molecules in the signal transduction pathway and
prevent cell activation (see Burshtyn et al., immunity 4:77-85,
1996). The KIRs commonly have two of these motifs spaced apart by
26 amino acids [YxxL/V(x).sub.26YxxL/V]. At least two NK cell
receptors, each specific for a human leukocyte antigen (HLA) C
allele (an MHC class I molecule), exist as an inhibitory and an
activatory receptor. These receptors are highly homologous in the
extracellular portions, but have major differences in their
transmembrane and cytoplasmic portions. One of the differences is
the appearance of the ITIM motif in the inhibitory receptor and the
lack of the ITIM motif in the activating receptor (see Biassoni et
al., Journal. Exp. Med, 183:645-650, 1996).
[0007] An immunoreceptor expressed by mouse mast cells, gp49B1,
also a member of the immunoglobulin superfamily, is known to
downregulate cell activation signals and contains a pair of ITIM
motifs. gp49B1 shares a high degree of homology with human KIRs
(Katz et al., Cell Biology, 93: 10809-10814, 1996). Mouse NK cells
also express a family of immunoreceptors, the Ly49 family, which
contain the ITIM motif and function in a manner similar to human
KIRs. However, the Ly49 immunoreceptors have no structural homology
with human KIRs and contain an extracellular C-type lectin domain,
making them a member of the lectin superfamily of molecules (see
Lanier et al., Immunology Today 17:86-91, 1996).
[0008] Clearly, the immune system activatory and inhibitory signals
mediated by opposing kinases and phosphatases are very important
for maintaining balance in the immune system. Systems with a
predominance of activatory signals will lead to autoimmunity and
inflammation. Immune systems with a predominance of inhibitory
signals are less able to challenge infected cells or cancer cells.
Isolating new activatory or inhibitory receptors is highly
desirable for studying the biological signal(s) transduced via the
receptor. Additionally, identifying such molecules provides a means
of regulating and treating diseased states associated with
autoimmunity, inflammation and infection.
[0009] For example engaging a newly discovered cell surface
receptor having ITIM motifs with an agonistic antibody or ligand
can be used to downregulate a cell function in disease states in
which the immune system is overactive and excessive inflammation or
immunopathology is present. On the other hand, using an
antagonistic antibody specific to the receptor or a soluble form of
the receptor can be used to block the interaction of the cell
surface receptor with the receptor's ligand to activate the
specific immune function in disease states associated with
suppressed immune function. Conversely, since receptors lacking the
ITIM motif send activatory signals once engaged as described above,
the effect of antibodies and soluble receptors is the opposite of
that just described.
SUMMARY OF THE INVENTION
[0010] The present invention provides a new family of
immunoreceptor molecules of the immunoglobulin superfamily,
designated herein as the Leukocyte Immunoglobulin-Like Receptor
(LIR) polypeptides. Within the scope of the present invention are
DNA sequences encoding LIR family members and their deduced amino
acid sequences disclosed herein. Further included in the present
invention are polypeptides encoded by DNA that hybridize to
oligonucleotide probes having defined sequences or to DNA or RNA
complementary to the probes. The present invention also includes
recombinant expression vectors comprising DNA encoding LIR family
members. Also within the scope of the present invention are
nucleotide sequences which, due to the degeneracy of the genetic
code, encode polypeptides substantially identical or substantially
similar to polypeptides encoded by the nucleic acid sequences
described above, and sequences complementary to those nucleotide
sequences.
[0011] Further, the present invention includes processes for
producing polypeptides of the LIR family by culturing host cells
transformed with a recombinant expression vector that contains an
LIR family member encoding DNA sequence under conditions
appropriate for expressing an LIR polypeptide family member, then
recovering the expressed LIR polypeptide from the culture. The
invention also provides agonistic and antagonistic antibodies to
LIR and LIR family members.
[0012] Further still within the present invention are fusion
proteins which include a soluble portion of an LIR family member
and the Fc portion of Ig.
[0013] Disorders mediated by autoimmune disease associated with
failure of a negative signaling LIR to downregulate cell function
may be treated by administering a therapeutically effective amount
of an agonistic antibody or ligand of LIR family members to a
patient afflicted with such a disorder. Disorders mediated by
disease states associated with suppressed immune function can be
treated by administering a soluble form of the negative signaling
LIR. Conversely, disorders mediated by diseases associated with
failure of a activatory signaling LIR can be treated by
administering an agonistic antibody of the activatory receptor.
Disorders mediated by states associated with autoimmune function
can be treated by administering a soluble form of the activatory
receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0014] A viral glycoprotein having a sequence similarity to MHC
class I antigens has been used to isolate and identify a new
polypeptide, designated LIR-P3G2, and a new family of cell surface
polypeptides designated the LIR polypeptide family. The LIR
polypeptide family members possess extracellular regions having
immunoglobulin-like domains, placing the members in a new subfamily
of the immunoglobulin superfamily. While, the LIR family members
are characterized as having very similar extracellular portions,
the family includes three groups of polypeptides which are
distinguishable by their transmembrane regions and their
cytoplasmic regions. One group of the LIR polypeptides has a
transmembrane region that includes a positively charged residue and
a short cytoplasmic tail and a second group has a nonpolar
transmembrane region and a long cytoplasmic tail. A third group
includes a polypeptide expressed as a soluble protein having no
transmembrane region or cytoplasmic tail. LIR-P3G2 is expressed by
a variety of cells and recognizes HLA-B44 and HLA-A2 MHC molecules
and, by analogy with known molecules, LIR-P3G2 has a role in immune
recognition and self/nonself discrimination.
[0015] Examples 1-3 below describe isolating cDNA encoding P3G2
(LIR-P3G2) and a substantially identical polypeptide designated
18A3 (LIR-18A3). Briefly, the LIR-P3G2 family member was isolated
by first expressing UL18, a Class I MHC-like molecule and using
UL18 to isolate and identify P3G2 and 18A3. The nucleotide
sequences of the isolated P3G2 cDNA and 18A3 cDNA are presented in
SEQ ID NO:1 and SEQ ID NO:3, respectively. The amino acid sequences
encoded by the cDNA presented in SEQ ID NO:1 and SEQ ID NO:3 are
presented in SEQ ID NO:2 and SEQ ID NO:4, respectively. The P3G2
amino acid sequence (SEQ ID NO:2) has a predicted extracellular
domain of 458 amino acids (1-458) including a signal peptide of 16
amino acids (amino acids 1-16); a transmembrane domain of 25 amino
acids (amino acids 459-483) and, a cytoplasmic domain of 167 amino
acids (amino acids 484-650). The extracellular domain includes four
immunoglobulin-like domains. Ig-like domain I includes
approximately amino acids 17-118; Ig-like domain II includes
approximately amino acids 119-220; Ig-like domain III includes
approximately amino acids 221-318; and Ig-like domain IV includes
approximately amino acids 319-419. Significantly, the cytoplasmic
domain of this polypeptide includes four ITIM motifs, each having
the consensus sequence of YxxL/V. The first ITIM motif pair is
found at amino acids 533-536 and 562-565 and the second pair is
found at amino acids 614-617 and 644-647. This feature is identical
to the ITIM motifs found in KIRs except that KIRs contain only one
pair of ITIM motifs.
[0016] The 18A3 amino acid sequence has a predicted extracellular
region of 459 amino acids (1-459) including a signal peptide of 16
amino acids (amino acids 1-16); a transmembrane domain of 25 amino
acids (amino acids 460-484) and a cytoplasmic domain of 168 amino
acids (485-652). The 18A3 amino acids sequence (SEQ ID NO:4) is
substantially identical to that of P3G2 (SEQ ID NO:2) except that
18A3 has two additional amino acids (at amino acid 438 and 552) and
18A3 possesses an isoleucine residue at amino acid 142 in contrast
to a threonine residue for P3G2. Additionally, 18A3 has a serine
residue at amino acid 155 and P3G2 has an isoleucine at 155.
Finally, the 18A3 polypeptide has a glutamic acid at amino acid 627
and P3G2 has a lysine at 625 which is aligned with the 627 residue
of the 18A3 polypeptide. ITIM motifs in the 18A3 cytoplasmic domain
are at amino acids 534-537 and 564-567 and at 616-619 and 646-649.
Glycosylation sites occur at the amino acid triplet Asn-X-Y, where
X is any amino acid except Pro and Y is Ser or Thr. Thus, potential
glycosylation sites on LIR-P3G2 occur at amino acids 140-142;
281-283; 302-304; and 341-343. Sites on LIR-18A3 are at 281-283;
302-304; and 341-343. The features of these encoded polypeptides
are consistent with type I transmembrane glycoproteins.
[0017] Example 8-10 describe isolating and identifying eight
additional LIR polypeptide family members by probing cDNA libraries
for plasmids that hybridize to a probe obtained from DNA encoding
the extracellular region of LIR-P3G2. The nucleotide sequences
(cDNA) of the isolated LIR family members are presented in SEQ ID
NO:7 (designated pbm25), SEQ ID NO:9 (designated pbm8), SEQ ID
NO:11 (designated pbm36-2), SEQ ID NO:13 (designated pbm36-4); SEQ
ID NO:15 (designated pbmhh); SEQ ID NO: 17 (designated pbm2), SEQ
ID NO: 19 (designated pbm17) and SEQ ID NO:21 (designated pbmnew).
The amino acid sequences encoded thereby are presented in SEQ ID
NO:8 (designated pbm25), SEQ ID NO: 10 (designated pbm8), SEQ ID
NO: 12 (designated pbm36-2), SEQ ID NO:14 (designated pbm36-4), SEQ
ID NO:16 (designated pbmhh); SEQ ID NO: 18 (designated pbm2); SEQ
ID NO: 20 (designated pbm17), and SEQ ID NO:22 (designated pbmnew),
respectively.
[0018] The identified extracellular, transmembrane and cytoplasmic
regions for the LIR family members of SEQ ID NO: 10, 12, 14, 16,
18, 20, and 22 are presented below. The polypeptide presented in
SEQ ID NO:8 is a soluble protein having no transmembrane and
cytoplasmic regions. As will be understood by the skilled artisan,
the transmembrane region of P3G2 and 18A3 described above and those
of LIR polypeptide family members presented below are identified in
accordance with conventional criteria for identifying hydrophobic
domains associated with such regions. Accordingly, the precise
boundaries of any selected transmembrane region may vary from those
presented above. Typically, the transmembrane domain does not vary
by more than five amino acids on either end of the domain. Computer
programs known in the art and useful for identifying such
hydrophobic regions in proteins are available.
[0019] The polypeptide presented in SEQ ID NO:8 (LIR-pbm25) has a
an extracellular domain that includes the entire amino acid
sequence of amino acids 1-439 and a signal peptide of amino acids
1-16. The amino acid sequence presented in SEQ ID NO: 10 (LIR-pbm8)
has a predicted extracellular region of 458 amino acids (1-458)
including a 16 amino acid signal peptide (amino acids 1-16); a
transmembrane domain that includes amino acids 459-483; and a
cytoplasmic domain that includes amino acids 484-598. The
extracellular domain includes four immunoglobulin-like domains and
the cytoplasmic domain includes an ITIM motif at amino acids
533-536 and 562-565.
[0020] The amino acid sequence presented in SEQ ID NO: 12
(LIR-pbm36-2) has a predicted extracellular domain of amino acids
including a 16 amino acid signal peptide of from amino acids 1-16;
a transmembrane domain which includes amino acids 262-280 and a
cytoplasmic domain of from amino acids 281-289. The transmembrane
domain includes a charged arginine residue at 264 and the
cytoplasmic domain is short, having only a length of only 9 amino
acids.
[0021] The amino acid sequence presented in SEQ ID NO: 14
(LIR-pbm36-4) has a predicted extracellular domain of amino acids
1-461 including a signal peptide from amino acids 1-16; a
transmembrane domain that includes amino acids 462-480 and
possesses a charged arginine residue at amino acid 464; and a
cytoplasmic domain that includes amino acids 481-489. SEQ ID NO:14
is nearly identical to that of SEQ ID NO: 12 except that it
possesses four immunoglobulin domains in contrast to the two
domains found in the extracellular region SEQ ID NO:12. The amino
acid sequences presented in SEQ ID NO: 12 and SEQ ID NO: 14 are
likely proteins encoded by alternatively spliced transcripts from
the same gene.
[0022] The amino acid sequence presented in SEQ ID NO: 16
(LIR-pbmhh) has a predicted extracellular domain that includes
amino acids 1-449 and a signal peptide from amino acids 1-16; a
transmembrane domain that includes amino acids 450-468 with a
charged arginine residue at amino acid 452; and a cytoplasmic
domain that includes amino acids 469-483. The cytoplasmic domain is
short with a length of 15 amino acids. The extracellular domain
includes four immunoglobulin-like domains.
[0023] The amino acid sequence presented in SEQ ID NO: 18
(LIR-pbm2) has a predicted extracellular region that includes amino
acids 1-259 and a signal peptide of amino acids 1-16; a
transmembrane domain that includes amino acids 260-280; and a
cytoplasmic domain that includes amino acids 281-448. This LIR
family member has cytoplasmic domain which includes an ITIM motif
at amino acids 412-415 and 442-445. The extracellular domain
includes two immunoglobulin-like domains.
[0024] The amino acid sequence presented in SEQ ID NO:20 (LIR-pbml
7) has a predicted extracellular domain of amino acids 1-443 that
includes a signal peptide of amino acids 1-16; a transmembrane
domain which includes amino acids 444-464; and a cytoplasmic domain
of amino acids 465-631. The extracellular domain has four
immunoglobulin-like domains. SEQ ID NO:20 has two pairs of ITIM
YxxL/V motifs in the cytoplasmic domain. A first pair is at amino
acids 514-517 and 543-546, and a second pair is at amino acids
595-598 and 625-628.
[0025] The amino acid sequence presented in SEQ ID NO:22
(LIR-pbmnew) has a predicted extracellular domain of amino acids
1-456 including a signal peptide of amino acids 1-16; a
transmembrane domain which includes amino acids 457-579; and a
cytoplasmic domain of amino acids 580-590. The extracellular
includes four immunoglobulin-like domains. SEQ ID NO:22 has an ITIM
motif at amino acid 554-557 and 584-587.
[0026] The sequences presented in SEQ ID NO: 2, 4, 8, 10, 12, 14,
16, 18, 20, and 22 reveal that the LIR family includes three groups
of polypeptides. One group includes the polypeptides of SEQ ID
NO:12, 14 and 16 which are distinguishable by a charged arginine
residue in their transmembrane regions and their short cytoplasmic
regions. A second group includes SEQ ID NO: 2, 4, 10, 18, 20 and 22
which are distinguishable by the hydrophobic cytoplasmic domains
and the presence of the ITIM motif in their cytoplasmic regions.
The third group includes the polypeptide of SEQ ID NO: 8 which is
expressed as a soluble polypeptide and has no transmembrane region.
This soluble polypeptide may function to block the interactions of
cell surface family members with their receptors. Alternatively,
this soluble polypeptide may act as an activatory signal when it
binds to its receptor. The LIR polypeptides are characterized
generally by the ability of their encoding DNA to hybridize to DNA
encoding the P3G2 extracellular region.
[0027] The extracellular regions of the LIR family members
presented in SEQ ID NO:2, 4, 8, 10, 12, 14, 16, 18, 20, and 22 have
a high degree of homology which varies from 59%-84%. The
extracellular regions of SEQ ID NO: 12 and SEQ ID NO:14 share
sequence homology which is close to 100% since these polypeptides
are from the same gene. Similarly, SEQ ID NO:2 and SEQ ID NO:4
share sequence homology that is in excess of 95% and it is thought
that these may be alleles of the same gene. While sharing some
structural similarities with other members of the immunoglobulin
superfamily, the LIR family members have limited homology to these
members of the immunoglobulin superfamily. Molecules having the
closest structural similarity are the human KIRs and mouse gp49.
However, LIR extracellular regions share only a 38-42% identity
with the extracellular regions of NKAT3 and p58 C1-39,
respectively. The extracellular regions of the LIR family members
are only 35-47% homologous with that of mouse gp49. In contrast,
KIRs in general are known to share at least a 80% amino acid
identity, with NKAT3 and p58 CL-39 being 81% homologous.
Additionally, none of the known KIR molecules has four
extracellular immunoglobulin domains which is characteristic of all
but two of the known LIR family members. In view of the high
sequence homology among the LIR related polypeptides disclosed
herein and their relatively low homology with KIRs, the LIR
polypeptides are members of a new family of immunoregulators.
[0028] An analysis of the amino acid sequences of the LIR
polypeptides reveals that specific stretches of amino acids of the
LIR polypeptides are highly conserved. One conserved region is the
sequence of amino acids 5-50. A data base search determined that
the LIR family members differ substantially from the most
structurally similar prior art polypeptides in this LIR conserved
region. The data base search and structural analysis was performed
using BLAST NB1, a local alignment search tool for searching data
bases and aligning amino acid sequences to determine identities and
variations in a given sequence. The BLAST NB1 software is
accessible on the internet at
http://www3.ncbl.nlm.nih.gov/entrez/blast. The BLAST NB1 search for
sequences having homology to the sequence of amino acids 5 to 50 of
SEQ ID NO:2 found that the most structurally similar proteins are
Fc.gamma. IIR, gp49B form 2, and gp49B form 1 having identities
with amino acids 5 to 50 of SEQ ID NO:2 of 63%, 67%, and 67%
respectively. This contrasts with an LIR family identity with amino
acids 5 to 50 of SEQ ID NO:2 which ranges from 77% to 100%.
Specifically, LIR family members of the present invention have the
following identities with amino acids 5-50 of SEQ ID NO:2: SEQ ID
NO: 8 has a 96%; SEQ ID NO: 10 has a 90% identity; SEQ ID NO: 12
has a 96% identity; SEQ ID NO:14 has a 91% identity; SEQ ID NO:16
has a 97% identity; SEQ ID NO:18 has a 77% identity; SEQ ID NO:20
has an 80% identity; and, SEQ ID NO:22 has an 80% identity.
[0029] Sequence identity as used herein is the number of aligned
amino acids which are identical, divided by the total number of
amino acids in the shorter of the two sequences being compared. A
number of computer programs are available commercially for aligning
sequences and determining sequence identities and variations. These
programs provide identity information based upon the above stated
definition of identity. One suitable computer program is the GAP
program, version 6.0, described by Devereux et al. (Nucl. Acids
Res. 12:387, 1984) and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The GAP program utilizes the
alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443,
1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,
1981). The preferred default parameters for the GAP program
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides, and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps. Another similar program, also
available from the University of Wisconsin as part of the GCG
computer package for sequence manipulation is the BESTFIT
program.
[0030] In another aspect, the polypeptides of the present invention
have conserved regions which are uniquely characterized as having
the amino acid sequence (SEQ ID NO:28):
[0031] Leu Xaa.sub.a Leu Ser Xaa.sub.b Xaa.sub.c Pro Arg Thr
Xaa.sub.d Xaa.sub.e Gln Xaa.sub.f Gly Xaa.sub.g Xaa.sub.h Pro
Xaa.sub.i Pro Thr Leu Trp Ala Glu Pro Xaa.sub.j Ser Phe Ile
Xaa.sub.j Xaa.sub.70 Ser Asp Pro Lys Leu Xaa.sub.k Leu Val
Xaa.sub.m Thr Gly
[0032] where Xaa.sub.a is Gly or Arg; Xaa.sub.b is Leu or Val;
Xaa.sub.c is Gly or Asp; Xaa.sub.d is His Arg or Cys; Xaa.sub.e is
Val or Met; Xaa.sub.f is Ala or Thr; Xaa.sub.g is His Pro or Thr;
Xaa.sub.h Leu Ile or Phe; Xaa.sub.i is Gly Asp or Ala; Xaa.sub.j is
Thr Ile Ser or Ala; Xaa.sub.k is Gly or Val; Xaa.sub.m is Met or
Ala; and Xaa.sub.70 is a sequence of 70 amino acids.
[0033] As mentioned above, certain LIR family have ITIM motifs
(YxxL/V.sub.25-26YxxL/V) in their cytoplasmic domains. It is known
that many immune regulating receptors such as KIRs, CD22, Fc.gamma.
RIIb1 also have ITIMs in their cytoplasmic domain and function to
send inhibitory signals which down regulate or inhibit cell
function. It has been shown that these receptors associate with
SHP-1 phosphatase via binding to the ITIM motifs. Recruitment of
the SHP-1 phosphatase by the receptor appears to be required for
intracellular signaling pathways that regulate the inhibitory
function of the receptors. The experiment described in Example 11
demonstrates that LIR-P3G2 associates with SHP-1 phosphatase. It is
known that many immune regulating receptors such as KIRs, CD22,
Fc.gamma. RIIb1 have ITIMs in their cytoplasmic domain and function
to send inhibitory signals which down regulate or inhibit cell
function. Thus, by analogy with KIRs, CD22 and Fc.gamma. RIIb1 ,
LIR family members presented in SEQ ID NO:2, 4, 10, 18,20, and 22
that have ITIM motifs, deliver an inhibitory signal via the
interaction of its ITIM with SHP-1 tyrosine phosphatase, or other
tyrosine phosphatases, when the LIR is coligated with an
appropriate receptor. Also by analogy with immunoregulatory
receptors possessing ITIMs, LIR family members have a regulatory
influence on humoral, inflammatory and allergic responses.
[0034] The LIR family members presented in SEQ ID NO: 12, 14, and
16 have relatively short cytoplasmic domains, have transmembrane
regions possessing at least one charged residue, and do not possess
the ITIM motif. By analogy with membrane proteins that lack ITIM
motifs and have charged transmembrane regions, these family members
mediate stimulatory or activatory signals to cells. For example,
membrane bound proteins containing a charged residue in the
transmembrane regions are known to associate with other
membrane-bound proteins that possess cytoplasmic tails having
motifs known as immunoreceptor tyrosine-based activation motifs
(ITAM). Upon association, the ITAMs become phosphorylated and
propagate an activation signal.
[0035] The LIR polypeptide designated LIR-P3G2 is expressed on the
surface of transfected or normal cells. This is evidenced by the
results of the experiments described in Example 3 and Example 5 in
which flow cytometry and precipitation techniques demonstrate that
LIR-P3G2 is found on monocytes, a subpopulation of NK cells, and B
cells. P3G2 was not detected on T cells. P3G2 is expressed as a
110-120 kDa glycoprotein. Since P3G2 has four potential
glycosylation sites, the molecular size will vary with the degree
of its glycosylation. Glycosylation sites occur at the amino acid
triplet Asn-X-Y, where X is any amino acid except Pro and Y is Ser
or Thr. Potential glycosylation sites on P3G2 occur at amino acids
139-141; 280-282; 302-304; and 340-342.
[0036] P3G2-LIR isolated as described in Example 3 was tested for
its ability to bind to cell surface ligands distinct from UL18. As
demonstrated by the experimental results detailed in Example 7,
P3G2 binds HLA-B44 and HLA-A2, class I MHC antigens. Since Class I
MHC molecules play a central role in immune surveillance,
self/non-self discrimination, the immune response to infection
etc., the LIR-P3G2 polypeptide has a role in regulation of immune
responses. It is known that NK cytolytic activity for killing tumor
cells and cells infected with a virus is regulated by a delicate
modulation of activatory and inhibitory signals. It has been shown
that receptors specific for the same HLA class I molecules to which
P3G2 binds may be activatory or inhibitory in their triggering
mechanism. By analogy, P3G2 which binds MHC class I molecules,
plays a role in balancing immune system cell activity and is useful
in treating disease states in which the immune system balance is
disrupted.
[0037] Within the scope of the present invention are polypeptides
which include amino acid sequences encoded by DNA that hybridizes
to LIR-P3G2 extracellular DNA probes under moderate to highly
stringent conditions as taught herein. Probes which hybridize to
DNA that encode polypeptides of the present invention include
probes which encompass nucleotides 310-1684 of SEQ ID NO:1 or
fragments thereof. Fragments of SEQ ID NO:1 utilized as
hybridization probes are preferably greater than 17 nucleotides in
length and may include nucleotides 358-1684; nucleotides 322-459
(encoding LIR conserved sequence); or DNA or RNA sequences
complementary to SEQ ID NO:5, 6, 23, 24, 27 and I or fragments
thereof. Fragments of SEQ ID NO:5, 6, 23, 24 and 27 include these
sequences without the restrictions sites. Conditions for
hybridization may be moderately stringent conditions described in,
for example, Sambrook et al, Molecular Cloning. A Laboratory
Manual, 2nd ed., Vol. 1, pp 1.101-104, Cold Spring Harbor
Laboratory Press, 1989. Conditions of moderate stringency, as
defined by Sambrook et al., include use of a prewashing solution of
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization
conditions of about 55.degree. C., 5.times.SSC, overnight. Highly
stringent conditions include higher temperatures of hybridization
and washing. The skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as the length of the probe.
Preferred embodiments include amino acid sequences encoded by DNA
that hybridizes to probes of the extracellular region of LIR-P3G2
having at least 17 nucleotides. Preferred hybridizing conditions
include a temperature of 63.degree. C. for 16 hours in a
hybridizing solution of Denhart's Solution, 0.05 M TRIS at pH 7.5,
0.9 M NaCl, 0.1% sodium pyrophosphate, 1% SDS and 200 .mu.g/mL
salmon sperm DNA, followed by washing with 2.times.SSC at
63.degree. C. for one hour and the a wash with 1.times.SSC at
63.degree. C. for one hour.
[0038] The present invention includes polypeptides having amino
acid sequences that differ from, but are highly homologous to,
those presented in SEQ ID NO:2, 4, 8, 10, 12, 14, 16, 18, 20 and
22. Examples include, but are not limited to, homologs derived from
other mammalian species, variants (both naturally occurring
variants and those generated by recombinant DNA technology), and
LIR P3G2 and LIR family member fragments that retain a desired
biological activity. Preferably, such polypeptides exhibit a
biological activity associated with the LIR polypeptides described
in SEQ ID Nos:2, 4, 8, 10, 12, 14, 16, 18 20 and 22 and comprise an
amino acid sequence that is at least 80% identical to any of the
amino acid sequences of the signal peptide and extracellular
domains of the polypeptides presented in SEQ ID NOS:2, 4, 8, 10,
12, 14, 16, 18, 20 and 22. Preferably such polypeptides are at
least 90% identical to any of the amino acid sequences of the
signal peptide and extracellular domains of the polypeptides
presented in SEQ ID NOS: 2, 4, 8, 10, 12, 14, 16, 18, 20 and 22.
Determining the degree of identity between polypeptides can be
achieved using any algorithms or computer programs designed for
analyzing protein sequences. The commercially available GAP program
described below is one such program. Other programs include the
BESTFIT and GCG programs which are also commercially available.
[0039] Within the scope of the present invention are LIR
polypeptide fragments that retain a desired biological property of
an LIR polypeptide family member such as binding to MHC class I or
other ligand. In one such embodiment, LIR polypeptide fragments are
soluble LIR polypeptides comprising all or part of the
extracellular domain, but lacking the transmembrane region that
would cause retention of the polypeptide on a cell membrane.
Soluble LIR polypeptides are capable of being secreted from the
cells in which they are expressed. Advantageously, a heterologous
signal peptide is fused to the N-terminus such that the soluble LIR
is secreted upon expression. Soluble LIR polypeptides include
extracellular domains incorporating the signal peptide and those in
which the signal peptide is cleaved signal peptide.
[0040] The use of soluble forms of a LIR family member is
advantageous for certain applications. One such advantage is the
ease of purifying soluble forms from recombinant host cells. Since
the soluble proteins are secreted from the cells, the protein need
not be extracted from cells during the recovery process.
Additionally, soluble proteins are generally more suitable for
intravenous administration and can be used to block the interaction
of cell surface LIR family members with their ligands in order to
mediate a desirable immune function.
[0041] Soluble LIR polypeptides include the entire extracellular
domain or any desirable fragment thereof, including extracellular
domains that exclude signal peptides. Thus, for example, soluble
LIR polypeptides include amino acids x.sub.1-458 of SEQ ID NO:2,
where x.sub.1 is amino acids 1 or 17; amino acids x.sub.2-459 of
SEQ ID NO:4, where x.sub.2 is amino acid 1 or 17; amino acids
x.sub.3-439 of SEQ ID NO:8, where x.sub.3 is amino acid 1 or 17;
amino acids x.sub.4-458 of SEQ ID NO: 10, where x.sub.4 is amino
acid 1 or 17; amino acids x.sub.5-241 of SEQ ID NO:12, where amino
acid x.sub.5 is amino acid 1 or 17, amino acids x6-461 of SEQ ID
NO:14, where x.sub.6 is amino acid 1 or 17; amino acids x7-449 of
SEQ ID NO: 16, where x.sub.7 is amino acid 1 or 17; amino acids
x.sub.8-259 of SEQ ID NO:18, where x.sub.8 is amino acid 1 or 17;
amino acids x.sub.9-443 of SEQ ID NO:20, where x.sub.9 is amino
acid 1 or 17; and amino acids x.sub.10-456 of SEQ ID NO:22, where
x.sub.10 is amino acid 1 or 17. The above identified soluble LIR
polypeptides include LIR extracellular regions that include and
exclude signal peptides. Additional soluble LIR polypeptides
include fragments of the extracellular domains of family members
that retain a desired biological activity, such as binding to
ligands that include MHC class I molecules.
[0042] LIR family member fragments, including soluble polypeptides,
may be prepared by any of a number of conventional techniques. A
DNA sequence encoding a desired LIR polypeptide encoding fragment
may be subcloned into an expression vector for production of the
LIR polypeptide fragment. The selected encoding DNA sequence
advantageously is fused to a sequence encoding a suitable leader or
signal peptide. The desired LIR member encoding DNA fragment may be
chemically synthesized using known DNA synthesis techniques. DNA
fragments also may be produced by restriction endonuclease
digestion of a full length cloned DNA sequence, and isolated by
electrophoresis on an appropriate gel. If necessary,
oligonucleotides that reconstruct the 5' or 3' terminus to a
desired point may be ligated to a DNA fragment generated by
restriction enzyme digestion. Such oligonucleotides may
additionally contain a restriction endonuclease cleavage site
upstream of the desired coding sequence, and position an initiation
codon (ATG) at the N-terminus of the coding sequence.
[0043] Another technique useful for obtaining a DNA sequence
encoding a desired protein fragment is the well known polymerase
chain reaction (PCR) procedure. Oligonucleotides which define the
termini of the desired DNA are used as probes to synthesize
additional DNA from a desired DNA template. The oligonucleotides
may also contain recognition sites for restriction endonucleases,
to facilitate inserting the amplified DNA fragment into an
expression vector. PCR techniques are described in Saiki et al.,
Science 239:487(1988): Recombinant DNA Methodology, Wu et al.,
eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR
Protocols: A Guide to Methods and Applications, Innis et al., eds.,
Academic Press, Inc. (1990).
[0044] DNA of LIR family members of the present invention include
cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA,
and combinations thereof. Genomic LIR family DNA may be isolated by
hybridization to the LIR family cDNA disclosed herein using
standard techniques. RNA transcribed from LIR family DNA is also
encompassed by the present invention.
[0045] Within the scope of the present invention are DNA fragments
such as LIR polypeptide coding regions and DNA fragments that
encode soluble polypeptides. Examples of DNA fragments that encode
soluble polypeptides include DNA that encodes entire extracellular
regions of LIR family members and DNA that encodes extracellular
region fragments such as regions lacking the signal peptide. More
specifically, the present invention includes nucleotides 310-2262
of SEQ ID NO:I (P3G2 coding region); nucleotides x.sub.1-1683 of
SEQ ID NO:1, where x.sub.1 is 310 or 358 (encoding the P3G2
extracellular domain); nucleotides 168-2126 of SEQ ID NO:3 (the
18A3 coding region) and nucleotides x.sub.2-1544 of SEQ ID NO:3,
where x.sub.2 is 168 or 216 (the 18A3 extracellular domain coding
region); nucleotides x.sub.3-1412 of SEQ ID NO:7, where x.sub.3 is
93 or 141 (the pbm25 coding region and extracellular region);
nucleotides 184-1980 of SEQ ID NO:9, (the pbm8 coding region) and
nucleotides x.sub.4-1557 of SEQ ID NO:9, where x.sub.3 is 184 or
232 (the pmb8 extracellular domain coding region); nucleotides
171-1040 of SEQ ID NO:11(pbm36-2 coding region) and nucleotides
x.sub.5-878 of SEQ ID NO: 11, where x.sub.5 is 171 or 219 (encoding
the pbm36-2 extracellular domain); nucleotides 183-1652 of SEQ ID
NO: 13 (coding region for pbm36-4) and nucleotides x.sub.6-1565 of
SEQ ID NO: 13, where x.sub.6 is 183 or 231 (encoding the pbm36-4
extracellular domain); nucleotides 40-1491 of SEQ ID NO: 15 (the
pbmhh coding region) and nucleotides x.sub.7-1386 of SEQ ID NO: 15,
where x.sub.7 is 40 or 88 (encoding the pbmhh extracellular
domain); nucleotides 30-1376 of SEQ ID NO: 17 (the pbm2 coding
region) and nucleotides x.sub.8-806 of SEQ ID NO: 17, where x.sub.8
is 30 or 78 (encoding the pbm2 extracellular region); nucleotides
66-1961 of SEQ ID NO:19 (the pbm17 coding region) and nucleotides
x.sub.9-1394 of SEQ ID NO:19, where x.sub.9 is 66 or 114 (encoding
the pbm17 extracellular domain); and nucleotides 67-1839 of SEQ ID
NO:21 (the pbmnew coding region) and nucleotides x.sub.10-1434 of
SEQ ID NO:21, where x.sub.10 is 67 or 115 (encoding the pbmnew
extracellular domain).
[0046] Included in the present invention are DNAs encoding
biologically active fragments of the LIR family members presented
in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18, 20, and 22.
[0047] The present invention encompasses nucleotide sequences
which, due to the degeneracy of the genetic code, encode
polypeptides substantially identical or substantially similar to
polypeptides encoded by the nucleic acid sequences described above,
and sequences complementary to them. Accordingly, within the
present invention are DNA encoding biologically active LIR family
members which include the coding region of a native human LIR
family member cDNA, or fragments thereof, and DNA which is
degenerate as a result of the genetic code to the native LIR
polypeptide DNA sequence or the DNA of native LIR family members
described herein.
[0048] In another aspect, the present invention includes LIR
variants and derivatives as well as variants and derivatives of LIR
family polypeptides, both recombinanat and non-recombinant, that
retain a desired biological activity. An LIR variant, as referred
to herein, is a polypeptide substantially homologous to a native
LIR polypeptide, as described herein, except the variant amino acid
sequence differs from that of the native polypeptide because of one
or more deletions, insertions or substitutions.
[0049] LIR family variants may be obtained from mutations of native
LIR nucleotide sequences. Within the present invention are such DNA
mutations or variants which include nucleotide sequences having one
or more nucleotide additions, nucleotide deletions, or nucleotide
substitutions compared to native DNA of LIR family members and
which encode variant LIR polypeptides or variant LIR family members
having a desired biological activity. Preferably the biological
activity is substantially the same as that of the native LIR
polypeptide.
[0050] Variant amino acid sequences and variant nucleotide
sequences of the present invention preferably are at least 80%
identical to that of a native LIR family member sequence. One
method for determining the degree of homology or identity between a
native amino acid or nucleotide sequence and a variant amino acid
or nucleotide sequence is to compare the sequences using computer
programs available for such purposes. One suitable computer program
is the GAP program, version 6.0, described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl.
Math 2.482, 1981). Briefly, the GAP program defines identity as the
number of aligned symbols (i.e., nucleotides or amino acids) which
are identical, divided by the total number of symbols in the
shorter of the two sequences being compared. The preferred default
parameters for the GAP program include: (1) a unary comparison
matrix (containing a value of I for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix
of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
[0051] Alterations of native LIR amino acid sequences may be
provided by using any of a number of known techniques. As described
above, mutations can be introduced at selected sequence sites by
synthesizing oligonucleotides containing a mutant coding sequence,
flanked by restriction sites enabling its ligation to fragments of
the native sequence. After ligating the synthesized
oligonucleotides to the native sequence fragments, the resulting
reconstructed nucleotide sequence will encode an analog or variant
polypeptide having the desired amino acid insertion, substitution,
or deletion. Another procedure suitable for preparing variant
polypeptides is oligonucleotide-directed site-specific mutagenesis
procedures which provide genes having specific codons altered in
accordance with the desired substitution, deletion, or insertion.
Techniques for making such alterations include those disclosed in
the following references: Walder et al. Gene, 42:133, 1986; Bauer
et al., Gene 37:73, 1985; Craik, BioTechniques, 12-19 Jan., 1985;
Smith et al. Genetic Engineering: Principles and Methods, Plenum
Press, 1981; and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of
which are incorporated herein by reference.
[0052] Variant polypeptides of the present invention may have amino
acid sequences which are conservatively substituted, meaning that
one or more amino acid residues of a native LIR polypeptide family
member is replaced by different residues, such that the variant
polypeptide retains a desired biological activity that is
essentially equivalent to that of a native LIR family member. In
general, a number of approaches to conservative substitutions are
well known in the art and can be applied in preparing variant of
the present invention. For example, amino acids of the native
polypeptide sequence may be substituted for amino acids which do
not alter the secondary and/or tertiary structure of the LIR
polypeptide. Other suitable substitutions include those which
involve amino acids outside of the ligand-binding domain of
interest. One approach to conservative amino acid substitutions
involves replacing one or amino acids with those having similar
physiochemical characteristics, e.g. substituting one aliphatic
residue for another such as Ile, Val, Leu, or Ala for one another);
substituting one polar residue for another (such as between Lys and
Arg; Glu and Asp; or Gln and Asn); or substituting entire regions
having similar hydrophobicity or hydrophilic characteristics.
[0053] LIR polypeptide variants can be tested for binding to cells
as described in Examples 5 and 6 and for phosphatase binding
activity as described in Example 11 to confirm biological activity.
Other LIR variants within the present invention include
polypeptides which are altered by changing the nucleotide sequence
encoding the polypeptide so that selected polypeptide Cys residues
are deleted or replaced with one or more alternative amino acids.
These LIR variants will not form intramolecular disulfide bridges
upon renaturation. Naturally occurring LIR polypeptides selected
for alteration by deleting or altering Cys residues preferably do
not have biological activities which depend upon disulfide bridges
formed by the Cys residue. Other possible variants are prepared by
techniques which cause the modification of adjacent dibasic amino
acid residues to enhance expression in yeast systems in which KEX2
protease activity is present. EP 212,914 discloses site-specific
mutagenesis techniques for inactivating KEX2 protease processing
sites in a protein. KEX2 protease processing sites are inactivated
by deleting, adding or substituting residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these
adjacent basic residues. Lys-Lys and pairings are considerably less
susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg
to Lys-Lys represents a conservative and preferred approach to
inactivating KEX2 sites.
[0054] Naturally occurring LIR variants are also encompassed by the
present invention. Examples of such variants are proteins that
result from alternative mRNA splicing events or from proteolytic
cleavage of an LIR polypeptide. Alternative splicing of mRNA may
yield a truncated but biologically active LIR polypeptide such as a
naturally occurring soluble form of the protein. Variations
attributable to proteolysis include difference in the N- or
C-termini upon expression in different types of host cells, due to
proteolytic removal of one or more terminal amino acids from the
LIR polypeptide. In addition, proteolytic cleavage may release a
soluble form of LIR from a membrane-bound form of the polypeptide.
Other naturally occurring LIR variations are those in which
differences from the amino acid sequence of SEQ ID Nos:2, 4, 8, 10,
12, 14, 16, 18, 20 and 22 are attributable to genetic polymorphism,
the allelic variation among individuals.
[0055] Within the scope of the present invention are derivative LIR
family polypeptides which include native or variant LIR
polypeptides modified to form conjugates with selected chemical
moieties. The conjugates can be formed by covalently linking
another moiety to a native or variant LIR or by non-covalently
linking another moiety to a native or variant LIR. Suitable
chemical moieties include but are not limited to glycosyl groups,
lipids, phosphates, acetyl groups, and other proteins or fragments
thereof. Techniques for covalently linking chemical moieties to
proteins are well known in the art and are generally suitable for
preparing derivative LIR polypeptides. For example, active or
activated functional groups on amino acid side chains can be used
as reaction sites for covalently linking a chemical moiety to a LIR
polypeptide. Similarly, the N-terminus or C-terminus can provide a
reaction site for a chemical moiety. LIR polypeptides or fragments
conjugated with other proteins or protein fragments can be prepared
in recombinant culture as N-terminal or C-terminal fusion products.
For example, the conjugate or fusion portions may include a signal
or leader sequence attached to an LIR molecule at its N-terminus.
The signal or leader peptide co-translationally or
post-translationally directs transfer of the conjugate from its
site of synthesis to a site inside or outside of the cell
membrane.
[0056] One useful LIR polypeptide conjugate is one incorporating a
poly-His or the antigenic identification peptides described in U.S.
Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1124, 1988.
For example, the FLAG.RTM. peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(DYKDDDDK) is highly antigenic and provides an epitope reversibly
bound by a specific monoclonal antibody, thus enabling rapid assay
and facile purification of expressed recombinant protein. This
sequence is specifically cleaved by bovine mucosal enterokinase at
the residue immediately following the Asp-Lys pairing. Fusion
proteins capped with this peptide may be resistant to intracellular
degradation in E. coli. Murine hybridoma designated 4E11 produced a
monoclonal antibody that binds the peptide DYKDDDDK in the presence
of certain divalent metal cations, and has been deposited with the
American Type Culture Collection under accession no HB9259.
Expression systems useful for producing recombinant proteins fused
to the FLAG.RTM. peptide, and monoclonal antibodies that bind the
peptide and are useful in purifying the recombinant proteins, are
available from Eastman Kodak Company, Scientific Imaging Systems,
New Haven, Conn.
[0057] Particularly suitable LIR fusion proteins are those in which
an LIR polypeptide is in the form of an oligomer. Oligomers may be
formed by disulfide bonds between cysteine residues on more than
one LIR polypeptide, or by noncovalent interactions between LIR
polypeptide chains. In another approach, LIR oligomers can be
formed by joining LIR polypeptides or fragment thereof via covalent
or noncovalent interactions between peptide moieties fused to the
LIR polypeptide. Suitable peptide moieties include peptide linkers
or spacers, or peptides that have the property of promoting
oligomerization. Leucine zippers and certain polypeptides derived
from antibodies are among the peptides that can promote
oligomerization of LIR polypeptides attached thereto.
[0058] Other LIR fusion proteins which promote oligomer formation
are fusion proteins having heterologous polypeptides fused to
various portions of antibody-derived polypeptides (including the Fc
domain). Procedures for preparing such fusion proteins are
described in Ashkenazi et al. PNAS USA 88:10535, 1991; Byrne et al.
Nature 344:667, 1990, and Hollenbaugh and Aruffo Current Protocols
in Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992; all of
which are incorporated herein by reference. Example 1 and Example 5
below describe methods for preparing UL18:Fc and P3G2:Fc fusion
proteins, respectively, by fusing P3G2 and UL18 to an Fc region
polypeptide derived from an antibody. This is accomplished by
inserting into an expression vector a gene fusion encoding the
P3G2:Fc fusion protein and expressing the P3G2:Fc fusion protein.
The fusion proteins are allowed to assemble much like antibody
molecules, whereupon interchain disulfide bonds form between the Fc
polypeptides, yielding divalent P3G2 polypeptide. In a similar
approach, P3G2 or any LIR polypeptide may be substituted for the
variable portion of an antibody heavy or light chain. If fusion
proteins are made with heavy and light chains of an antibody, it is
possible to form a LIR oligomer with as many as four LIR
regions.
[0059] As used herein, a Fc polypeptide includes native and mutein
forms, as well as truncated Fc polypeptides containing the hinge
region that promotes dimerization. One suitable Fc polypeptide is
the native Fc region polypeptide derived from a human IgG1, which
is described in PCT application WO 93/10151, hereby incorporated
herein by reference. Another useful Fc polypeptide is the Fc mutein
described in U.S. Pat. 5,457,035. The amino acid sequence of the
mutein is identical to that of the native Fc sequence presented in
WO 93/10151, except that amino acid 19 has been changed from Leu to
Ala, amino acid 20 has been changed from Leu to Glu, and amino acid
22 has been changed from Gly to Ala. This mutein Fc exhibits
reduced affinity for immunoglobulin receptors.
[0060] Alternatively, oligomeric LIR polypeptide variants may
include two or more LIR peptides joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627, incorporated herein by reference. Fusion proteins which
include multiple LIR polypeptides separated by peptide linkers may
be produced conventional recombinant DNA technology.
[0061] Another method for preparing oligomeric LIR polypeptide
variants involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were first identified in several
DNA-binding proteins (Landschulz et al. Science 240:1759, 1988).
Among the known leucine zippers are naturally occurring peptides
and peptide derivatives that dimerize or trimerize. Examples of
leucine zipper domains suitable for producing soluble oligomeric
LIR polypeptides or oligomeric polypeptides of the LIR family are
those described in PCT application WO 94/10308, incorporated herein
by reference. Recombinant fusion proteins having a soluble LIR
polypeptide fused to a peptide that dimerizes or trimerizes in
solution may be expressed in suitable host cells, and the resulting
soluble oligomeric LIR polypeptide recovered from the culture
supernatant.
[0062] Numerous reagents useful for cross-linking one protein
molecule to another are known. Heterobifunctional and
homobifunctional linkers are available for this purpose from Pierce
Chemical Company, Rockford, Ill., for example. Such linkers contain
two functional groups (e.g., esters and/or maleimides) that will
react with certain functional groups on amino acid side chains,
thus linking one polypeptide to another.
[0063] One type of peptide linker that may be employed in the
present invention separates polypeptide domains by a distance
sufficient to ensure that each domain properly folds into the
secondary and tertiary structures necessary for the desired
biological activity. The linker also should allow the extracellular
portion to assume the proper spatial orientation to form the
binding sites for ligands.
[0064] Suitable peptide linkers are known in the art, and may be
employed according to conventional techniques. Among the suitable
peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and
4,935,233, which are hereby incorporated by reference. A peptide
linker may be attached to LIR polypeptides by any of the
conventional procedures used to attach one polypeptide to another.
The cross-linking reagents available from Pierce Chemical Company
as described above are among those that may be employed. Amino
acids having side chains reactive with such reagents may be
included in the peptide linker, e.g., at the termini thereof.
Preferably, a fusion proteins formed via a peptide linker are
prepared by recombinant DNA technology.
[0065] The fusion proteins of the present invention include
constructs in which the C-terminal portion of one protein is fused
to the linker which is fused to the N-terminal portion of another
protein. Peptides linked in such a manner produce a single protein
which retains the desired biological activities. The components of
the fusion protein are listed in their order of occurrence (i.e.,
the N-terminal polypeptide is listed first, followed by the linker
and then the C-terminal polypeptide).
[0066] A DNA sequence encoding a fusion protein is constructed
using recombinant DNA techniques to insert separate DNA fragments
encoding the desired proteins into an appropriate expression
vector. The 3' end of a DNA fragment encoding one protein is
ligated (via the linker) to the 5' end of the DNA fragment encoding
another protein with the reading frames of the sequences in phase
to permit translation of the mRNA into a single biologically active
fusion protein. A DNA sequence encoding an N-terminal signal
sequence may be retained on the DNA sequence encoding the
N-terminal polypeptide, while stop codons, which would prevent
read-through to the second (C-terminal) DNA sequence, are
eliminated. Conversely, a stop codon required to end translation is
retained on the second DNA sequence. DNA encoding a signal sequence
is preferably removed from the DNA sequence encoding the C-terminal
polypeptide.
[0067] A DNA sequence encoding a desired polypeptide linker may be
inserted between, and in the same reading frame as, the DNA
sequences encoding the two proteins using any suitable conventional
technique. For example, a chemically synthesized oligonucleotide
encoding the linker and containing appropriate restriction
endonuclease cleavage sites may be ligated between the sequences
encoding Fc and a P3G2 polypeptide.
[0068] Within the scope of the present invention are recombinant
expression vectors for expressing polypeptides of the LIR family,
and host cells transformed with the expression vectors. Expression
vectors of the invention include DNA encoding LIR family members
operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or
enhancers, an mRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are operably linked when the
regulatory sequence functionally relates to the LIR DNA sequence.
Thus, a promoter nucleotide sequence is operably linked to a LIR
DNA sequence if the promoter nucleotide sequence controls the
transcription of the LIR DNA sequence. An origin of replication
that confers the ability to replicate in the desired host cells,
and a selection gene by which transformants are identified, are
generally incorporated in the expression vector.
[0069] In addition, a sequence encoding an appropriate signal
peptide can be incorporated into expression vectors. A DNA sequence
for a signal peptide (secretory leader) may be fused in frame to
the LIR sequence so that the LIR is initially translated as a
fusion protein comprising the signal peptide. A signal peptide that
is functional in the intended host cells promotes extracellular
secretion of the LIR polypeptide. The signal peptide is cleaved
from the LIR polypeptide upon secretion of the LIR polypeptide from
the cell.
[0070] Suitable host cells for expression of LIR polypeptides
include prokaryotes, yeast or higher eukaryotic cells. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described, for example, in
Pouwels et al. Coning Vectors. A Laboratory Manual, Elsevier, N.Y.,
(1985). Cell-free translation systems could also be employed to
produce P3G2 polypeptides using RNAs derived from DNA constructs
disclosed herein.
[0071] Prokaryote host cells suitable in the practice of the
present invention include gram negative or gram positive organisms,
for example, E. coli or Bacilli. Suitable prokaryotic host cells
for transformation include, for example, E. coli, Bacillus
subtilis, Salmonella typhimurium, and various other species within
the general Pseudomonas, Streptomyces, and Staphylococcus. In a
prokaryotic host cell, such as E. coli, a P3G2 polypeptide may
include an N-terminal methionine residue to facilitate expression
of the recombinanat polypeptide. The N-terminal Met may be cleaved
from the expressed recombinant LIR polypeptide.
[0072] Expression vectors for use in prokaryotic host cells
generally include one or more phenotypic selectable marker genes. A
phenotypic selectable marker gene is, for example, a gene encoding
a protein that confers antibiotic resistance or that supplies an
autotrophic requirement. Examples of useful expression vectors for
prokarytoic host cells include those derived from commercially
available plasmids such as the cloning vector pBR322 (ATCC 37017).
pBR322 contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed cells.
An appropriate promoter and a LIR family DNA may be inserted into
the pBR322 vector. Other commercially available vectors include,
for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden)
and pGEM1 (Promega Biotec, Madison, Wis., USA).
[0073] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al. Nature
75.615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan
(trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057,
1980); and EP-A-36776) and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression
system employs a phase .lamda.P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lamda.P.sub.L promoter include plastid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1,
ATCC 53082).
[0074] Alternatively, LIR polypeptides may be expressed in yeast
host cells, preferably from the Saccharomyces genus (e.g., S.
cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces
may also be employed. Yeast vectors will often contain an origin of
replication sequence from a 2.mu. yeast plasmid, an autonomously
replicating sequence (ARS), a promoter region, sequences for
polyadenylation, sequences for transcription termination, and a
selectable marker gene. Suitable promoter sequences for yeast
vectors include, among others, 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, phospho-glucose isomerase, and
glucokinase. Other suitable vectors and promoters for use in yeast
expression are further described in Hitzeman, EPA-73,675. Another
alternative is the glucose-repressible ADH2 promoter described by
Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al.
(Nature 300:724, 1982). Shuttle vectors replicable in both yeast
and E. coli may be constructed by inserting DNA from pBR322 for
selection and replication in E. coli (Amp.sup.r gene and origin of
replication) into the above-described yeast vectors.
[0075] The yeast .alpha.-factor leader sequence may be employed to
direct secretion of the LIR polypeptide. The .alpha.-factor leader
sequence is often inserted between the promoter sequence and the
structural gene sequence. See, e.g., Kurjan et al., Cell
30:933,1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,
1984. Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence may be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0076] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 .mu.g/mL adenine and 20 .mu.g/mL
uracil.
[0077] Yeast host cells transformed by vectors containing an ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one having 1% yeast extract,
2% peptone, and 1% glucose supplemented with 80 .mu.g/mL uracil.
Derepression of the ADH2 promoter occurs when glucose is exhausted
from the medium.
[0078] Mammalian or insect host cell culture systems may be used to
express recombinant LIR polypeptides. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, Bio/Technology 6:47 (1988). Established cell
lines of mammalian origin also may be employed. Examples of
suitable mammalian host cell lines include the COS-7 line of monkey
kidney cells (ATCC CRL 1651)(Gluzman et al., Cell 23:175, 1981), L
cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary
(CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the
CVI/EBNA cell cline derived from the African green monkey cell line
CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10:2821,
1991). COS-1 (ATCC CRL-1650).
[0079] Transcriptional and translational control sequences for
mammalian host cell expression vectors may be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from Polyoma virus, Adenovirus 2, Simian Virus 40
(SV40), and human cytomegalovirus. DNA derived from the SV40 viral
genome, for example, SV40 origin, early and late promoter,
enhancer, splice, and polyadenylation sites may be used to provide
other genetic elements for expression of a structural gene sequence
in a mammalian host cell. Viral early and late promoters are
particularly useful because both are easily obtained from a viral
genome as a fragment which may also contain a 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 Bg/I site
located in the SV40 viral origin of replication site is
included.
[0080] Suitable expression vectors for use in mammalian host cells
can be constructed as disclosed by Okayama and Berg (Mol. Cell.
Biol. 3.280, 1983). One 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 high expression
vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768,
1984 has been deposited as ATCC 39890. Additional mammalian
expression vectors are described in EP-A-0367566, and in WO
91/18982. Still additional expression vectors for use in mammalian
host cells include pDC201 (Sims et al., Science 241:585, 1988),
pDC302 (Mosley et al. Cell 59:335, 1989), and pDC406 (McMahan et
al., EMBO J 10:2821, 1991). Vectors derived from retroviruses also
may be employed. One preferred expression system employs pDC409 as
discussed in Example 5 below.
[0081] For expression of LIR polypeptides the expression vector may
comprise DNA encoding a signal or leader peptide. In place of the
native signal sequence, a heterologous signal sequence may be
added, such as the signal sequence for interleukin-7 (IL-7)
described in U.S. Pat. No. 4,965,195; the signal sequence for
interleukin-2 receptor described in Cosman et al., Nature 312:768,
1984); the interleukin-4 signal peptide described in EP 367,566;
the type I interleukin-1 receptor signal peptide described in U.S.
Pat. No. 4,968,607; and the type II interleukin-1 receptor signal
peptide described in EP 460,846.
[0082] Further contemplated within the present invention are
purified LIR family polypeptides. The purified polypeptides of the
present invention may be purified from recombinant expression
systems as described above or purified from naturally occurring
cells. The desired degree of purity may depend on the intended use
of the protein with a relatively high degree of purity preferred
when the protein is intended for in vivo use. Preferably, LIR
polypeptide purification processes are such that no protein bands
corresponding to proteins other than the desired LIR protein are
detectable by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It
will be recognized by one skilled in the art that multiple bands
corresponding to any LIR polypeptide my be detected by SDS-PAGE,
due to differential glycosylation, variations in post-translational
processing, and the like, as discussed above. Most preferably, any
specific LIR polypeptide is purified to substantial homogeneity, as
indicated by a single protein band upon analysis by SDS-PAGE. The
protein band may be visualized by silver staining, Coomassie blue
staining, or by autoradiography or fluorescence if the protein is
appropriately labeled.
[0083] One process for providing purified LIR polypeptides includes
first culturing a host cell transformed with an expression vector
comprising a DNA sequence that encodes the desired polypeptide
under conditions that promote expressing the desired LIR
polypeptide and then recovering the LIR polypeptide. As the skilled
artisan will recognize, procedures for recovering the polypeptide
will vary according to such factors as the type of host cells
employed and whether the polypeptide is secreted in the culture
medium is extracted from cells.
[0084] When the expression system secretes the polypeptide into the
culture medium, the medium may be first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a suitable
purification matrix such as a gel filtration medium. Alternatively,
an anion exchange resin can be employed, such as a resin matrix or
resin substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Similarly, a
purification matrix having cation exchange groups such as
sulfopropyl or carboxymethyl functionalities on an insoluble matrix
can be used. Sulfopropyl groups are preferred. Still other
purification matrices and methods suitable for providing purified
LIR are high performance liquid chromatography using hydrophobic
reversed phase media (RP-HPLC). One skilled in the art will
recognized the any or all of the foregoing purification steps, in
various combinations, can be employed to provide a purified LIR
polypeptide.
[0085] Alternatively, LIR polypeptides can be purified by
immunoaffinity chromatography. An affinity column containing an
antibody that binds a LIR polypeptide may be prepared by
conventional procedures and employed in purifying LIR. Example 5
describes a procedures for generating monoclonal antibodies
directed against P3G2 which may be utilized in immunoaffinity
chromatography.
[0086] Recombinant protein produced in bacterial culture may be
isolated by first disrupting the host cells by any convenient
method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents and then extracting the
polypeptide from cell pellets if the polypeptide is insoluble, or
from the supernatant fluid if the polypeptide is soluble. After the
initial isolation step, the purification process may include one or
more concentrating, salting out, ion exchange, affinity, or size
exclusion chromatography purification steps. For many application a
final RP-HPLC purification step is beneficial.
[0087] Additional methods for providing LIR polypeptides and
purified LIR polypeptides involves fermenting yeast which express
proteins as a secreted protein. 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), involving two sequential, reversed-phase HPLC steps
for purification of a recombinant protein on a preparative HPLC
column.
[0088] LIR-P3G2 DNA in pDC406 vector was deposited with the
American Type Culture Collection on Apr. 22, 1997 and assigned
accession No. ______. The deposit was made under the terms of the
Budapest Treaty.
[0089] As described above, LIR-P3G2 is a MHC class I receptor
molecule found on the surface of certain monocytes, B cells, and NK
cells. Certain LIR family members have the ITIM motif and by
analogy with the structure and function of known MHC class I
receptor molecules, are inhibitory receptors mediating negative
signaling. Other LIR family members lack the ITIM motif and by
analogy with the structure and function of known MHC class I
receptors are activatory receptors. Failure of a receptor that
mediates negative signaling could result in autoimmune diseases.
Thus, engaging an LIR family member having ITIM motifs with an
agonistic antibody or ligand can be used to downregulate a cell
function in disease states in which the immune system is overactive
and excessive inflammation or immunopathology is present. On the
other hand, using an antagonistic antibody specific to the ITIM
possessing LIR receptor or a soluble form of the receptor can be
used to block the interaction of the cell surface receptor with the
receptor's ligand to activate the specific immune function in
disease states associated with suppressed immune function. Since
receptors lacking the ITIM motif send activatory signals once
engaged as described above, failure of a receptor that mediates an
activatory signal could result in suppressed immune function.
Engaging the receptor with its agonistic antibody or ligand can be
used to treat diseases associated with the suppressed immune
function. Using an antagonistic antibody specific to the activatory
LIR receptor or a soluble form of the receptor can be used to block
the interaction of the activatory receptor with the receptor's
ligand to downregulate the activatory signaling.
[0090] Since LIR-P3G2 binds to various cells, LIR-P3G2 may be used
to purify or isolate these cells from heterogeneous preparations.
Additionally, P3G2 probes can be used to isolate and identify
related molecules.
[0091] LIR polypeptides of the present invention may be used in
developing treatments for any disorder mediated directly or
indirectly by defective or insufficient amounts of any of the LIR
polypeptides. A therapeutically effective amount of purified LIR
protein is administered by a patient afflicted with such a
disorder. Alternatively, LIR DNA may be employed in developing a
gene therapy approach to treating such disorders. Disclosure herein
of native LIR nucleotide sequence permits the detection of
defective LIR genes, and the replacement thereof with normal
LIR-encoding genes. Defective genes may be detected in vitro
diagnostic assays, and by comparison of the native LIR nucleotide
sequence disclosed herein with that of an LIR gene derived from a
person suspected of harboring a defect in the gene.
[0092] The present invention also provides pharmaceutical
compositions which may include an LIR polypeptide, or fragments or
variants thereof with a physiologically acceptable carrier or
diluent. Such carriers and diluents will be nontoxic to recipients
at the dosages and concentrations employed. Such compositions may
further include buffers, antioxidants such as ascorbic acid, low
molecular weight (less than about ten residues) polypeptides,
proteins, amino acids, carbohydrates including glucose, sucrose or
dextrins, chelating agents such as EDTA, glutathione and other
stabilizers and excipients commonly used in pharmaceutical
compositions. The pharmaceutical compositions of the present
invention may be formulated as a lyophilizate using appropriate
excipient solutions as diluents. The pharmaceutical compositions
may include an LIR polypeptide in any for described herein,
including but not limited to active variants, fragments, and
oligomers. LIR polypeptides may be formulated according to known
methods that are used to prepare pharmaceutically useful
compositions. Components that are commonly employed in
pharmaceutical formulations include those described in Remington 's
Pharmaceutical Sciences, 16th ed. (Mack Publishing Company, Easton,
Pa., 1980).
[0093] The pharmaceutical preparations of the present invention may
be administered to a patient, preferably a human, in a manner
appropriate to the indication. Thus, for example, the compositions
can be administered by intravenous injection, local administration,
continuous infusion, sustained release from implants, etc.
Appropriate dosages and the frequency of administration will depend
on such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient and so
forth.
[0094] In preferred embodiments an LIR polypeptide used in the
pharmaceutical compositions of the present invention is purified
such that the LIR polypeptide is substantially free of other
proteins of natural or endogenous origin, desirably containing less
than about 1% by mass of protein contaminants residual of the
production processes. Such compositions, however, can contain other
proteins added as stabilizers, carriers, excipients or
co-therapeutics.
[0095] LIR encoding DNAs and DNA fragments disclosed herein find
use in the production of LIR polypeptides, as described above. In
one embodiment, such fragments comprise at least about 17
consecutive nucleotides, more preferably at least 30 consecutive
nucleotides, of LIR DNA. DNA and RNA complements of the fragments
have similar utility. Among the uses of LIR nucleic acid fragments
are as probes or primers in polymerase chain reactions. For
example, a probe corresponding to a fragment of DNA encoding the
extracellular domain of LIR may be employed to detect the presence
of LIR nucleic acids in in vitro assays and in other probing assays
such as Northern Blot and Southern blot assays. Cell types
expressing an LIR polypeptide can be identified using LIR family
nucleic acid probes using probing procedures well known in the art.
Those skilled in the art have the knowledge to choose a probe of
suitable length and apply conventional PCR techniques to isolate
and amplify a DNA sequence.
[0096] Nucleic acid fragments may also be used as a probe in cross
species hybridization procedures to isolate LIR DNA from other
mammalian species. As one example, a probe corresponding to the
extracellular domain of an LIR polypeptide may be employed. The
probes may be labeled (e.g., with .sup.32P) by conventional
techniques.
[0097] Other useful fragments of LIR nucleic acids are antisense or
sense oligonucleotides which include a single-stranded nucleic acid
sequence (either RNA or DNA) capable of binding to a target LIR
mRNA (sense) or P3G2 DNA (antisense) sequences. Such fragments are
generally at least about 14 nucleotides, preferably from about 14
to about 30 nucleotides. 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.
[0098] Binding antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means. The
antisense oligonucleotides thus may be used to block LIR
expression.
[0099] In one embodiment antisense or sense LIR oligonucleotides
used in binding procedures may encompass oligonucleotides having
modified sugar-phosphodiester backbones (or other sugar linkages,
such as those described in WO91/06629) and wherein such sugar
linkages are resistant to endogenous nucleases. Oligonucleotides
having sugar linkages resistant to endogenous nucleases are stable
in vivo (i.e., capable of resisting enzymatic degradation) but
retain sequence specificity to be able to bind to target nucleotide
sequences. Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10448, and other
moieties that increase affinity of the oligonucleotide for a target
nucleic acid sequence, such as poly-(L-lysine). Further still,
intercalating agents, such as ellipticine, and alkylating agents or
metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0100] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by inserting he antisense or sense oligonucleotide
into a suitable retroviral vector, then contacting the cell with
the retroviral vector containing the inserted sequence, either in
vivo or ex vivo. Suitable retroviral vectors include, but are not
limited to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see PCT Application US
90/02656).
[0101] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugating the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind its corresponding molecule or receptor, or block
entry of the sense of antisense oligonucleotide or its conjugated
version into the cell.
[0102] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0103] In still a further aspect, the present invention provides
antibodies that specifically bind LIR polypeptides, i.e.,
antibodies bind to LIR polypeptides via an antigen-binding site of
the antibody (as opposed to non-specific binding). Antibodies of
the present invention may be generated using LIR polypeptides or
immunogenic fragments thereof. Polyclonal and monoclonal antibodies
may be prepared by conventional techniques. See, for example,
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Kennet et al. (eds.), Plenum Press, New York 1980; and
Antibodies. A Laboratory Manual, Harlow and Land (eds.), Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988. An
exemplary procedure for producing monoclonal antibodies
immunoreactive with P3G2-LIR is further illustrated in Example 5
below.
[0104] Included within the scope of the present invention are
antigen binding fragments of antibodies which specifically bind to
an LIR polypeptide. Such fragments include, but are not limited to,
Fab, F(ab'), and F(ab').sub.2. Antibody variants and derivatives
produced by genetic engineering techniques are contemplated as
within the presented invention.
[0105] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such antibodies may be prepared by known techniques and
offer the advantage of reduced immunogenicity when the antibodies
are administered to humans. In one embodiment a humanized
monoclonal antibody comprises the variable region of a murine
antibody (or just the antigen binding site thereof) and a constant
region derived from a human antibody. Alternatively, a humanized
antibody fragment may comprise the antigen binding site of a murine
monoclonal antibody and a variable region fragment (lacking the
antigen-binding site) derived from a human antibody. Procedures for
the production of chimeric and further engineered monoclonal
antibodies include those described in Riechmann et al., Nature
332:232, 1988; Lie et al. PNAS 84:3439, 1987; Larrick et al.
Bio/Technology 7:934, 1989; and Winter and Harris TIPS 14:139,
1993.
[0106] As mentioned above, antibodies of the present invention are
useful in in vitro or in vivo assays to detect the presence of LIR
polypeptides and in purifying an LIR polypeptide by affinity
chromatography.
[0107] Additionally, antibodies capable of blocking an LIR from
binding to target cells may be used to inhibit a biological
activity of an LIR polypeptide. More specifically, therapeutic
compositions of an antibody antagonistic to one or more LIR family
members having the ITIM motif may be administered to an individual
in order to block the interaction of a cell surface LIR with its
ligand. The result is an activation of immune function and is
particularly beneficial in disease states in which the immune
system is hyporesponsive or suppressed. Conversely, therapeutic
compositions of an antibody antagonistic to one or more LIR family
members lacking the ITIM motif may be used to obtain the opposite
effect and be beneficial in disease states in which the immune
system is overactive and excessive inflammation or immunopathology
is present.
[0108] Pharmaceutical compositions which include at least one
antibody that is immunoreactive with an LIR polypeptide and a
suitable diluent, excipient, or carrier, are considered with the
present invention. Suitable diluents, excipients, and carriers are
described in the context of pharmaceutical compositions which
include polypeptides of the present invention.
[0109] The following examples are provided to illustrate certain
embodiments of the invention, and are not to be construed as
limiting the scope of the invention.
EXAMPLES
Example 1
Isolating and Expressing Viral Protein
[0110] DNA encoding P3G2 polypeptide of the present invention was
identified by isolating and expressing a viral glycoprotein, UL18,
known to be expressed on cells infected with HCMV, and then
expressing and using a UL18/Fc fusion protein to search for UL18
receptors. DNA encoding UL18 and its amino acid sequence are known
and described in Beck, S., B. G. Barrell, Nature 331:269-272, 1988.
The following describes isolating UL18 and preparing the UL18/Fc
fusion protein.
[0111] Using standard techniques, total RNA was isolated from Human
Foreskin Fibroblasts infected with HCMV (AD169) at three different
transcription stages-immediate early (1E, 8 p.i.h.), early (24
p.i.h.) and late (48 p.i.h.). Because UL18 is known to be
transcribed early in the infection, the IE total RNA was polyA+
selected and used to construct an HCMV-IE cDNA library using a cDNA
kit according to the manufacturer's instructions (Pharmacia TIME
SAVER cDNA Kit). In order to isolate the full length UL18 gene, two
oligonucleotide primers known to include the terminal sequences of
the UL18 gene were synthesized and used to isolate and amplify the
UL18 gene from the HCMV-IE cDNA library. The primers had the
following sequences and included Not I restriction sites which
incorporate into the PCR product. TABLE-US-00001 Not I 5'- TAT GCG
GCC GCC ATG ATG ACA (SEQ ID NO: 23) ATG TGG T -3' 5'- TAT GCG GCC
GCC CCT TGC GAT (SEQ ID NO: 24) AGC G -3' Not I
The PCR conditions included one 5 minute 95.degree. C. cycle
followed by 30 cycles of 45 seconds at 95.degree., 45 seconds at
58.degree. and 45 seconds at 72.degree., and then one cycle for 5
minutes at 72.degree. C. The PCR product was electrophoresed on a
1% agarose gel and sized using ethidium bromide to visualize the
separated DNA products. The presence of DNA of having the expected
size of approximately 1.1 kb was confirmed.
[0112] The pDC409 expression vector, a vector derived from pDC406
(McMahan et al., EMBO J. 10:2821, 1991) but having a single Bgl II
site was selected for the cloning process. The PCR product was
subcloned into a pDC409 expression vector through the Not I sites,
sequenced and the amino acid sequence deduced from the DNA
sequence. The determined nucleotide sequence and amino acid
sequence were identical to the previously published sequences
(ibid.).
[0113] A fusion protein of the extracellular region of UL18 and a
mutein human IgG1 Fc region (UL18:Fc) was prepared by first
isolating cDNA encoding the extracellular region of UL18 using
primers which flank the extracellular region of UL18. The primers
were synthesized with Sal I and Bgl II restriction sites inserted
at the 5' and 3' termini so that the PCR amplified cDNA introduced
Sal I and Bgl II restriction sites at the 5' and 3' ends,
respectively. The primers had the following sequences:
TABLE-US-00002 5'- ATA GTC GAC AAC GCC ATG ATG (SEQ ID NO: 25) ACA
ATG TGG TG -3' Sal I 5'- TAA AGA TCT GGG CTC GTT AGC (SEQ ID NO:
26) TGT CGG GT -3' Bgl II
The conditions for the PCR reaction were as described above except
that the template was the full length gene isolated as just
described.
[0114] To prepare a vector construct for expressing fusion protein,
sUL18:Fc, for use in cell binding studies, a DNA fragment encoding
the Fc region of a human IgG1 antibody was isolated from a plasmid
using Bgl II and Not I restriction enzymes. The encoded Fc portion
was the mutein Fc described in U.S. Pat. No. 5,457,035 having
reduced affinity for immunoglobulin receptors. The Bgl II site on
the sUL18 gene was used to ligate the sUL18 gene DNA to the Bgl II
site on the Fc gene to form a sUL18:Fc fusion DNA construction
having an N-terminal Sal I restriction site and a C-terminal Not I
restriction site. This fusion sUL18:Fc DNA construct was then
ligated into pDC409 expression vector at its Sal I and Not I sites
to form a 409/sUL18/Fc DNA construct.
[0115] The monkey cell line COS-1 (ATCC CRL-1650) was used to
confirm expression of the fusion protein. COS-1 cells in 6-well
plates (2.times.10.sup.5 cells per well) were transfected with
about 2 .mu.g of the DNA construct 409/sUL18/Fc per well. The cells
were cultured for 2-3 days in 5% FBSDMEM/F12 (available from
GIBCO), then washed twice with PBS, starved for 1 hour in
cysteine/methionine depleted RPMI (available from GIBCO as RPMI
1640) and metabolically labeled with 100 .mu.Ci/mL of
.sup.35S-Met/Cys for 4 hours. The supernatant was spun clear to
remove loose cells and 150 .mu.L of the supernatant was incubated
with 100 .mu.L of RIPA (0.05% Tween 20, 0.1% SDS, 1% Triton X-100,
0.5% deoxycholate in PBS) buffer and 50 .mu.L of 50% Protein
A-Sepharose solid support beads at 4.degree. C. for 1 hour. Protein
A-Sepharose is a Sepharose solid support (available from Pharmacia)
having immobilized Protein A which binds the Fc portion of the
fusion protein. After washing the solid support with RIPA to remove
unbound material, fusion protein bound to the Protein A-Sepharose
solid support was eluted from the Protein A-Sepharose using 35
.mu.L of SDS -PAGE reducing sample buffer and then heated at
100.degree. C. for 5 minutes. The eluant was then electrophoresed
on a 4-20% SDS polyacrylamide gradient gel with .sup.14C labeled
protein molecular weight markers. After electrophoresis the gel was
fixed with 8% acetic acid and enhanced at room temperature for 20
minutes with Amplifier available from Amersham. After drying the
gel under vacuum it was exposed to x-ray film. Film analysis
confirmed that the expected protein, a 100-120 kDa protein which
includes the mutein Fc region of IgG and UL18 extracellular domains
fused to the Fc, was expressed.
[0116] Once cells expressing the fusion protein were identified
large scale cultures of transfected cells were grown to accumulate
supernatant from cells expressing the fusion protein. This
procedure involved transfecting COS-1 cells in T175 flasks with 15
.mu.g of the ULI8/Fc/409 fusion DNA per flask. After 7 days of
culture in medium containing 0.5% low immunoglobulin bovine serum,
a solution of 0.2% azide was added to the supernatant and the
supernatant was filtered through a 0.22 .mu.m filter. Then
approximately 1 L of culture supernatant was passed through a
BioCad Protein A HPLC protein purification system using a
4.6.times.100 mm Protein A column (POROS 20A from PerSeptive
Biosystems) at 10 mL/min. The Protein A column binds the Fc portion
of the sUL18/Fc fusion protein in the supernatant, immobilizing the
fusion protein and allowing other components of the supernatant to
pass through the column. The column was washed with 30 mL of PBS
solution and bound sUL18/Fc was eluted from the HPLC column with
citric acid adjusted to pH 3.0. Eluted purified sUL18/Fc was
neutralized as it eluted using 1M Hepes solution at pH 7.4. The
pooled eluted protein was analyzed using SDS PAGE with silver
staining, confirming expression of the 100-120 kDa UL18/Fc fusion
protein.
Example 2
Screening Cell Lines for Binding to UL18
[0117] The sUL18/Fc protein isolated as described in Example I was
used to screen cells lines to which it binds using quantitative
binding studies according to standard flow cytometry methodologies.
For each cell line screened, the procedure involved incubating
approximately 100,000 of the cells blocked with 2% FCS (fetal calf
serum), 5% normal goat serum and 5% rabbit serum in PBS for 1 hour.
Then the blocked cells were incubated with 5pg/mL of sUL18/Fc
fusion protein in 2% FCS, 5% goat serum and 5% rabbit serum in PBS.
Following the incubation the sample was washed 2 times with FACS
buffer (2% FCS in PBS) and then treated with mouse anti human
Fc/biotin (purchased from Jackson Research) and SAPE
(streptavidin-phycoerythrin purchased from Molecular Probes). This
treatment causes the anti human Fc/biotin to bind to any bound
sUL18/Fc and the SAPE to bind to the anti human Fc/biotin resulting
in a fluorescent identifying label on sUL18/Fc which is bound to
cells. The cells were analyzed for any bound protein using
fluorescent detection flow cytometry. The results indicated that
ULI8 binds well to B cell lines CB23, RAJI and MP-1; monocytic cell
lines Thp-1 and U937; and primary B cell and primary monocytes.
UL18 does not bind detectably to T cell lines nor does it bind to
primary T cells.
Example 3
Isolating a P3G2 cDNA and Polypeptide
[0118] The following describes screening cDNA of one of the cell
lines found to bind UL18 and the isolation of a novel polypeptide
expressed by the cell line. A CB23 cDNA library in the mammalian
expression vector pDC406, prepared as described in U.S. Pat. No.
5,350,683 (incorporated herein by reference) was obtained and
plasmid DNA was isolated from pools consisting of approximately
2,000 clones per pool. The isolated DNA was transfected into
CV1-EBNA cells (ATCC CRL 10478) using DEAE-dextran followed by
chloroquine treatment. The CV1-EBNA cells were maintained in
complete medium (Dulbecco's modified Eagles' media containing 10%
(v/v) fetal calf serum, 50 U/mL penicillin, 50 U/mL streptomycin,
and 2 mM L-glutamine) and were plated to a density of approximately
2.times.10.sup.5 cells/well in single-well chambered slides. The
slides had been pre-treated with 1 mL of a solution of 10 .mu.g/mL
human fibronectin in PBS for 30 minutes followed by a single
washing with PBS. Media was removed from adherent cells growing in
a layer and replaced with 1.5 mL complete medium containing 66.6
.mu.M chloroquine sulfate. About 0.2 mL of a DNA solution (2 .mu.g
DNA, 0.5 mg/mL DEAE-dextran in complete medium containing
chloroquine) was added to the cells and the mixture was incubated
at 37 C for about five hours. Following incubation, the media was
removed and the cells were shocked by addition of complete medium
containing 10% DMSO (dimethylsulfoxide) for 2.5 minutes. Shocking
was followed by replacing the solution with fresh complete medium.
The cells were grown in culture for two to three days to permit
transient expression of the inserted DNA sequences. These
conditions led to a 30% to 80% transfection frequency in surviving
CV1-EBNA cells.
[0119] Each slide was incubated with 1 mL of UL18:Fc at a
concentration of 1 .mu.g/mL in binding buffer (RPMI 1640 containing
25 mg/mL bovine serum albumin, 2 mg/mL sodium azide, 20 mM Hepes at
pH 7.2, and 50 mg/mL nonfat dry milk) at room temperature for I
hour. The incubated slides were washed with the binding buffer and
then incubated with Fc specific .sup.125I-mouse anti-human IgG (see
Goodwin et al., Cell 73:447-456, 1993). This was followed by a
second wash with buffer after which the slides were fixed with a
2.5% glutaraldehyde/PBS solution, washed with PBS solution and
allowed to air dry. The dried slides were dipped in Kodak GTNB-2
photographic emulsion (6x dilution in water). After air drying, the
slides were placed in a dark box and refrigerated. After three days
the slides were developed in Kodak D19 developer, rinsed in water
and fixed in Agfa G433C fixer. The fixed slides were individually
examined under a microscope at 25-40.times. magnification. Positive
cells demonstrating binding of sUL18:Fc were visualized by the
presence of autoradiographic silver grains against the film
background. Two positive pools were identified. Bacterial clones
from each pool were titered and plated to provide plates containing
approximately 200 colonies each. Each plate was scraped to provide
pooled plasmid DNA for transfection into CV1-EBNA cells and
screening as described above. Following subsequent breakdowns and
screenings, two positive individual colonies were obtained. The
cDNA inserts of the two positive clones were 2922 and 2777
nucleotides in length as determined by automated DNA sequences. The
coding regions of the two inserts, designated P3G2 and 18A3 were
1953 (nucleotides 310-2262) and 1959 (nucleotides 168-2126)
nucleotides, respectively. The two cDNA clones encode proteins that
are substantially similar and probably represent different alleles
of the same gene.
[0120] The cDNA sequence and encoded amino acid of P3G2 are
presented in SEQ ID NO: 1 and SEQ ID NO:2, respectively. The cDNA
sequence and encoded amino acid of 18A3 are presented in SEQ ID
NO:3 and SEQ ID NO:4, respectively. The P3G2 amino acid sequence
(SEQ ID NO:2) has a predicted signal peptide of 16 amino acids
(amino acids 1-16); an extracellular domain of 442 amino acids
(amino acids 17-458); a transmembrane domain of 25 amino acids
(amino acids 459-483) and, a cytoplasmic domain of 167 amino acids
(amino acids 484-650. The extracellular domain includes four
immunoglobulin-like domains. Ig-like domain I includes
approximately amino acids 17-118; Ig-like domain II includes
approximately amino acids 119-220; Ig-like domain III includes
approximately amino acids 221-318; and Ig-like domain IV includes
approximately amino acids 319-419. Significantly, the cytoplasmic
domain of this polypeptide includes four ITIM motifs, each having
the consensus sequence of YxxL/V. The first ITIM motif pair is
found at amino acids 533-536 and 562-565 and the second pair is
found at amino acids 614-617 and 644-647. The amino acid sequence
of 18A3 is nearly identical having the features describes
above.
[0121] The features of these encoded polypeptides are consistent
with a type I transmembrane glycoprotein.
Example 4
Preparing P3G2 Fusion Protein
[0122] The following describes procedures used to generate a P3G2
fusion protein which was then used to identify cell lines to which
it binds and finally isolate a normal cell-surface P3G2 ligand
which is distinct from UL18. A fusion protein of the extracellular
region of P3G2 and the mutein human Fc region (sP3G2:Fc) was
prepared by first isolating cDNA encoding the extracellular region
of P3G2 using primers which flank the extracellular region of P3G2.
The primers were synthesized with Sal I and Bgl II restriction
sites inserted at the 5' and 3' termini so that the PCR amplified
cDNA introduced Sal I and Bgl II restriction sites at the 5' and 3'
ends, respectively. The primers had the following sequences:
TABLE-US-00003 Sal I (SEQ ID NO: 5) 5'- TAT GTC GAC CAT GAC CCC CAT
CCT CAC GGT -3' Bgl II Xa (SEQ ID NO:6) 5'- TAT GGG CTC TGC TCC AGG
AGA AGA TCT TCC TTC TAT AAC CCC GAG GTG CCT T
[0123] The conditions for the PCR reaction were as described above
and the template was the full length gene P3G2 gene isolated as
described in Example 3 above.
[0124] To prepare a vector construct for expressing fusion protein
sP3G2:Fc for use in cell binding studies, the mutein human Fc
region of IgG1 was cut from the plasmid described above in Example
1 using Bgl II and Not I restriction enzymes. The Bgl II site on
the sP3G2 gene was used to ligate the sP3G2 gene DNA to the Bgl II
site on the human mutein Fc gene to form a sP3G2/Fc fusion DNA
construction having an N-terminal Sal I restriction site and a
terminal Not I restriction site. This fusion sP3G2:Fc DNA construct
was then ligated into pDC409 expression vector at its Sal I and Not
I sites to form a 409/sP3G2/Fc DNA construct.
[0125] The monkey cell line COS-1 (ATCC CTL-1650) was used to
confirm expression of the fusion protein. COS-1 cells in 6-well
plates (2.times.10.sup.5 cells per well) were transfected with
about 2 .mu.g of the DNA construct 409/sP3G2/Fc per well. The cells
were cultured in 5% FBS/DMEM/F12 (available from GIBCO) and at day
two or three following transfection, the cells were starved for 1
hour in cysteine/methionine depleted RPMI and the transfected cells
were metabolically labeled with 100 .mu.Ci/mL of .sup.35S-Met/Cys
for 4 hours. The supernatant was spun clear to removed loose cells
and debris and 150 .mu.L of the supernatant was incubated with 100
.mu.L of RIPA buffer and 50 .mu.L of 50% Protein A-Sepharose solid
support beads at 4.degree. C. for 1 hour. After washing the solid
support with RIPA to remove unbound material, fusion protein bound
to the Protein A-Sepharose solid support was eluted from the
Protein A-Sepharose using 30 .mu.L of SDS-PAGE reducing sample
buffer and then heated at 1000.degree. C. for 5 minutes. The eluant
was then electrophoresed on a 4-20% SDS polyacrylamide gradient gel
with .sup.14C labeled protein molecular weight markers. After
electrophoresis the gel was fixed with 8% acetic acid and enhanced
at room temperature for 20 minutes with Amplifier available from
Amersham. After drying the gel under vacuum it was exposed to x-ray
film. Film analysis confirmed that the expected protein, having a
molecular weight of 120-130 kDa, was expressed.
[0126] Once fusion protein expression was verified, large scale
cultures of transfected cells were grown to accumulate supernatant
from COS-1 cells expressing the fusion protein as described in
Example 1 above. The P3G2/Fc fusion protein was purified according
to the procedure described in Example 3 above using the BioCad
system and the POROS 20A column from PerSeptive Biosystems. The
pooled eluted protein was analyzed using SDS PAGE with silver
staining, confirming expression.
Example 5
Generating LIR-P3G2 Antibody
[0127] The following example describes generating monoclonal
antibody to P3G2 that was used in flow cytometry analysis to
identify cells on which P3G2 is expressed. Purified P3G2/Fc fusion
protein was prepared by COS-1 cell expression and affinity
purification as described in Example 4. The purified protein or
cells transfected with an expression vector encoding the full
length protein can generate monoclonal antibodies against P3G2
using conventional techniques, for example those techniques
described in U.S. Pat. No. 4,411,993. Briefly BALB-C mice were
immunized at 0, 2 and 6 weeks with 10 .mu.g P3G2/Fc. The primary
immunization was prepared with TITERMAX adjuvant, from Vaxcell,
Inc., and subsequent immunization were prepared with incomplete
Freund's adjuvant (IFA). At 11 weeks, the mice were IV boosted with
3-4 .mu.g P3G2 in PBS. Three days after the IV boost, splenocytes
were harvested and fused with an Ag8.653 myeloma fusion partner
using 50% aqueous PEG 1500 solution. Hybridoma supernatants were
screened by ELISA using P3G2 transfected COS-1 cells in PBS at
2.times.10.sup.3 cells per well and dried to polystyrene 96-well
microtiter plates as the platecoat antigen. Positive supernatants
were subsequently confirmed by FACS analysis and RIP using P3G2
transfected COS-1 cells. Hybridomas were cloned and followed using
the same assays. Monoclonal cultures were expanded and supernatants
purified by affinity chromatography using BioRad Protein A
agarose.
[0128] The monoclonal antibodies to P3G2/Fc were used to screen
cells and cell lines using standard flow cytometry procedures to
identify cells on which P3G2 is expressed. Cell lines and cells
screened in the flow cytometry analyses were CB23, CB39, RAJI,
AK778, K299, PS-1, U937, THP-1, JURKAT and HSB2. For each cell line
or cell sample screened, the procedure involved incubating
approximately 100,000 of the cells blocked with 2% FCS (fetal calf
serum), 5% normal Goat serum and 5% rabbit serum in PBS with 5
.mu.g of FITC conjugated mouse anti-P3G2 antibody for 1 hour.
Following the incubation the sample was washed 2 times with FACS
buffer (2% FCS in PBS). The cells were analyzed for any bound
protein using fluorescent detection flow cytometry to detect FITC.
The results indicated that LIR-P3G2 antibody binds well to B cell
lines CB23 and RAJI1; monocytic cell lines THP-1 and U937; and
primary B cell and primary monocytes. The highest expression of
LIR-P3G2 was shown on monocytes that stained brightly for CD 16 and
less brightly for CD 14 and CD64. The antibody does not bind
detectably to T cell lines nor does it bind detectably to primary T
cells.
[0129] In a related experiment, the P3G2 antibody generated as
described above was used in immunoprecipitation experiments. The
immunoprecipitation analyses involved first surface biotinylating
2.5.times.10.sup.6 monocytes by washing the cells with PBS and
suspending the cells in a biotinylation buffer of 10 mM sodium
borate and 150 mM NaCl at pH 8.8, followed by adding 5 .mu.L of a
10 mg/mL solution of biotin-CNHS-ester (D-biotinoyl-e-aminocaproic
acid-N-hydroxysuccinimide ester purchased from Amersham) in DMSO to
the cells. After quenching the reaction with 10 .mu.L of 1 M
ammonium chloride per 1 mL of cells and washing the cells in PBS,
the cells were lysed in 1 mL of 0.5% NP40-PBS and the lysate was
recovered following centrifugation. Then 100 .mu.L of 0.5%NP40-PBS
was added to 150 .mu.L of the lysate and the resulting mixture was
incubated with 2 .mu.g/mL of antibody, at 4.degree. C. for 16
hours. Fifty microliters of 50% Protein A-Sepharose slurry was
added to the antibody mixture and the slurry was shaken at
4.degree. C. for 1 hour. The slurry was centrifuged and the
resulting pellet was washed with 0.75 mL of 0.5% NP40 in PBS six
times. Protein bound to the Protein A-Sepharose was eluted with 30
.mu.L of SDS-PAGE reducing sample buffer and heating at 100.degree.
C. for five minutes.
[0130] The eluted proteins were analyzed using 4-20% gradient
SDS-PAGE with enhanced chemiluminescence (ECL) protein markers.
Then the electrophoreses samples were transferred in a Western Blot
onto nitrocellulose membranes. The membranes were treated with
blocking reagent (0.1% Tween-20 and 3% nonfat dry milk in PBS) for
one hour at room temperature and then they were washed once for 15
minutes followed and twice for 5 minutes with 0.1% Tween-20 in PBS.
The washed membranes were incubated with 10 mL of 1:100
HRP-Streptavidin for 30 minutes and then washed 1 times for 15
minutes followed by 4 times for 5 minutes with 0.1% Tween-20 in
PBS.
[0131] Bound streptavidin HRP was detected with ECL Detection
Reagents purchased from Amersham and used according to
manufacturer's instructions. The developed membranes were exposed
to x-ray film and then visualized. The results showed that LIR-P3G2
was immunoprecipitated from CB23 cells and P3G2 transfected COS-1
cells, indicating that P3G2 is expressed by these cells.
Example 6
Screening Cells and Cell Lines for Binding to P3G2
[0132] The following describes flow cytometry analyses used to
identify cells and cell lines which bind to P3G2. The cells and
cell lines tested were CB23, HSB2, MP-1, Jurkat, primary T cells,
primary B cells, and primary NK cells. For each cell line or cell
line tested the procedure involved washing the cells three times
with FACS buffer (2% FCS in PBS with 0.2% azide) and incubating
each sample (105 cells) in 100 .mu.L blocking buffer (2% FCS, 5%
NGS, 5% rabbit serum in PBS) for one hour. For each cell line 4
test samples were prepared, one each having 0, 2, 5, or 10 .mu.g of
W6/32 (ATCC HB-95) in 100 .mu.L blocking buffer added to the
samples, respectively. W6/32 is an antibody against MHC Class I
heavy chains (an anti HLA-A, B, and C molecule). Following the
addition of the W6/32 solution, the samples were incubated on ice
for 1 hour and then washed three times with 200 .mu.L of FACS
buffer. Then 5 .mu.g of P3G2/Fc in blocking buffer was added to
each sample and they were incubated on ice for one hour. The
P3G2/Fc competes with W6/32 for binding sites on the cells.
[0133] Following the incubation, the cells were washed three times
with 200 .mu.L of FACS buffer and treated with mouse anti human
Fc/biotin and SAPE for 45 minutes. This treatment causes the anti
human Fc/biotin to bind to any cell bound sP3G2/Fc and the SAPE to
bind to the anti human F/Biotin. Since the SAPE is a fluorescing
compound its detection using appropriate excitation and emission
conditions positively identifies cell bound P3G2/Fc. Finally the
treated cells were washed three times with FACS buffer and
subjected to flow cytometry to identify cells bound to protein.
[0134] The results demonstrated that W6/32 competed with P3G2 for
binding to all cells and cell lines tested. The P3G2 binding was
totally blocked at 5 .mu.g W6/32 indicating that W6/32 and P3G2 are
binding to the same or overlapping sites on the MHC Class I heavy
chains.
Example 7
Screening HSB2 cDNA Library to Isolate a P3G2 Binding Ligand
[0135] The following describes screening a cDNA library from of one
of the cell lines, HSB-2, a T lymphoblastic leukemia cell line,
found to bind P3G2, and identifying a P3G2 binding ligand. An HSB2
cDNA library in the mammalian expression vector pDC302, was
prepared as generally described in U.S. Pat. No. 5,516,658 and
specifically in Kozlosky et al. Oncogene 10.299-306, 1995. Briefly,
mRNA was isolated from sorted HSB-2 cells and a first cDNA strand
was synthesized using 5 .mu.g polyA.sup.+ and the reverse
transcriptase AMV RTase from Life Science. The second cDNA strand
was synthesized using DNA polymerase I from BRL at concentration of
1.5 U/.mu.L. Using standard techniques as described in Haymerle et
al., Nucl. Acids Res. 14:8615, 1986, the cDNA was ligated into the
appropriate site of the pDC302 vector.
[0136] E.coli. strain DH5.alpha. cells were transformed with the
cDNA library in pDC302. After amplifying the library a titer check
indicated that there was a total of 157,200 clones. The transformed
cells were plated into 15 different plates. Plasmid DNA was
isolated from pools consisting of approximately 2,000 clones per
pool. The isolated DNA was transfected into CV1-EBNA cells (ATCC
CRL 10478) using DEAE-dextran followed by chloroquine treatment.
The CV1-EBNA cells were maintained in complete medium (Dulbecco's
modified Eagles' media containing 10% (v/v) fetal calf serum, 50
U/mL penicillin, 50 U/mL streptomycin, and 2 mM L-glutamine) and
were plated to a density of approximately 2.times.10.sup.5
cells/well in single-well chambered slides. The slides had been
pre-treated with 1 mL of a solution of 10 .mu.g/mL human
fibronectin in PBS for 30 minutes followed by a single washing with
PBS. Media was removed from adherent cells growing in a layer and
replaced with 1.5 mL complete medium containing 66.6 .mu.M
chloroquine sulfate. About 0.2 mL of a DNA solution (2 .mu.g DNA,
0.5 mg/mL DEAE-dextran in complete medium containing chloroquine)
was added to the cells and mixture was incubated at 37 C for about
five hours. Following incubation media was removed and the cells
were shocked by adding complete medium containing 10% DMSO for 2.5
minutes. After shocking the cells the complete medium was replaced
with fresh complete medium and the cells were grown in culture for
three days to permit transient expression of the inserted DNA
sequences. These conditions led to a 30% to 80% transfection
frequency in surviving CV1-EBNA cells.
[0137] Each slide was incubated with 1 mL of P3G2:Fc at a
concentration of 0.45 .mu.g/mL in binding buffer (RPMI 1640
containing 25 mg/mL bovine serum albumin, 2 mg/mL sodium azide, 20
mM Hepes at pH 7.2, and 50 mg/mL nonfat dry milk) at room
temperature for 1 hour. After incubating the slides, they were
washed with binding buffer and then incubated with Fc specific
.sup.125I-mouse anti-human IgG (see Goodwin et al. Cell 73.447-456,
1993). This was followed by a second wash with buffer after which
the slides were fixed with a 2.5% glutaraldehyde/PBS solution,
washed in PBS and allowed to air dry. The slides were dipped in
Kodak GTNB-2 photographic emulsion (6.times. dilution in water).
After air drying the slides were placed in a dark box and
refrigerated. After three days the slides were developed in Kodak
D19 developer, rinsed in water and fixed in Agfa G433C fixer. The
fixed slides were individually examined under a microscope at
25-40.times. magnification. Positive pools demonstrating binding of
sP3G2:Fc were visualized by the presence of autoradiographic silver
grains against the film background. Two positive pools were titered
and plated to provide plates containing approximately 200 colonies
each. Each plate was scraped to provide pooled plasmid DNA for
transfection into CV1-EBNA cells and screening as described above.
Following subsequent breakdowns and screenings, one positive
individual colony was obtained for each pool. The cDNA insert of
the positive clones were identified as HLA-B44 and HLA-A2, class I
MHC antigens.
Example 8
Northern Blot Analysis
[0138] Since the experiments described in Example 4 resulted in the
detection of LIR-P3G2 surface expression on a number of cell lines,
conventional Northern Blot analysis procedures were used to study
the expression of LIR-P3G2 and any LIR-P3G2 related mRNAs in
different tissue types. The cell lines selected for Northern Blot
analysis were RAJI, PBT, PBM, YT, HEP3B, HELA, KB, KG-1, IMTLH,
HPT, HFF, THP-1, and U937. The following describes the Northern
Blot analysis and the analysis results.
[0139] The cDNA encoding the extracellular region of P3G2 was
isolated using primers which flank the extracellular region of P3G2
and having the following sequences: TABLE-US-00004 Sal I 5'- TAT
GTC GAC CAT GAC CCC CAT (SEQ ID NO: 5) CCT CAC GGT -3' BgI II 5'-
TAT AGA TCT ACC CCC AGG TGC (SEQ ID NO: 27) CTT CCC AGA CCA
The PCR template was the full length P3G2 gene isolated as
described in Example 3 above. The conditions for the PCR reaction
were as follows: One cycle at 95.degree. C. for 5 minutes; 30
cycles which included 95.degree. C. for 45 seconds, 64.degree. C.
for 45 seconds and 72.degree. C. for 45 seconds; and, one cycle at
72.degree. C. for 5 minutes. The PCR product was cloned into PCR II
vector, purchased from Invitrogen, in accordance with the
supplier's instructions. The isolated DNA encoding the
extracellular region of P3G2 was used to make a riboprobe with the
Ambion MAXISCRIPT Kit according to the manufacturer's
instructions.
[0140] Northern blots containing poly A+selected RNA or total RNA
from a variety of human cell lines were prepared by resolving RNA
samples on a 1.1% agarose-formaldehyde gel, blotting onto Hybond-N
as recommended by the manufacturer (Amersham Corporation) and
staining with methylene blue to monitor RNA concentrations. The
blots were prepared using 1 .mu.g of the PolyA+RNA or 10 .mu.g of
total RNA and each blot was probed with 10.sup.6 cpm/mL RNA
extracellular P3G2 riboprobe, prepared as just described, at
63.degree. C. for 16 hours. The probed blots were washed with
2.times.SSC at 63.degree. C. for 30 minutes 2 times; 1.times.SSC at
63.degree. C. for 30 minutes 2 times; and, 0.1.times.SSC at
63.degree. C. for 5 minutes 2 times.
[0141] The probed blots were autoradiographically developed. The
developed blots showed that the P3G2 RNA hybridized to a 3.5 kb RNA
expressed by RAJI, CB23 and U937; an approximately 1.5 kb RNA
expressed by THP-1; and multiple RNAs ranging from 1.5 kb to 3.5 kb
expressed by PBM. These results suggest that different genes having
extracellular domains similar in structure to that of P3G2 may be
expressed by peripheral blood monocytes.
Example 9
Probing PBM cDNA Library to Isolate LIR Polypeptides
[0142] The following describes steps taken to screen a peripheral
blood monocyte cDNA library to isolate polypeptides relating to the
P3G2 polypeptide using conventional Southern Blot methodologies. A
peripheral blood monocyte cDNA library was prepared using
substantially the same procedures described in Example 7.
[0143] DNA from an initial 15 pools of cDNA having 10,000 clones
per pool was digested with Bgl II restriction enzyme and
electrophoresed on a I % agarose gel at 100 V for 2 hours. Southern
Blots were prepared by electroblotting the electrophoresed DNA in
0.55% TBE buffer onto Hybond membranes. The blotted DNA was
denatured in 0.5 M NaOH in 0.6M NaCl solution for 5 minutes and
then neutralized in 0.5 M TRIS in 1.5 M NaCl at pH 7.8 for 5
minutes. The membranes were placed in a STRATALINKER UV crosslinker
for 20 seconds to crosslink the blotted DNA to the membrane. The
membrane and bound DNA were placed in pre-hybridization solution of
10.times. Denhart's Solution, 0.05M TRIS at pH 7.5, 0.9M NaCl, 0.1%
sodium pyrophosphate, 1% SDS and 200 .mu.g/mL salmon sperm DNA at
63.degree. C. for 2 hours and then the bound DNA was probed with
.sup.32P labeled probe of DNA encoding the extracellular region of
LIR-P3G2, including the signal peptide and Sal I and Bgl II
restriction sites. The concentration of the DNA probe in
hybridization solution was 10.sup.6 CPM per mL of hybridization
solution. The probed blots were incubated for 16 hours at
63.degree. C. and then washed with 2.times.SSC at 63.degree. C. for
1 hour with one solution change; 1.times. with SSC at 63.degree. C.
for one hour with one solution change; and, with 0.1.times.SSC at
68.degree. C. for 45 minutes with one solution change. After drying
the blots they were autoradiographically developed and visualized
for DNA bands which hybridized to the P3G2 extracellular DNA
probe.
[0144] The results of the autoradiography visualization indicated
that all pools contained DNA which hybridized to the probe. One
pool showing 7 positive DNA bands was selected and subsequently
subdivided to 10 pools having 3,000 clones per pool. Applying
subsequent Southern Blotting methodologies to the 10 pools resulted
in one pool showing 9 positively hybridizing DNA sequences. Single
hybridizing clones were isolated by standard colony hybridization
techniques.
[0145] Duplicate bacterial colonies on filters were probed with the
P3G2 extracellular probe described above at a concentration of
500,000 cpm/mL at 63.degree. C. for 16 hours. The hybridized
filters were washed with 2.times.SSC at 63.degree. C. for 30
minutes; with 1.times.SSC at 63.degree. C. for 30 minutes; and
finally with 0.1.times.SSC at 68.degree. C. for 15 minutes.
[0146] Forty-eight clones were visualized as hybridizing on
duplicate filters by autoradiography and DNA obtained from these
clones using standard DNA preparation methodologies was digested
with Bgl II. Then Southern Blots of the digests were obtained and
probed with the P3G2 extracellular probe described above. Seven
different sized cloned inserts were identified as positively
hybridizing to the P3G2 probe. The nucleotide sequence of each of
the inserts was obtained using automated sequencing technology. Of
the 8 different cloned inserts, one was identical in sequence to
LIR-P3G2. The others were identified as DNA encoding polypeptides
of the new LIR family of polypeptides. The nucleotide sequences
(cDNA) of the isolated LIR family members are presented in SEQ ID
NO:7 (designated pbm25), SEQ ID NO:9 (designated pbm8), SEQ ID
NO:11 (designated pbm36-2), SEQ ID NO: 13 (designated pbm36-4); SEQ
ID NO: 15 (designated pbmhh); SEQ ID NO: 17 (designated pbm2) and
SEQ ID NO: 19 (designated pbml7). The amino acid sequences encoded
thereby are presented in SEQ ID NO:8 (designated pbm25), SEQ ID NO:
10 (designated pbm8), SEQ ID NO: 12 (designated pbm36-2), SEQ ID
NO: 14 (designated pbm36-4), SEQ ID NO:16 (designated pbmhh); SEQ
ID NO:18 (designated pbm2); and SEQ ID NO:20 (designated
pbm17).
Example 10
Screening a Human Dendritic Cell cDNA Library for LIR cDNA
Sequences
[0147] The following describes the isolation and identification of
an LIR family member by screening a human bone marrow-derived
dendritic cell cDNA library in the .lamda. Zap vector with a
radiolabeled Hh0779 cDNA fragment. The Hh0779 cDNA fragment is a
0.7 kb insert of the Hh0779 clone previously isolated from a human
dendritic cell cDNA library and obtained by restriction digestion
with the enzymes PstI and SpeI. The Hh0779 cDNA fragment was
labeled with [a-.sup.32P]dCTP using the DECAprime II DNA labeling
kit purchased from Ambion.
[0148] The .lamda. Zap cDNA library was plated at a density of
20,000 pfu per plate to provide a total of 480,000 plagues for the
initial screening. The .lamda. Zap cDNA was blotted in duplicate
onto Hybond membranes, purchased from Amersham, and then denatured
in a solution of 0.5N NaOH and 0.5M NaCl for 5 minutes. The
membranes were neutralized in a solution of 0.5M Tris (pH 7.8) and
1.5M NaCl for 5 minutes, and then washed in 2x SSC for 3 minutes.
The cDNA was crosslinked to the Hybond membranes using a
STRATALINKER UV crosslinker in the auto setting.
[0149] The membranes were pre-hybridized at 65.degree. C. for 2.25
hours in hybridization buffer containing 10.times. Denhardt's,
0.05M Tris (pH 7.5), 0.9M NaCl, 0.1% sodium pyrophosphate, 1% SDS
and 4 mg/mL heat denatured salmon sperm DNA. After the
pre-hybridization, the radiolabeled Hh0779 cDNA was added to the
hybridization buffer to a final concentration of
0.54.times.10.sup.6 cpm/mL. After 24 hours of hybridization, the
membranes were washed in 0.25.times.SSC, 0.25% SDS at 65.degree. C.
for 1.5 hours. The blots were then exposed to autoradiographic film
to visual positive clones.
[0150] A total of 146 positive clones showing hybridization signals
in both membranes of a duplicate set were identified, isolated, and
saved for future use. Of the 146 clones, 35 were selected for
secondary screening. The selected clones were plated at low density
and single clones were isolated after hybridization to the HH0779
probe using the hybridization conditions described above. The
plasmids were then isolated from the .lamda. Zap clones using the
VCSM13 helper phage purchased from Stratagene. The plasmid DNA was
analyzed by restriction digestion and PCR, and the clones
containing the 24 largest inserts were selected and sequenced. Of
the 24 sequenced clones, 6 encoded LIR-P3G2, 3 encoded LIR-pbm2, 8
encoded LIR-pbm36-4 and LIR-pbm36-2, I encoded LIR-pbm8, 2 encoded
LIR-pmbhh, and 1 encoded a novel sequence designated LIR-pbmnew.
Three clones were identified as encoding amino acid sequences that
are not relevant to the LIR polypeptide family.
Example 11
Association of LIR-P3G2 and Tyrosine Phosphatase, SHP-1
[0151] The following describes the tests performed to demonstrate
that LIR-P3G2 and SHP-1 associate. CB23 cells were cultured in RPMI
medium supplemented with 10% FBS, concentrated by centrifugation
and finally subdivided into two aliquots. One aliquot was
stimulated with a solution of 50 mM/mL sodium pervanadate for 5
minutes. The second aliquot was not stimulated. After stimulation,
the cells in each aliquot were immediately lysed in RIPA buffer
containing 1% NP-40, 0.5% sodium deoxycholate, 50 mM Tris pH8, 2 mM
EDTA, 0.5 mM sodium orthovanadate, 5 mM sodium fluoride, 25 mM
.beta.-glycerol phosphate, and protease inhibitors. Samples of
24.times.10.sup.6 cell equivalents were incubated for 2 hours at
4.degree. C. with either 5 .mu.g/mL of anti-SHP-1 antibody
purchased from Transduction Laboratories, or 5 .mu.g/mL of an
isotype-matched antibody control (anti-Flag-M5 IgG1). The resulting
immunocomplexes were precipitated by incubation with protein
G-agarose (Boehringer Mannheim), washed, and resuspended in 40 mL
of 2.times.SDS-PAGE sample buffer. Twenty microliters of each
immunoprecipitate were loaded onto electrophoresis gels,
electrophoresed under reducing conditions, and transferred to
nitrocellulose membranes purchased from Amersham. Western blots
were probed with anti-LIR-P3G2 polyclonal antisera and the
immunocomplexes were detected by enhanced chemiluminescence
(NEN).
[0152] A protein having a molecular weight of approximately 120kDa
and corresponding to LIR-P3G2 was readily detected in SHP-1
immunoprecipitates, but not in anti-Flag-M5 antibody. The LIR-P3G2
band was not seen in the absence of sodium pervanadate treatment,
showing that tyrosine phosphorylation of LIR-P3G2 is essential for
the association of LIR-1 and SHP-1 . This data demonstrates that
LIR-P3G2, by analogy with molecules possesses the ITIM motif, sends
an inhibitory signal intracellularly when it interacts with its
counterstructures, viral or cellular MHC class I molecules.
Example 12
Generating Antibodies Immunoreactive with LIR Polypeptides
[0153] The following describes generating monoclonal antibody
immunoreactive with LIR family members. A purified LIR polypeptide
is prepared by COS-1 cell expression and affinity purification as
described in Example 4. The purified protein or cells transfected
with an expression vector encoding the full length protein can
generate monoclonal antibodies against the LIR polypeptide using
conventional techniques, for example those techniques described in
U.S. Pat. No. 4,411,993. Briefly BALB-C mice are immunized at 0, 2
and 6 weeks with 10 .mu.g of the LIR polypeptide. The primary
immunization is prepared with TITERMAX adjuvant and subsequent
immunizations are prepared with incomplete Freund's adjuvant (IFA).
At 11 weeks, the mice are IV boosted with 3-4 .mu.g the LIR
polypeptide in PBS. Three days after the IV boost, splenocytes are
harvested and fused with an Ag8.653 myeloma fusion partner using
50% aqueous PEG 1500 solution. Hybridoma supernatants are screened
by ELISA using the LIR transfected cells in PBS at 7.times.10.sup.3
cells per well and dried to polystyrene 96-well microtiter plates
as the platecoat antigen. Positive supernatants are subsequently
confirmed by FACS analysis and RIP using LIR transfected cells.
Hybridomas are cloned and followed in the same manner of screening.
Monoclonal cultures are expanded and supernatants purified by
affinity chromatography.
Sequence CWU 1
1
* * * * *
References