U.S. patent application number 11/559281 was filed with the patent office on 2007-03-22 for human tumor necrosis factor receptor.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Francis J. Grant, Jane A. Gross, Randal M. Henne, Wenfeng Xu.
Application Number | 20070066805 11/559281 |
Document ID | / |
Family ID | 27500224 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070066805 |
Kind Code |
A1 |
Gross; Jane A. ; et
al. |
March 22, 2007 |
HUMAN TUMOR NECROSIS FACTOR RECEPTOR
Abstract
Tumor necrosis factors and their receptors have proven
usefulness in both basic research and as therapeutics. The present
invention provides a new human tumor necrosis factor receptor
designated as "Ztnfr12."
Inventors: |
Gross; Jane A.; (Seattle,
WA) ; Xu; Wenfeng; (Mukilteo, WA) ; Henne;
Randal M.; (Seattle, WA) ; Grant; Francis J.;
(Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
27500224 |
Appl. No.: |
11/559281 |
Filed: |
November 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10008063 |
Nov 5, 2001 |
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11559281 |
Nov 13, 2006 |
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60246449 |
Nov 7, 2000 |
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60257131 |
Dec 20, 2000 |
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60301715 |
Jun 28, 2001 |
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60315565 |
Aug 29, 2001 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 2319/30 20130101; A61P 35/00 20180101; A61P 37/02 20180101;
A61P 35/02 20180101; C07K 14/7151 20130101 |
Class at
Publication: |
530/350 ;
530/388.22; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C07K 14/715 20060101
C07K014/715; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 16/28 20060101 C07K016/28 |
Claims
1. An isolated polypeptide, comprising amino acid residues 7 to 71
of the amino acid sequence of SEQ ID NO:2.
2. The isolated polypeptide of claim 1, wherein the isolated
polypeptide comprises amino acids 1 to 71 of the amino acid
sequence of SEQ ID NO:2.
3. The isolated polypeptide of claim 1, wherein the isolated
polypeptide comprises the amino acid sequence of SEQ ID NO:2.
4. The isolated polypeptide of claim 1, wherein the isolated
polypeptide has an amino acid sequence consisting of the amino acid
sequence of SEQ ID NO:2.
5. An isolated nucleic acid molecule, wherein the nucleic acid
molecule encodes an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of amino acid residues 7
to 71 of SEQ ID NO:2, (b) the amino acid sequence of amino acid
residues 1 to 71 of SEQ ID NO:2, and (c) the amino acid sequence of
SEQ ID NO:2.
6. The isolated nucleic acid molecule of claim 5, wherein the
nucleic acid molecule encodes an amino acid sequence comprising
amino acid residues 7 to 71 of SEQ ID NO:2.
7. The isolated nucleic acid molecule of claim 5, comprising the
nucleotide sequence of nucleotides 27 to 578 of SEQ ID NO:1, or
nucleotides 45 to 239 of SEQ ID NO:1.
8. A vector, comprising the isolated nucleic acid molecule of claim
7.
9. An expression vector, comprising a nucleic acid molecule that
encodes a polypeptide comprising amino acid residues 7 to 71 of SEQ
ID NO:2, a transcription promoter, and a transcription terminator,
wherein the promoter is operably linked with the nucleic acid
molecule, and wherein the nucleic acid molecule is operably linked
with the transcription terminator.
10. A recombinant host cell comprising the expression vector of
claim 9, wherein the host cell is selected from the group
consisting of bacterium, yeast cell, avian cell, fungal cell,
insect cell, mammalian cell, and plant cell.
11. A method of producing a polypeptide that comprises amino acid
residues 7 to 71 of SEQ ID NO:2, the method comprising culturing
recombinant host cells that comprise the expression vector of claim
9, and that produce the polypeptide.
12. The method of claim 11, further comprising isolating the
polypeptide from the cultured recombinant host cells.
13. An antibody or antibody fragment that specifically binds with
the polypeptide of claim 4.
14. The antibody of claim 13, wherein the antibody is selected from
the group consisting of: (a) polyclonal antibody, (b) murine
monoclonal antibody, (c) humanized antibody derived from (b), and
(d) human monoclonal antibody.
15. A fusion protein, comprising the polypeptide of claim 1.
16. The fusion protein of claim 15, wherein the fusion protein
comprises amino acid residues 1 to 71 of SEQ ID NO:2.
17. The fusion protein of claim 15, wherein the fusion protein
further comprises an immunoglobulin moiety.
18. The fusion protein of claim 16, wherein the fusion protein
further comprises an immunoglobulin moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/008,063 filed Nov. 5, 2001, which claims the benefit of U.S.
Provisional Application Ser. No. 60/315,565, filed Aug. 29, 2001,
U.S. Provisional Application Ser. No. 60/301,715, filed Jun. 28,
2001, U.S. Provisional Application Ser. No. 60/257,131, filed Dec.
20, 2000, and U.S. Provisional Application Ser. No. 60/246,449,
filed Nov. 7, 2000, all of which are herein incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates generally to a new protein
expressed by human cells. In particular, the present invention
relates to a novel gene that encodes a receptor, designated as
"Ztnfr12," and to nucleic acid molecules encoding Ztnfr12
polypeptides.
BACKGROUND OF THE INVENTION
[0003] Cytokines are soluble, small proteins that mediate a variety
of biological effects, including the regulation of the growth and
differentiation of many cell types (see, for example, Arai et al.,
Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol.
3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Proteins that
constitute the cytokine group include interleukins, interferons,
colony stimulating factors, tumor necrosis factors, and other
regulatory molecules. For example, human interleukin-17 is a
cytokine which stimulates the expression of interleukin-6,
intracellular adhesion molecule 1, interleukin-8, granulocyte
macrophage colony-stimulating factor, and prostaglandin E2
expression, and plays a role in the preferential maturation of
CD34+ hematopoietic precursors into neutrophils (Yao et al., J.
Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593
(1996)).
[0004] Receptors that bind cytokines are typically composed of one
or more integral membrane proteins that bind the cytokine with high
affinity and transduce this binding event to the cell through the
cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains. For
example, the receptor chains responsible for binding and/or
transducing the effect of interferons are members of the type II
cytokine receptor family, based upon a characteristic 200 residue
extracellular domain.
[0005] Cellular interactions, which occur during an immune
response, are regulated by members of several families of cell
surface receptors, including the tumor necrosis factor receptor
(TNFR) family. The TNFR family consists of a number of integral
membrane glycoprotein receptors many of which, in conjunction with
their respective ligands, regulate interactions between different
hematopoietic cell lineages (see, for example, Cosman, Stem Cells
12:440 (1994); Wajant et al., Cytokine Growth Factor Rev. 10:15
(1999); Yeh et al., Immunol. Rev. 169:283 (1999); Idriss and
Naismith, Microsc. Res. Tech. 50:184 (2000)).
[0006] One such receptor is TACI, transmembrane activator and
CAML-interactor (von Bulow and Bram, Science 228:138 (1997); PCT
publication WO 98/39361). TACI is a membrane bound receptor, which
has an extracellular domain containing two cysteine-rich
pseudo-repeats, a transmembrane domain and a cytoplasmic domain
that interacts with CAML (calcium-modulator and cyclophilin
ligand), an integral membrane protein located at intracellular
vesicles which is a co-inducer of NF-AT activation when
overexpressed in Jurkat cells. TACI is associated with B cells and
a subset of T cells.
[0007] The demonstrated in vivo activities of tumor necrosis factor
receptors illustrate the clinical potential of, and need for, other
such receptors, as well as tumor necrosis factor receptor agonists,
and antagonists.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides a novel tumor necrosis factor
receptor, designated "Ztnfr12." The present invention also provides
Ztnfr12 polypeptides and Ztnfr12 fusion proteins, as well as
nucleic acid molecules encoding such polypeptides and proteins, and
methods for using these nucleic acid molecules and amino acid
sequences.
BRIEF DESCRIPTION OF THE DRAWING
[0009] FIG. 1 is a schematic diagram of an immunoglobulin of the
IgG1 subclass. C.sub.L: light chain constant region; C.sub.H1,
C.sub.H2, C.sub.H3: heavy chain constant regions; V.sub.L: light
chain variable region; V.sub.H: heavy chain variable region; CHO:
carbohydrate; N: amino terminus; C: carboxyl terminus.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0010] ZTNF4 is a member of the tumor necrosis factor (TNF) ligand
family (SEQ ID NO:5). This molecule has also been designated as
"BAFF," "BLyS," "TALL-1," and "THANK" (Moore et al., Science
285:269 (1999); Mukhopadhyay et al., J. Biol. Chem. 274:15978
(1999); Schneider et al., J. Exp. Med. 189:1747 (1999); Shu et al.,
J. Leukoc. Biol. 65:680 (1999)). Two receptors have been identified
that bind with ZTNF4: the transmembrane activator and CAML
interactor (TACI) and the B-cell maturation receptor (BCMA) (Gross
et al., Nature 404:995 (2000)). Biotinylated ZTNF4 was used to
identify potential new receptors for this ligand. In these studies,
the binding of biotinylated ZTNF4 to a panel of tumor cells was
measured using flow cytometry. Surprisingly, ZTNF4 was found to
bind to a human B-lymphoid precursor cell line (REH cells), even
though there was little binding of the cells with either monoclonal
antibodies to TACI, or polyclonal antibodies to BCMA. Similar
results were found with BJAB cells, derived from a human lymphoma.
These observations suggested that ZTNF4 bound with a receptor other
than TACI or BCMA.
[0011] To investigate this possibility further, I.sup.125-labeled
ZTNF4 was bound to the B-lymphoid precursor cells, and crosslinked
to cell surface molecules. Treatment with anti-ZTNF4 polyclonal
antibodies produced a radioactive precipitate, whereas treatment
with anti-TACI or anti-BCMA polyclonal antibodies did not produce a
radioactive precipitate. Thus, this data supported the hypothesis
that a new receptor accounted for the binding of ZTNF4 to the
cells. The receptor, designated as "Ztnfr12," was isolated as
described in Example 1. Binding studies indicated that the new
receptor binds ZTNF4, but not a ligand designated as "ZTNF2" (SEQ
ID NO:6). ZTNF2 has also been designated as "APRIL" and "TNRF death
ligand-1" (Hahne et al., J. Exp. Med. 188:1185 (1998); Kelly et
al., Cancer Res. 60:1021 (2000)).
[0012] The binding characteristics of Ztnfr12 were also
investigated using recombinant host cells. Baby hamster kidney
cells were transfected with an expression vector that comprised
Ztnfr12 encoding sequences, and the transfected cells were used in
a binding study with I.sup.125-labeled ZTNF4. Binding studies and
Scatchard analyses indicated that the Kd of ZTNF4 for Ztnfr12 is
1.0 nM, which is comparable to the Kd of ZTNF4 for TACI receptor
(1.25 nM) expressed by baby hamster kidney cells transfected with a
TACI expression vector. The transfected cells expressed
approximately 150.times.10.sup.6 Ztnfr12 cell surface receptors per
cell.
[0013] ZTNF4 appears to bind to virtually all mature CD19.sup.+
peripheral B cells, weakly to immature B cells in the bone marrow,
and to most transformed B-cell lines. However, several B lymphomas,
REH and BJAB for example, bind ZTNF4 but do not express appreciable
levels of either TACI or BCMA. In addition, TACI and BCMA surface
expression and ZTNF4 binding were determined on cells isolated from
human tonsil, peripheral blood, and bone marrow using flow
cytometry. The results indicate that TACI and BCMA are expressed at
the highest levels on the most immature B cell population,
IgM.sup.+IgD.sup.lo, in human tonsil and peripheral blood.
Expression levels of both receptors decrease on the surface of the
more mature IgM.sup.+IgD.sup.+ B cells and are found at very low
levels on IgM.sup.-IgD.sup.+ B cells, a population that represents
the most mature stage of B cell maturation. However, ZTNF4 ligand
binds to virtually all mature B cells at high levels. Taken
together these data implicate the presence of additional receptors
for ZTNF4 on some B cell tumors and peripheral human B cells. These
data suggest that Ztnfr12 is expressed at highest levels on the
most mature IgM.sup.-IgD.sup.+B cells and may account for the high
levels of zTNF4 binding to this population.
[0014] An illustrative nucleotide sequence that encodes Ztnfr12 is
provided by SEQ ID NO:1. The encoded polypeptide has the following
amino acid sequence: MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT
PRPKPAGASS PAPRTALQPQ ESVGAGAGEA ALPLPGLLFG APALLGLALV LALVLVGLVS
WRRRQRRLRG ASSAEAPDGD KDAPEPLDKV IILSPGISDA TAPAWPPPGE DPGTTPPGHS
VPVPATELGS TELVTTKTAG PEQQ (SEQ ID NO:2). Features of the Ztnfr12
polypeptide include an extracellular domain that comprises amino
acid residues 1 to 69 of SEQ ID NO:2 or amino acid residues 1 to 79
of SEQ ID NO:2, a transmembrane domain that comprises amino acid
residues 70 to 100 of SEQ ID NO:2 or amino acid residues 80 to 100
of SEQ ID NO:2, and an intracellular domain at about amino acid
residues 101 to 184 of SEQ ID NO:2.
[0015] A nucleotide sequence that includes the Ztnfr12 gene is
provided by SEQ ID NO:9. The Ztnfr12 gene comprises three exons.
With reference to the amino acid sequence of SEQ ID NO:2, exon 1
encodes amino acid residues 1 to the first nucleotide of the codon
for amino acid 46, exon 2 encodes the remainder of amino acid 46 to
the first nucleotide of the codon for amino acid 123, and exon 3
encodes the remainder of amino acid 123 to amino acid 184. The
3'-untranslated region includes nucleotides 2405 to about 5720 of
SEQ ID NO:9. Table 1 provides further features of this nucleotide
sequence. TABLE-US-00001 TABLE 1 Corresponding region Feature SEQ
ID NO: 9 of SEQ ID NO: 1 Exon 1 1001-1136 27-162 Intron 1 1137-1442
Exon 2 1443-1673 163-393 Intron 2 1674-2219 Exon 3 2220-2404
394-578
[0016] The Ztnfr12 gene resides in chromosome 22q13.2, and Ztnfr12
is expressed in most lymph tissues (e.g. lymphoid node tissue),
B-cell tumors, and germinal center B-cells. Northern and dot blot
analyses revealed that Ztnfr12 gene expression is detectable in
spleen, lymph node, peripheral blood lymphocytes, kidney, heart,
liver, skeletal muscle, pancreas, adrenal gland, testis, brain,
uterus, stomach, bone marrow, trachea thymus, placenta, fetal liver
and Raji cells. The strongest signals were associated with spleen
and lymph node tissues, whereas weak signals were associated with
brain, uterine, and placental tissue. Accordingly, Ztnfr12
antibodies and nucleic acid probes can be used to differentiate
between these tissues.
[0017] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 80%, or at least 90% identical to a reference amino
acid sequence selected from the group consisting of: (a) amino acid
residues 7 to 69 of SEQ ID NO:2, (b) amino acid residues 7 to 79 of
SEQ ID NO:2, (c) amino acid residues 7 to 39 of SEQ ID NO:2, (d)
amino acid residues 19 to 35 of SEQ ID NO:2, (e) amino acid
residues 1 to 69 of SEQ ID NO:2, (f) amino acid residues 1 to 79 of
SEQ ID NO:2, (g) amino acid residues 1 to 39 of SEQ ID NO:2, (h)
amino acid residues 1 to 71 of SEQ ID NO:2, (i) amino acid residues
7 to 71 of SEQ ID NO:2, (j) amino acid residues 70 to 100 of SEQ ID
NO:2, (k) amino acid residues 80 to 100 of SEQ ID NO:2, (1) amino
acid residues 101 to 184 of SEQ ID NO:2, and (m) the amino acid
sequence of SEQ ID NO:2. Certain Ztnfr12 polypeptides specifically
bind with an antibody that specifically binds with a polypeptide
consisting of the amino acid sequence of SEQ ID NO:2. Certain
Ztnfr12 polypeptides specifically bind ZTNF4, while other
polypeptides specifically bind ZTNF4 but do not specifically bind
ZTNF2. Illustrative Ztnfr12 polypeptides include polypeptides
comprising, or consisting of, amino acid residues 7 to 69 of SEQ ID
NO:2, amino acid residues 7 to 79 of SEQ ID NO:2, amino acid
residues 7 to 39 of SEQ ID NO:2, amino acid residues 19 to 35 of
SEQ ID NO:2, amino acid residues 1 to 69 of SEQ ID NO:2, amino acid
residues 1 to 79 of SEQ ID NO:2, amino acid residues 1 to 71 of SEQ
ID NO:2, amino acid residues 7 to 71 of SEQ ID NO:2, amino acid
residues 1 to 39 of SEQ ID NO:2, amino acid residues 80 to 100 of
SEQ ID NO:2, amino acid residues 70 to 100 of SEQ ID NO:2, amino
acid residues 101 to 184 of SEQ ID NO:2, and the amino acid
sequence of SEQ ID NO:2. The present invention also provides
isolated polypeptides comprising at least 15, or at least 30,
contiguous amino acid residues of amino acid residues 7 to 69 of
SEQ ID NO:2, amino acid residues 7 to 79 of SEQ ID NO:2, amino acid
residues 7 to 39 of SEQ ID NO:2, amino acid residues 19 to 35 of
SEQ ID NO:2, amino acid residues 1 to 69 of SEQ ID NO:2, amino acid
residues 1 to 79 of SEQ ID NO:2, amino acid residues 1 to 71 of SEQ
ID NO:2, amino acid residues 7 to 71 of SEQ ID NO:2, or amino acid
residues 1 to 39 of SEQ ID NO:2.
[0018] The present invention further provides polypeptides encoded
by at least one Ztnfr12 exon. For example, such polypeptides can
consist of the following amino acid sequences of SEQ ID NO:2: amino
acid residues 1 to 45, amino acid residues 47 to 122, and amino
acid residues 124 to 184.
[0019] The present invention also includes variant Ztnfr12
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with amino acid residues 7 to 69 of
SEQ ID NO:2, amino acid residues 7 to 79 of SEQ ID NO:2, amino acid
residues 7 to 39 of SEQ ID NO:2, amino acid residues 19 to 35 of
SEQ ID NO:2, amino acid residues 1 to 69 of SEQ ID NO:2, amino acid
residues 1 to 79 of SEQ ID NO:2, amino acid residues 1 to 39 of SEQ
ID NO:2, amino acid residues 1 to 71 of SEQ ID NO:2, amino acid
residues 7 to 71 of SEQ ID NO:2, or amino acid residues 1 to 184 of
SEQ ID NO:2, selected from the group consisting of at least 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, or greater than 95% identity, and wherein any
difference between the amino acid sequence of the variant
polypeptide and the corresponding amino acid sequence of SEQ ID
NO:2 is due to one or more conservative amino acid
substitutions.
[0020] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include polyclonal antibodies, murine
monoclonal antibodies, humanized antibodies derived from murine
monoclonal antibodies, and human monoclonal antibodies.
Illustrative antibody fragments include F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, scFv, and minimal recognition units. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, antibody, or anti-idiotype antibody described
herein.
[0021] The present invention also provides isolated nucleic acid
molecules that encode a Ztnfr12 polypeptide, wherein the nucleic
acid molecule is selected from the group consisting of: (a) a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:3, (b) a nucleic acid molecule encoding an amino acid sequence
that comprises amino acid residues 7 to 69 of SEQ ID NO:2, amino
acid residues 7 to 79 of SEQ ID NO:2, amino acid residues 7 to 39
of SEQ ID NO:2, amino acid residues 19 to 35 of SEQ ID NO:2, amino
acid residues 1 to 69 of SEQ ID NO:2, amino acid residues 1 to 79
of SEQ ID NO:2, amino acid residues 1 to 39 of SEQ ID NO:2, amino
acid residues 1 to 71 of SEQ ID NO:2, amino acid residues 7 to 71
of SEQ ID NO:2, or amino acid residues 1 to 184 of SEQ ID NO:2, and
(c) a nucleic acid molecule that remains hybridized following
stringent wash conditions to a nucleic acid molecule comprising the
nucleotide sequence of nucleotides 27 to 578 of SEQ ID NO:1, the
nucleotide sequence of nucleotides 27 to 233 of SEQ ID NO:1, the
complement of the nucleotide sequence of nucleotides 27 to 578 of
SEQ ID NO:1, or the complement of the nucleotide sequence of
nucleotides 27 to 233 of SEQ ID NO:1. Illustrative nucleic acid
molecules include those in which any difference between the amino
acid sequence encoded by nucleic acid molecule (c) and the
corresponding amino acid sequence of SEQ ID NO:2 is due to a
conservative amino acid substitution.
[0022] The present invention further contemplates isolated nucleic
acid molecules that comprise nucleotides 27 to 578 of SEQ ID NO:1
(which encodes amino acid residues 1 to 184 of SEQ ID NO:2),
nucleotides 27 to 233 of SEQ ID NO:1 (which encodes amino acid
residues 1 to 69 of SEQ ID NO:2), nucleotides 27 to 263 of SEQ ID
NO:1 (which encodes amino acid residues 1 to 79 of SEQ ID NO:2),
nucleotides 45 to 233 of SEQ ID NO:1 (which encodes amino acid
residues 7 to 69 of SEQ ID NO:2), nucleotides 45 to 263 of SEQ ID
NO:1 (which encodes amino acid residues 7 to 79 of SEQ ID NO:2),
nucleotides 45 to 143 of SEQ ID NO:1 (which encodes amino acid
residues 7 to 39 of SEQ ID NO:2), nucleotides 81 to 131 of SEQ ID
NO:1 (which encodes amino acid residues 19 to 35 of SEQ ID NO:2),
nucleotides 27 to 239 of SEQ ID NO:1 (which encodes amino acid
residues 1 to 71 of SEQ ID NO:2), nucleotides 45 to 239 of SEQ ID
NO:1 (which encodes amino acid residues 7 to 71 of SEQ ID NO:2),
and nucleotides 327 to 578 of SEQ ID NO:1 (which encodes amino acid
residues 101 to 184 of SEQ ID NO:2).
[0023] The present invention also provides nucleic acid molecules
that consist of the nucleotide sequence of a Ztnfr12 exon or
intron. The nucleotide sequences of these exons and introns are
identified in Table 1.
[0024] The present invention also includes vectors and expression
vectors comprising such nucleic acid molecules. Such expression
vectors may comprise a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator. The present
invention further includes recombinant host cells and recombinant
viruses comprising these vectors and expression vectors.
Illustrative host cells include bacterial, avian, yeast, fungal,
insect, mammalian, and plant cells. Recombinant host cells
comprising such expression vectors can be used to produce Ztnfr12
polypeptides by culturing such recombinant host cells that comprise
the expression vector and that produce the Ztnfr12 protein, and,
optionally, isolating the Ztnfr12 protein from the cultured
recombinant host cells. The present invention further includes the
products of such processes.
[0025] The present invention also provides polypeptides comprising
amino acid residues 1 to 69 of SEQ ID NO:13, polypeptides
comprising at least 10, at least 15, at least 20, at least 25, or
at least 30 consecutive amino acid residues of amino acid residues
1 to 69 of SEQ ID NO:13, polypeptides comprising amino acid
residues 21 to 38 of SEQ ID NO:13, fusion proteins comprising amino
acid residues 1 to 69 of SEQ ID NO:13, nucleic acid molecules
encoding such amino acid sequences, expression vectors comprising
such nucleic acid molecules, and recombinant host cells comprising
such expression vectors. The present invention further includes
methods for producing murine Ztnfr12 polypeptides using such
recombinant host cells.
[0026] An alignment of the amino acid sequences of TACI, BCMA,
human Ztnfr12, and murine Ztnfr12 revealed the following motif in
the extracellular domains:
C[NVPS][QPE][TAEN][EQ][CY][FW]D[PLS]L[VL][RGH][NHTA]C[VMI][SAP]C,
wherein acceptable amino acids for a given position are indicated
within square brackets (SEQ ID NO:46). The present invention
includes polypeptides having an amino acid sequence that consists
of this motif, wherein the polypeptides bind ZTNF4. The present
invention also includes antibodies that bind to a polypeptide
having an amino acid sequence that consists of this motif.
[0027] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors. The present invention further
includes pharmaceutical compositions, comprising a pharmaceutically
acceptable carrier and a polypeptide described herein.
[0028] The present invention also contemplates methods for
detecting the presence of Ztnfr12 RNA in a biological sample,
comprising the steps of (a) contacting a Ztnfr12 nucleic acid probe
under hybridizing conditions with either (i) test RNA molecules
isolated from the biological sample, or (ii) nucleic acid molecules
synthesized from the isolated RNA molecules, wherein the probe has
a nucleotide sequence comprising a portion of the nucleotide
sequence of SEQ ID NO:1, or its complement, and (b) detecting the
formation of hybrids of the nucleic acid probe and either the test
RNA molecules or the synthesized nucleic acid molecules, wherein
the presence of the hybrids indicates the presence of Ztnfr12 RNA
in the biological sample. For example, suitable probes consist of
the following nucleotide sequences: nucleotides 27 to 578 of SEQ ID
NO:1, and nucleotides 27 to 233 of SEQ ID NO:1. Other suitable
probes consist of the complement of these nucleotide sequences, or
a portion of the nucleotide sequences as described herein, or their
complements.
[0029] The present invention further provides methods for detecting
the presence of Ztnfr12 polypeptide in a biological sample,
comprising the steps of: (a) contacting the biological sample with
an antibody or an antibody fragment that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2,
wherein the contacting is performed under conditions that allow the
binding of the antibody or antibody fragment to the biological
sample, and (b) detecting any of the bound antibody or bound
antibody fragment. Such an antibody or antibody fragment may
further comprise a detectable label selected from the group
consisting of radioisotope, fluorescent label, chemiluminescent
label, enzyme label, bioluminescent label, and colloidal gold.
[0030] The present invention also provides kits for performing
these detection methods. For example, a kit for detection of
Ztnfr12 gene expression may comprise a container that comprises a
nucleic acid molecule, wherein the nucleic acid molecule is
selected from the group consisting of: (a) a nucleic acid molecule
comprising the nucleotide sequence of nucleotides 27 to 233 of SEQ
ID NO:1, (b) a nucleic acid molecule comprising the complement of
nucleotides 27 to 233 of the nucleotide sequence of SEQ ID NO:1,
and (c) a nucleic acid molecule that is a fragment of (a) or (b)
consisting of at least eight nucleotides. Such a kit may also
comprise a second container that comprises one or more reagents
capable of indicating the presence of the nucleic acid molecule. On
the other hand, a kit for detection of Ztnfr12 protein may comprise
a container that comprises an antibody, or an antibody fragment,
that specifically binds with a polypeptide consisting of the amino
acid sequence of SEQ ID NO:2.
[0031] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
An exemplary anti-idiotype antibody binds with an antibody that
specifically binds a polypeptide consisting of amino acid residues
1 to 69 of SEQ ID NO:2, amino acid residues 1 to 79 of SEQ ID NO:2,
amino acid residues 7 to 69 of SEQ ID NO:2, amino acid residues 7
to 79 of SEQ ID NO:2, amino acid residues 1 to 71 of SEQ ID NO:2,
or amino acid residues 7 to 71 of SEQ ID NO:2.
[0032] The present invention also provides fusion proteins,
comprising a Ztnfr12 polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.c
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0033] The present invention further includes methods for
inhibiting, in a mammal, the activity of a ligand that binds
Ztnfr12 (e.g., ZTNF4), comprising administering to the mammal a
composition comprising at least one of: (a) soluble Ztnfr12
receptor, (b) an antibody or antibody fragment which specifically
binds with the extracellular domain of Ztnfr12, and (c) a fusion
protein comprising the extracellular domain of Ztnfr12. As an
illustration, such a composition can be used to treat disorders and
diseases associated with B lymphocytes, activated B lymphocytes, or
resting B lymphocytes. Examples of B cell lymphomas that may be
treated with the molecules described herein include Burkitt's
lymphoma, Non-Burkitt's lymphoma, Non-Hodgkin's lymphoma, multiple
myeloma, follicular lymphoma, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, large cell lymphoma, marginal zone lymphoma,
mantle cell lymphoma, large cell lymphoma (e.g., immunoblastic
lymphoma), small lymphocytic lymphoma, and other B cell lymphomas.
Such compositions can also be used to treat T cell lymphomas,
including lymphoblastic lymphoma, anaplastic large cell lymphoma,
cutaneous T cell lymphoma, peripheral T cell lymphomas,
angioimmunoblastic lymphoma, angiocentric lymphoma, intestinal T
cell lymphoma, adult T cell lymphoma, adult T cell leukemia, and
the like.
[0034] For example, the present invention includes methods for
inhibiting the proliferation of tumor cells (e.g., B cell lymphoma
cells or T cell lymphoma cells), comprising administering to the
tumor cells a composition that comprises at least one of: (a)
soluble Ztnfr12 receptor, (b) an antibody or antibody fragment
which specifically binds with the extracellular domain of Ztnfr12,
and (c) a fusion protein comprising the extracellular domain of
Ztnfr12. Such a composition can be administered to cells cultured
in vitro. Alternatively, the composition can be a pharmaceutical
composition, and wherein the pharmaceutical composition is
administered to a subject, which has a tumor.
[0035] One example of a fusion protein is a Ztnfr12-immunoglobulin
fusion protein that comprises the extracellular domain of Ztnfr12
is a Ztnfr12 polypeptide the comprises a fragment of a polypeptide
comprising amino acid residues 1 to 69 of SEQ ID NO:2, and an
immunoglobulin moiety comprising a constant region of an
immunoglobulin. An illustrative immunoglobulin moiety comprises a
heavy chain constant region. A Ztnfr12-immunoglobulin fusion
protein can be a monomer, a dimer, or other configuration, as
discussed below.
[0036] In another example, a composition that comprises an
anti-Ztnfr12 antibody component is administered to tumor cells to
inhibit the proliferation of the cells. The composition can be
administered to cells cultured in vitro, or the composition can be
a pharmaceutical composition that is administered to a subject,
which has a tumor. Such compositions can comprise an anti-Ztnf12
antibody component that is a naked Ztnf12 antibody, or such
compositions can comprise an anti-Ztnf12 antibody component that is
a naked Ztnf12 antibody fragment. Moreover, the composition can
comprise an immunoconjugate that comprises an anti-Ztnf12 antibody
component and a therapeutic agent. Illustrative therapeutic agents
include a chemotherapeutic drug, cytotoxin, immunomodulator,
chelator, boron compound, photoactive agent, photoactive dye, and
radioisotope. Such compositions may comprise an antibody fusion
protein that comprises an anti-Ztnfr12 antibody component and
either an immunomodulator or a cytotoxic polypeptide. Another form
of composition is a multispecific antibody, which comprises an
anti-Ztnf12 naked antibody component, and at least one of an
anti-BCMA naked antibody component, and an anti-TACI naked antibody
component. An illustrative multispecific antibody composition
comprises bispecific antibodies that bind Ztnfr12, and at least one
of BCMA and TACI. Multispecific antibody compositions can further
comprise a therapeutic agent. Moreover, a multispecific antibody
composition can comprise: (a) an anti-Ztnfr12 antibody fusion
protein that comprises either an immunomodulator or a cytotoxic
polypeptide, and (b) at least one of an anti-BCMA antibody
component or an anti-TACI antibody component.
[0037] Polypeptides comprising a Ztnfr12 extracellular domain or
anti-Ztnfr12 antibodies can be used to treat an autoimmune disease.
Examples of autoimmune diseases include systemic lupus
erythomatosis, myasthenia gravis, multiple sclerosis, insulin
dependent diabetes mellitus, and rheumatoid arthritis. Polypeptides
comprising a Ztnfr12 extracellular domain or anti-Ztnfr12
antibodies can also be used to treat asthma, bronchitis, emphysema,
and end stage renal failure or renal disease. Illustrative renal
diseases include glomerulonephritis, vasculitis, chronic lymphoid
leukemia, nephritis, and pyelonephritis. Polypeptides comprising a
Ztnfr12 extracellular domain or anti-Ztnfr12 antibodies can further
be used to treat renal neoplasms, multiple myelomas, lymphomas,
light chain neuropathy, or amyloidosis.
[0038] The present invention also includes methods for inhibiting
ZTNF4 activity, wherein the ZTNF4 activity is associated with
effector T cells. Within a related method, the ZTNF4 activity is
associated with regulating immune response. Within another method,
the ZTNF4 activity is associated with immunosuppression. Within yet
another method, the immunosuppression is associated with graft
rejection, graft verses host disease, or inflammation. Within still
another method, the immunosuppression is associated with autoimmune
disease. As an illustration, the autoimmune disease may be
insulin-dependent diabetes mellitus or Crohn's disease. In yet
other methods, immunosuppression is associated with inflammation.
Such inflammation can be associated with, for example, joint pain,
swelling, anemia, or septic shock.
[0039] The present invention also includes methods for detecting a
chromosome 22q13.2 abnormality in a subject by (a) amplifying, from
genomic DNA isolated from a biological sample of the subject,
nucleic acid molecules that either (i) comprise a nucleotide
sequence that encodes at least one of Ztnfr12 exons 1 to 3, or that
(ii) comprise a nucleotide sequence that is the complement of (i),
and (b) detecting a mutation in the amplified nucleic acid
molecules, wherein the presence of a mutation indicates a
chromosome 22q13.2 abnormality. In variations of these methods, the
detecting step is performed by comparing the nucleotide sequence of
the amplified nucleic acid molecules to the nucleotide sequence of
SEQ ID NO:1 or SEQ ID NO:9.
[0040] The present invention further provides methods for detecting
a chromosome 22q13.2 abnormality in a subject comprising: (a)
amplifying, from genomic DNA isolated from a biological sample of
the subject, a segment of the Ztnfr12 gene that comprises either
the nucleotide sequence of at least one of intron 1 and intron 2,
or the complementary nucleotide sequence of at least one of intron
1 and intron 2, and (b) detecting a mutation in the amplified
nucleic acid molecules, wherein the presence of a mutation
indicates a chromosome 22q13.2 abnormality. In variations of these
methods, the detecting step is performed by binding the amplified
Ztnfr12 gene segments to a membrane, and contacting the membrane
with a nucleic acid probe under hybridizing conditions of high
stringency, wherein the absence of hybrids indicates an abnormality
associated with the expression of Ztnfr12, or a mutation in
chromosome 22q13.2. As an illustration, a suitable nucleic acid
probe can comprise the nucleotide sequence (or the complement of
the nucleotide sequence) of any one of introns 1 and 2.
[0041] Examples of mutations or alterations of the Ztnfr12 gene or
its gene products include point mutations, deletions, insertions,
and rearrangements. Another example of a Ztnfr12 gene mutation is
aneuploidy. The present invention also includes methods for
detecting a chromosome 22q13.2 abnormality in a subject comprising
the identification of an alteration in the region upstream of the
first exon of the Ztnfr12 gene (e.g., within nucleotides 1 to 1000
of SEQ ID NO:9) using the detection methods described herein.
[0042] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. Definitions
[0043] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0044] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0045] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0046] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0047] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid molecule that encodes a
polypeptide. Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0048] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0049] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0050] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0051] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0052] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0053] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SPI, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0054] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0055] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0056] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0057] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0058] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0059] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0060] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0061] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0062] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, which has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0063] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0064] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces Ztnfr12 from an expression vector. In
contrast, Ztnfr12 can be produced by a cell that is a "natural
source" of Ztnfr12, and that lacks an expression vector.
[0065] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0066] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a Ztnfr12-immunoglobulin fusion protein
comprises a Ztnfr12 receptor moiety and an immunoglobulin moiety.
As used herein, a "Ztnfr12 receptor moiety" is a portion of the
extracellular domain of the Ztnfr12 receptor that binds at least
one of ZTNF2 or ZTNF4. The phrase an "immunoglobulin moiety" refers
to a polypeptide that comprises a constant region of an
immunoglobulin. For example, the immunoglobulin moiety can comprise
a heavy chain constant region. The term "Ztnfr12-Fc" fusion protein
refers to a Ztnfr12-immunoglobulin fusion protein in which the
immunoglobulin moiety comprises immunoglobulin heavy chain constant
regions, C.sub.H2 and C.sub.H3.
[0067] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. In the context of
Ztnfr12 receptor binding, the phrase "specifically binds" or
"specific binding" refers to the ability of the ligand to
competitively bind with the receptor. For example, ZTNF4
specifically binds with the Ztnfr12 receptor, and this can be shown
by observing competition for the Ztnfr12 receptor between
detectably labeled ZTNF4 and unlabeled ZTNF4.
[0068] Receptors can be membrane bound, cytosolic or nuclear;
monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor).
Membrane-bound receptors are characterized by a multi-domain
structure comprising an extracellular ligand-binding domain and an
intracellular effector domain that is typically involved in signal
transduction. In certain membrane-bound receptors, the
extracellular ligand-binding domain and the intracellular effector
domain are located in separate polypeptides that comprise the
complete functional receptor.
[0069] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0070] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0071] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0072] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0073] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0074] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0075] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and synthetic analogs of these
molecules.
[0076] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of less than 10.sup.9 M.sup.-1.
[0077] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-Ztnfr12 antibody, and thus, an anti-idiotype antibody
mimics an epitope of Ztnfr12.
[0078] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-Ztnfr12
monoclonal antibody fragment binds with an epitope of Ztnfr12.
[0079] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0080] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0081] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0082] As used herein, a "therapeutic agent" is a molecule or atom,
which is conjugated to an antibody moiety to produce a conjugate,
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0083] A "detectable label" is a molecule or atom, which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0084] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith
and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). DNA molecules encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0085] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0086] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0087] A "bispecific antibody" has binding sites for two different
antigens within a single antibody molecule.
[0088] A "multispecific antibody composition" comprises antibody
components that have binding sites for two different antigens. For
example, a multispecific antibody composition can comprise
bispecific antibody components, or a multispecific antibody
composition can comprise at least two antibody components that bind
with different antigens.
[0089] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0090] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
Ztnfr12 polypeptide component. Examples of an antibody fusion
protein include a protein that comprises a Ztnfr12 extracellular
domain, and either an Fc domain or an antigen-biding region.
[0091] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0092] An "antigenic peptide" is a peptide, which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex,
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell, which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0093] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0094] An "anti-sense oligonucleotide specific for Ztnfr12" or a
"Ztnfr12 anti-sense oligonucleotide" is an oligonucleotide having a
sequence (a) capable of forming a stable triplex with a portion of
the Ztnfr12 gene, or (b) capable of forming a stable duplex with a
portion of an mRNA transcript of the Ztnfr12 gene.
[0095] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0096] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0097] The term "variant Ztnfr12 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2. Such variants include
naturally-occurring polymorphisms of Ztnfr12 genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:2. Additional variant forms
of Ztnfr12 genes are nucleic acid molecules that contain insertions
or deletions of the nucleotide sequences described herein. A
variant Ztnfr12 gene can be identified, for example, by determining
whether the gene hybridizes with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1, or its complement, under
stringent conditions.
[0098] Alternatively, variant Ztnfr12 genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0099] A variant Ztnfr12 gene or variant Ztnfr12 polypeptide, a
variant gene or polypeptide encoded by a variant gene may be
functionally characterized by at least one of: the ability to bind
specifically to an anti-Ztnfr12 antibody, the ability to
specifically bind ZTNF4, and the ability to specifically bind
ZTNF4, but not ZTNF2.
[0100] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0101] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0102] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0103] The present invention includes functional fragments of
Ztnfr12 genes. Within the context of this invention, a "functional
fragment" of a Ztnfr12 gene refers to a nucleic acid molecule that
encodes a portion of a Ztnfr12 polypeptide, which is a domain
described herein, or can be characterized by at least one of: the
ability to bind specifically to an anti-Ztnfr12 antibody, the
ability to specifically bind ZTNF4, and the ability to specifically
bind ZTNF4, but not ZTNF2.
[0104] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
3. Production of Nucleic Acid Molecules Encoding Ztnfr12
[0105] Nucleic acid molecules encoding a human Ztnfr12 gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NOs:1 or 9. These
techniques are standard and well-established.
[0106] As an illustration, a nucleic acid molecule that encodes a
human Ztnfr12 gene can be isolated from a cDNA library. In this
case, the first step would be to prepare the cDNA library by
isolating RNA from, for example, germinal center B-cells or lymph
node tissue, using methods well-known to those of skill in the art.
In general, RNA isolation techniques must provide a method for
breaking cells, a means of inhibiting RNase-directed degradation of
RNA, and a method of separating RNA from DNA, protein, and
polysaccharide contaminants. For example, total RNA can be isolated
by freezing tissue in liquid nitrogen, grinding the frozen tissue
with a mortar and pestle to lyse the cells, extracting the ground
tissue with a solution of phenol/chloroform to remove proteins, and
separating RNA from the remaining impurities by selective
precipitation with lithium chloride (see, for example, Ausubel et
al. (eds.), Short Protocols in Molecular Biology, 3.sup.rd Edition,
pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)"];
Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press,
Inc. 1997) ["Wu (1997)"]).
[0107] Alternatively, total RNA can be isolated by extracting
ground tissue with guanidinium isothiocyanate, extracting with
organic solvents, and separating RNA from contaminants using
differential centrifugation (see, for example, Chirgwin et al.,
Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu
(1997) at pages 33-41).
[0108] In order to construct a cDNA library, poly(A).sup.+ RNA must
be isolated from a total RNA preparation. Poly(A).sup.+ RNA can be
isolated from total RNA using the standard technique of
oligo(dT)-cellulose chromatography (see, for example, Aviv and
Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at
pages 4-11 to 4-12).
[0109] Double-stranded cDNA molecules are synthesized from
poly(A).sup.+ RNA using techniques well-known to those in the art.
(see, for example, Wu (1997) at pages 41-46). Moreover,
commercially available kits can be used to synthesize
double-stranded cDNA molecules. For example, such kits are
available from Life Technologies, Inc. (Gaithersburg, Md.),
CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Promega
Corporation (Madison, Wis.) and STRATAGENE (La Jolla, Calif.).
[0110] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lamda.gt10 vector.
See, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lamda.gt10 and .lamda.gt11," in DNA Cloning: A
Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52.
[0111] Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a PBLUESCRIPT vector
(STRATAGENE; La Jolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or
other commercially available vectors. Suitable cloning vectors also
can be obtained from the American Type Culture Collection
(Manassas, Va.).
[0112] To amplify the cloned cDNA molecules, the cDNA library is
inserted into a prokaryotic host, using standard techniques. For
example, a cDNA library can be introduced into competent E. coliDH5
cells, which can be obtained, for example, from Life Technologies,
Inc. (Gaithersburg, Md.).
[0113] A human genomic library can be prepared by means well-known
in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6;
Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing
tissue with the detergent Sarkosyl, digesting the lysate with
proteinase K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the lysate using
isopropanol, and purifying resuspended DNA on a cesium chloride
density gradient.
[0114] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well-known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0115] Alternatively, human genomic libraries can be obtained from
commercial sources such as ResGen (Huntsville, Ala.) and the
American Type Culture Collection (Manassas, Va.).
[0116] A library containing cDNA or genomic clones can be screened
with one or more polynucleotide probes based upon SEQ ID NO:1,
using standard methods (see, for example, Ausubel (1995) at pages
6-1 to 6-11).
[0117] Nucleic acid molecules that encode a human Ztnfr12 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the Ztnfr12 gene, as described
herein. General methods for screening libraries with PCR are
provided by, for example, Yu et al., "Use of the Polymerase Chain
Reaction to Screen Phage Libraries," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover,
techniques for using PCR to isolate related genes are described by,
for example, Preston, "Use of Degenerate Oligonucleotide Primers
and the Polymerase Chain Reaction to Clone Gene Family Members," in
Methods in Molecular Biology, Vol. 15: PCR Protocols: Current
Methods and Applications, White (ed.), pages 317-337 (Humana Press,
Inc. 1993).
[0118] Anti-Ztnfr12 antibodies, produced as described below, can
also be used to isolate DNA sequences that encode human Ztnfr12
genes from cDNA libraries. For example, the antibodies can be used
to screen .lamda.gt11 expression libraries, or the antibodies can
be used for immunoscreening following hybrid selection and
translation (see, for example, Ausubel (1995) at pages 6-12 to
6-16; Margolis et al., "Screening .lamda. expression libraries with
antibody and protein probes," in DNA Cloning 2: Expression Systems,
2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University
Press 1995)).
[0119] As an alternative, a Ztnfr12 gene can be obtained by
synthesizing nucleic acid molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)).
[0120] The nucleic acid molecules of the present invention can also
be synthesized with "gene machines" using protocols such as the
phosphoramidite method. If chemically-synthesized double stranded
DNA is required for an application such as the synthesis of a gene
or a gene fragment, then each complementary strand is made
separately. The production of short genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. For reviews on polynucleotide synthesis,
see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles and Applications of Recombinant DNA (ASM Press 1994),
Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and Climie et
al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0121] The sequence of a Ztnfr12 cDNA or Ztnfr12 genomic fragment
can be determined using standard methods. Ztnfr12 polynucleotide
sequences disclosed herein can also be used as probes or primers to
clone 5' non-coding regions of a Ztnfr12 gene. Promoter elements
from a Ztnfr12 gene can be used to direct the expression of
heterologous genes in lymph node tissue, for example, transgenic
animals or patients treated with gene therapy. Such a promoter
element can be provided by a fragment consisting of 50, 100, 200,
400, or 600 nucleotides of nucleotides 1 to 1000 of SEQ ID NO:9.
Alternatively, a Ztnfr12 gene promoter may be provided by
nucleotides 1 to 1000 of SEQ ID NO:9. The identification of genomic
fragments containing a Ztnfr12 promoter or regulatory element can
be achieved using well-established techniques, such as deletion
analysis (see, generally, Ausubel (1995)).
[0122] Cloning of 5' flanking sequences also facilitates production
of Ztnfr12 proteins by "gene activation," as disclosed in U.S. Pat.
No. 5,641,670. Briefly, expression of an endogenous Ztnfr12 gene in
a cell is altered by introducing into the Ztnfr12 locus a DNA
construct comprising at least a targeting sequence, a regulatory
sequence, an exon, and an unpaired splice donor site. The targeting
sequence is a Ztnfr12 5' non-coding sequence that permits
homologous recombination of the construct with the endogenous
Ztnfr12 locus, whereby the sequences within the construct become
operably linked with the endogenous Ztnfr12 coding sequence. In
this way, an endogenous Ztnfr12 promoter can be replaced or
supplemented with other regulatory sequences to provide enhanced,
tissue-specific, or otherwise regulated expression.
4. Production of Ztnfr12 Variants
[0123] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, which encode the
Ztnfr12 polypeptides disclosed herein. Those skilled in the art
will readily recognize that, in view of the degeneracy of the
genetic code, considerable sequence variation is possible among
these polynucleotide molecules. SEQ ID NO:3 is a degenerate
nucleotide sequence that encompasses all nucleic acid molecules
that encode the Ztnfr12 polypeptide of SEQ ID NO:2. Those skilled
in the art will recognize that the degenerate sequence of SEQ ID
NO:3 also provides all RNA sequences encoding SEQ ID NO:2, by
substituting U for T. Thus, the present invention contemplates
Ztnfr12 polypeptide-encoding nucleic acid molecules comprising
nucleotide 27 to nucleotide 578 of SEQ ID NO:1, and their RNA
equivalents.
[0124] Table 2 sets forth the one-letter codes used within SEQ ID
NO:3 to denote degenerate nucleotide positions. "Resolutions" are
the nucleotides denoted by a code letter. "Complement" indicates
the code for the complementary nucleotide(s). For example, the code
Y denotes either C or T, and its complement R denotes A or G, A
being complementary to T, and G being complementary to C.
TABLE-US-00002 TABLE 2 Nucleotide Resolution Complement Resolution
A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T
K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G
V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0125] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table 3.
TABLE-US-00003 TABLE 3 One Amino Letter Degenerate Acid Code Codons
Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA
ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E
GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA
CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT
ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe
F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter .cndot. TAA TAG
TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0126] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0127] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (See Table 3). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0128] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts from other
species (orthologs). These species include, but are not limited to
mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and invertebrate species. As an illustration, SEQ ID
NO:12, SEQ ID NO:13, and SEQ ID NO:14 provide the nucleotide, amino
acid, and degenerate nucleotide sequences, respectively, of murine
Ztnfr12. Features of the murine Ztnfr12 polypeptide include an
extracellular domain at amino acid residues 1 to 69 of SEQ ID
NO:13, a transmembrane domain at amino acid residues 70 to 96 of
SEQ ID NO:13, an intracellular domain at amino acid residues 97 to
175 of SEQ ID NO:13, and a cys-rich region at amino acid residues
21 to 138 of SEQ ID NO:13.
[0129] Of particular interest are Ztnfr12 polypeptides from other
mammalian species, including mouse, porcine, ovine, bovine, canine,
feline, equine, and other primate polypeptides. Orthologs of human
Ztnfr12 can be cloned using information and compositions provided
by the present invention in combination with conventional cloning
techniques. For example, a Ztnfr12 cDNA can be cloned using mRNA
obtained from a tissue or cell type that expresses Ztnfr12 as
disclosed herein. Suitable sources of mRNA can be identified by
probing northern blots with probes designed from the sequences
disclosed herein. A library is then prepared from mRNA of a
positive tissue or cell line.
[0130] A Ztnfr12-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
Ztnfr12 sequences disclosed herein. In addition, a cDNA library can
be used to transform or transfect host cells, and expression of the
cDNA of interest can be detected with an antibody to Ztnfr12
polypeptide.
[0131] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
Ztnfr12, and that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the
nucleotide sequences disclosed herein, including those containing
silent mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of the amino acid sequences
disclosed herein. cDNA molecules generated from alternatively
spliced mRNAs, which retain the properties of the Ztnfr12
polypeptide are included within the scope of the present invention,
as are polypeptides encoded by such cDNAs and mRNAs. Allelic
variants and splice variants of these sequences can be cloned by
probing cDNA or genomic libraries from different individuals or
tissues according to standard procedures known in the art.
[0132] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, to nucleic acid molecules
consisting of the nucleotide sequence of nucleotides 27 to 578 of
SEQ ID NO:1, or to nucleic acid molecules comprising a nucleotide
sequence complementary to SEQ ID NO:1, or nucleotides 27 to 578 of
SEQ ID NO:1. In general, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe.
[0133] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0134] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Conditions that
influence the T.sub.m include, the size and base pair content of
the polynucleotide probe, the ionic strength of the hybridization
solution, and the presence of destabilizing agents in the
hybridization solution. Numerous equations for calculating T.sub.m
are known in the art, and are specific for DNA, RNA and DNA-RNA
hybrids and polynucleotide probe sequences of varying length (see,
for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et
al., (eds.), Current Protocols in Molecular Biology (John Wiley and
Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular
Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit.
Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software
such as OLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0
(Premier Biosoft International; Palo Alto, Calif.), as well as
sites on the Internet, are available tools for analyzing a given
sequence and calculating T.sub.m based on user defined criteria.
Such programs can also analyze a given sequence under defined
conditions and identify suitable probe sequences. Typically,
hybridization of longer polynucleotide sequences, >50 base
pairs, is performed at temperatures of about 20-25.degree. C. below
the calculated T.sub.m. For smaller probes, <50 base pairs,
hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0135] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0136] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0137] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). Typically, hybridization
buffers contain from between 10 mM-1 M Na.sup.+. The addition of
destabilizing or denaturing agents such as formamide,
tetralkylammonium salts, guanidinium cations or thiocyanate cations
to the hybridization solution will alter the T.sub.m of a hybrid.
Typically, formamide is used at a concentration of up to 50% to
allow incubations to be carried out at more convenient and lower
temperatures. Formamide also acts to reduce non-specific background
when using RNA probes.
[0138] As an illustration, a nucleic acid molecule encoding a
variant Ztnfr12 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) at 42.degree. C. overnight in a solution comprising 50%
formamide, 5.times.SSC, 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution (100.times. Denhardt's solution: 2% (w/v)
Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine
serum albumin), 10% dextran sulfate, and 20 .mu.g/ml denatured,
sheared salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For example, the
hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0139] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. As an illustration, nucleic acid molecules
encoding a variant Ztnfr12 polypeptide remain hybridized with a
nucleic acid molecule comprising the nucleotide sequence of
nucleotides 27 to 578 of SEQ ID NO:1 (or its complement) following
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0140] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. For example, nucleic acid
molecules encoding a variant Ztnfr12 polypeptide remain hybridized
with a nucleic acid molecule comprising the nucleotide sequence of
nucleotides 27 to 578 of SEQ ID NO:1 (or its complement) following
highly stringent washing conditions, in which the wash stringency
is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS at
50-65.degree. C., including 0.1.times.SSC with 0.1% SDS at
50.degree. C., or 0.2.times.SSC with 0.1% SDS at 65.degree. C.
[0141] The present invention also provides isolated Ztnfr12
polypeptides that have a substantially similar sequence identity to
the polypeptide of SEQ ID NO:2, or its orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95% or greater than 95% sequence identity to the sequences
shown in SEQ ID NO:2, or orthologs.
[0142] The present invention also contemplates Ztnfr12 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay, as described above. Such Ztnfr12 variants include nucleic
acid molecules (1) that remain hybridized with a nucleic acid
molecule comprising the nucleotide sequence of nucleotides 27 to
578 of SEQ ID NO:1 (or its complement) following stringent washing
conditions, in which the wash stringency is equivalent to
0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95% sequence identity to
the amino acid sequence of SEQ ID NO:2. Alternatively, Ztnfr12
variants can be characterized as nucleic acid molecules (1) that
remain hybridized with a nucleic acid molecule comprising the
nucleotide sequence of nucleotides 27 to 578 of SEQ ID NO:1 (or its
complement) following highly stringent washing conditions, in which
the wash stringency is equivalent to 0.1.times.-0.2.times.SSC with
0.1% SDS at 50-65.degree. C., and (2) that encode a polypeptide
having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% sequence identity to the amino acid sequence of
SEQ ID NO:2.
[0143] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
TABLE-US-00004 TABLE 4 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0144] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative Ztnfr12 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then rescored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0145] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as described above.
[0146] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a Ztnfr12 amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a Ztnfr12 amino acid sequence, a sulfur-containing
amino acid is substituted for a sulfur-containing amino acid in a
Ztnfr12 amino acid sequence, a hydroxy-containing amino acid is
substituted for a hydroxy-containing amino acid in a Ztnfr12 amino
acid sequence, an acidic amino acid is substituted for an acidic
amino acid in a Ztnfr12 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a Ztnfr12 amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a Ztnfr12 amino acid
sequence. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine.
[0147] The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62
substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid
sequences of the present invention. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably refers to a substitution represented
by a BLOSUM62 value of greater than -1. For example, an amino acid
substitution is conservative if the substitution is characterized
by a BLOSUM62 value of 0, 1, 2, or 3. According to this system,
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0148] Certain conservative amino acid substitutions can be
identified by aligning human and murine Ztnfr12 amino acid
sequences. For example, an alignment indicates the following amino
acid substitutions in the human Ztnfr12 amino acid sequence of SEQ
ID NO:2: Ala.sup.15 to Val.sup.15, Arg.sup.39 to His.sup.39, and
Ala.sup.71 to Leu.sup.71. Such an alignment identifies other
conservative amino acid substitutions of the human Ztnfr12 amino
acid sequence, or conservative amino acid substitutions of the
murine Ztnfr12 amino acid sequence.
[0149] Particular variants of Ztnfr12 are characterized by having
at least 70%, at least 80%, at least 90%, at least 95% or greater
than 95% sequence identity to the corresponding amino acid sequence
(e.g., SEQ ID NO:2), wherein the variation in amino acid sequence
is due to one or more conservative amino acid substitutions.
[0150] Conservative amino acid changes in a Ztnfr12 gene can be
introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1. Such "conservative amino acid"
variants can be obtained by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed Mutagenesis: A Practical Approach
(IRL Press 1991)). A variant Ztnfr12 polypeptide can be identified
by the ability to specifically bind anti-Ztnfr12 antibodies.
[0151] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0152] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0153] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for Ztnfr12 amino acid residues.
[0154] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity to
identify amino acid residues that are critical to the activity of
the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699
(1996).
[0155] Although sequence analysis can be used to further define the
Ztnfr12 ligand binding region, amino acids that play a role in
Ztnfr12 binding activity can also be determined by physical
analysis of structure, as determined by such techniques as nuclear
magnetic resonance, crystallography, electron diffraction or
photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., Science
255:306 (1992), Smith et al, J. Mol. Biol. 224:899 (1992), and
Wlodaver et al., FEBS Lett. 309:59 (1992).
[0156] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
Ztnfr12 labeled with biotin or FITC can be used for expression
cloning of new Ztnfr12 ligands.
[0157] Variants of the disclosed Ztnfr12 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0158] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-Ztnfr12 antibodies, can be
recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0159] The present invention also includes "functional fragments"
of Ztnfr12 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a Ztnfr12 polypeptide. As an
illustration, DNA molecules comprising the nucleotide sequence of
nucleotides 27 to 578 of SEQ ID NO:1 can be digested with Bal31
nuclease to obtain a series of nested deletions. The fragments are
then inserted into expression vectors in proper reading frame, and
the expressed polypeptides are isolated and tested for the ability
to bind anti-Ztnfr12 antibodies. One alternative to exonuclease
digestion is to use oligonucleotide-directed mutagenesis to
introduce deletions or stop codons to specify production of a
desired fragment. Alternatively, particular fragments of a Ztnfr12
gene can be synthesized using the polymerase chain reaction. An
example of a functional fragment is the extracellular domain of
Ztnfr12 (i.e., about amino acid residues 1 to 69 of SEQ ID NO:2, or
about amino acid residues 1 to 79 of SEQ ID NO:2).
[0160] This general approach is exemplified by studies on the
truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0161] The present invention also contemplates functional fragments
of a Ztnfr12 gene that have amino acid changes, compared with an
amino acid sequence disclosed herein. A variant Ztnfr12 gene can be
identified on the basis of structure by determining the level of
identity with disclosed nucleotide and amino acid sequences, as
discussed above. An alternative approach to identifying a variant
gene on the basis of structure is to determine whether a nucleic
acid molecule encoding a potential variant Ztnfr12 gene can
hybridize to a nucleic acid molecule comprising a nucleotide
sequence, such as SEQ ID NO:1.
[0162] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a Ztnfr12
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0163] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0164] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a Ztnfr12 polypeptide,
or by chemical peptide synthesis, as described herein. Moreover,
epitopes can be selected by phage display of random peptide
libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616
(1996)). Standard methods for identifying epitopes and producing
antibodies from small peptides that comprise an epitope are
described, for example, by Mole, "Epitope Mapping," in Methods in
Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992), Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0165] In addition to the uses described above, polynucleotides and
polypeptides of the present invention are useful as educational
tools in laboratory practicum kits for courses related to genetics
and molecular biology, protein chemistry, and antibody production
and analysis. Due to its unique polynucleotide and polypeptide
sequences, molecules of Ztnfr12 can be used as standards or as
"unknowns" for testing purposes. For example, Ztnfr12
polynucleotides can be used as an aid, such as, for example, to
teach a student how to prepare expression constructs for bacterial,
viral, or mammalian expression, including fusion constructs,
wherein Ztnfr12 is the gene to be expressed; for determining the
restriction endonuclease cleavage sites of the polynucleotides;
determining mRNA and DNA localization of Ztnfr12 polynucleotides in
tissues (i.e., by northern and Southern blotting as well as
polymerase chain reaction); and for identifying related
polynucleotides and polypeptides by nucleic acid hybridization. As
an illustration, students will find that PstI digestion of a
nucleic acid molecule consisting of the nucleotide sequence of
nucleotides 27 to 578 of SEQ ID NO:1 provides two fragments of
about 174 base pairs, and 378 base pairs, and that HinfI digestion
yields fragments of about 182 base pairs, 226 base pairs, and 144
base pairs.
[0166] Ztnfr12 polypeptides can be used as an aid to teach
preparation of antibodies; identifying proteins by western
blotting; protein purification; determining the weight of expressed
Ztnfr12 polypeptides as a ratio to total protein expressed;
identifying peptide cleavage sites; coupling amino and carboxyl
terminal tags; amino acid sequence analysis, as well as, but not
limited to monitoring biological activities of both the native and
tagged protein (i.e., protease inhibition) in vitro and in vivo.
For example, students will find that digestion of unglycosylated
Ztnfr12 with endopeptidase Lys C yields five fragments having
approximate molecular weights of 4870, 7691, 883, 4758, and 729,
whereas digestion of unglycosylated Ztnfr12 with BNPS or NCS/urea
yields fragments having approximate molecular weights of 10279,
4740, and 3877.
[0167] Ztnfr12 polypeptides can also be used to teach analytical
skills such as mass spectrometry, circular dichroism, to determine
conformation, especially of the four alpha helices, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing the Ztnfr12 can be given to the student to analyze.
Since the amino acid sequence would be known by the instructor, the
protein can be given to the student as a test to determine the
skills or develop the skills of the student, the instructor would
then know whether or not the student has correctly analyzed the
polypeptide. Since every polypeptide is unique, the educational
utility of Ztnfr12 would be unique unto itself.
[0168] The antibodies which bind specifically to Ztnfr12 can be
used as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify Ztnfr12, cloning and sequencing
the polynucleotide that encodes an antibody and thus as a practicum
for teaching a student how to design humanized antibodies. The
Ztnfr12 gene, polypeptide, or antibody would then be packaged by
reagent companies and sold to educational institutions so that the
students gain skill in art of molecular biology. Because each gene
and protein is unique, each gene and protein creates unique
challenges and learning experiences for students in a lab
practicum. Such educational kits containing the Ztnfr12 gene,
polypeptide, or antibody are considered within the scope of the
present invention.
[0169] For any Ztnfr12 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise
Ztnfr12 variants based upon the nucleotide and amino acid sequences
described herein. Accordingly, the present invention includes a
computer-readable medium encoded with a data structure that
provides at least one of the following sequences: SEQ ID NO:1, SEQ
ID NO:2, and SEQ ID NO:3. Suitable forms of computer-readable media
include magnetic media and optically-readable media. Examples of
magnetic media include a hard or fixed drive, a random access
memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk
cache, and a ZIP disk. Optically readable media are exemplified by
compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW),
and CD-recordable), and digital versatile/video discs (DVD) (e.g.,
DVD-ROM, DVD-RAM, and DVD+RW).
[0170] 5. Production of Ztnfr12 Polypeptides
[0171] The polypeptides of the present invention, including
full-length polypeptides, functional fragments, and fusion
proteins, can be produced in recombinant host cells following
conventional techniques. To express a Ztnfr12 gene, a nucleic acid
molecule encoding the polypeptide must be operably linked to
regulatory sequences that control transcriptional expression in an
expression vector and then, introduced into a host cell. In
addition to transcriptional regulatory sequences, such as promoters
and enhancers, expression vectors can include translational
regulatory sequences and a marker gene which is suitable for
selection of cells that carry the expression vector.
[0172] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a Ztnfr12
expression vector may comprise a Ztnfr12 gene and a secretory
sequence derived from any secreted gene.
[0173] Expression of Ztnfr12 can be achieved using nucleic acid
molecules that either include or do not include noncoding portions
of the Ztnfr12 gene. However, higher efficiency of production from
certain recombinant host cells may be obtained when at least one
Ztnfr12 intron sequence is included in the expression vector.
[0174] Ztnfr12 proteins of the present invention may be expressed
in mammalian cells. Examples of suitable mammalian host cells
include African green monkey kidney cells (Vero; ATCC CRL 1587),
human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO-KI; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell.
Molec. Genet. 12:555, 1986)), rat pituitary cells (GHI; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0175] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0176] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0177] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
Ztnfr12 gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.
Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res.
19:4485 (1991)).
[0178] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0179] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
selection is carried out in the presence of a neomycin-type drug,
such as G-418 or the like. Selection systems can also be used to
increase the expression level of the gene of interest, a process
referred to as "amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of the
selective agent and then increasing the amount of selective agent
to select for cells that produce high levels of the products of the
introduced genes. A suitable amplifiable selectable marker is
dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g., hygromycin resistance,
multi-drug resistance, puromycin acetyltransferase) can also be
used. Alternatively, markers that introduce an altered phenotype,
such as green fluorescent protein, or cell surface proteins such as
CD4, CD8, Class I MHC, placental alkaline phosphatase may be used
to sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0180] Ztnfr12 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, herpesvirus, vaccinia virus
and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA
virus, is currently the best studied gene transfer vector for
delivery of heterologous nucleic acid (for a review, see Becker et
al., Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel,
Science & Medicine 4:44 (1997)). Advantages of the adenovirus
system include the accommodation of relatively large DNA inserts,
the ability to grow to high-titer, the ability to infect a broad
range of mammalian cell types, and flexibility that allows use with
a large number of available vectors containing different
promoters.
[0181] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0182] Ztnfr12 can also be expressed in other higher eukaryotic
cells, such as avian, fungal, insect, yeast, or plant cells. The
baculovirus system provides an efficient means to introduce cloned
Ztnfr12 genes into insect cells. Suitable expression vectors are
based upon the Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV), and contain well-known promoters such as Drosophila
heat shock protein (hsp) 70 promoter, Autographa californica
nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and
the delayed early 39K promoter, baculovirus p10 promoter, and the
Drosophila metallothionein promoter. A second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, et al., J. Virol. 67:4566 (1993)).
This system, which utilizes transfer vectors, is sold in the
BAC-to-BAC kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, PFASTBAC (Life Technologies) containing
a Tn7 transposon to move the DNA encoding the Ztnfr12 polypeptide
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol.
71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), and
Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In
addition, transfer vectors can include an in-frame fusion with DNA
encoding an epitope tag at the C- or N-terminus of the expressed
Ztnfr12 polypeptide, for example, a Glu-Glu epitope tag
(Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using
a technique known in the art, a transfer vector containing a
Ztnfr12 gene is transformed into E. coli, and screened for bacmids,
which contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is then isolated using common techniques.
[0183] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed, which replace the
native Ztnfr12 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native Ztnfr12 secretory signal
sequence.
[0184] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0185] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0186] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp 17, YEp vectors such as YEp 13 and YCp
vectors, such as YCp 19. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No.
4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S.
Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, and
Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e.g., leucine). A suitable vector system for
use in Saccharomyces cerevisiae is the POT1 vector system disclosed
by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows
transformed cells to be selected by growth in glucose-containing
media. Additional suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et al., U.S. Pat. No.
4,615,974, and Bitter, U.S. Pat. No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154,
5,139,936, and 4,661,454.
[0187] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0188] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which can be
linearized prior to transformation. For polypeptide production in
P. methanolica, the promoter and terminator in the plasmid can be
that of a P. methanolica gene, such as a P. methanolica alcohol
utilization gene (AUG1 or AUG2). Other useful promoters include
those of the dihydroxyacetone synthase (DHAS), formate
dehydrogenase (FMD), and catalase (CAT) genes. To facilitate
integration of the DNA into the host chromosome, the entire
expression segment of the plasmid can be flanked at both ends by
host DNA sequences. A suitable selectable marker for use in Pichia
methanolica is a P. methanolica ADE2 gene, which encodes
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21),
and which allows ade2 host cells to grow in the absence of adenine.
For large-scale, industrial processes where it is desirable to
minimize the use of methanol, host cells can be used in which both
methanol utilization genes (AUG1 and AUG2) are deleted. For
production of secreted proteins, host cells can be deficient in
vacuolar protease genes (PEP4 and PRB1). Electroporation is used to
facilitate the introduction of a plasmid containing DNA encoding a
polypeptide of interest into P. methanolica cells. P. methanolica
cells can be transformed by electroporation using an exponentially
decaying, pulsed electric field having a field strength of from 2.5
to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t)
of from 1 to 40 milliseconds, most preferably about 20
milliseconds.
[0189] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0190] Alternatively, Ztnfr12 genes can be expressed in prokaryotic
host cells. Suitable promoters that can be used to express Ztnfr12
polypeptides in a prokaryotic host are well-known to those of skill
in the art and include promoters capable of recognizing the T4, T3,
Sp6 and T7 polymerases, the P.sub.R and P.sub.L promoters of
bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,
lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
subtilis, the promoters of the bacteriophages of Bacillus,
Streptomyces promoters, the int promoter of bacteriophage lambda,
the bla promoter of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters have
been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et
al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
1987), and by Ausubel et al. (1995).
[0191] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH41, DH5, DH51, DH51F',
DH51MCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0192] When expressing a Ztnfr12 polypeptide in bacteria such as E.
coli, the polypeptide may be retained in the cytoplasm, typically
as insoluble granules, or may be directed to the periplasmic space
by a bacterial secretion sequence. In the former case, the cells
are lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0193] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0194] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0195] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0196] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0197] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:2. As an illustration, polypeptides can
comprise at least six, at least nine, or at least 15 contiguous
amino acid residues of amino acid residues 1 to 69 of SEQ ID NO:2,
amino acid residues 1 to 79 of SEQ ID NO:2, amino acid residues 7
to 69 of SEQ ID NO:2, or amino acid residues 7 to 79 of SEQ ID
NO:2. Within certain embodiments of the invention, the polypeptides
comprise 20, 30, 40, 50, 100, or more contiguous residues of these
amino acid sequences. For example, polypeptides can comprise at
least 30 contiguous amino acid residues of an amino acid sequence
selected from the group consisting of: (a) amino acid residues 1 to
184 of SEQ ID NO:2, (b) amino acid residues 1 to 69 of SEQ ID NO:2,
(c) amino acid residues 1 to 79 of SEQ ID NO:2, (d) amino acid
residues 7 to 69 of SEQ ID NO:2, and (e) amino acid residues 7 to
79 of SEQ ID NO:2. Nucleic acid molecules encoding such peptides
and polypeptides are useful as polymerase chain reaction primers
and probes, and these peptides and polypeptides are useful to
produce antibodies to Ztnfr12.
6. Production of Ztnfr12 Fusion Proteins and Conjugates
[0198] One general class of Ztnfr12 analogs are variants having an
amino acid sequence that is a mutation of the amino acid sequence
disclosed herein. Another general class of Ztnfr12 analogs is
provided by anti-idiotype antibodies, and fragments thereof, as
described below. Moreover, recombinant antibodies comprising
anti-idiotype variable domains can be used as analogs (see, for
example, Monfardini et al., Proc. Assoc. Am. Physicians 108:420
(1996)). Since the variable domains of anti-idiotype Ztnfr12
antibodies mimic Ztnfr12, these domains can provide Ztnfr12 binding
activity. Methods of producing anti-idiotypic catalytic antibodies
are known to those of skill in the art (see, for example, Joron et
al., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et al., Appl.
Biochem. Biotechnol. 47:229 (1994), and Avalle et al., Ann. N Y
Acad. Sci. 864:118 (1998)).
[0199] Another approach to identifying Ztnfr12 analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0200] Ztnfr12 polypeptides have both in vivo and in vitro uses. As
an illustration, a soluble form of Ztnfr12 can be added to cell
culture medium to inhibit the effects of ZTNF4 either produced by
the cultured cells, or added to test medium.
[0201] Fusion proteins of Ztnfr12 can be used to express Ztnfr12 in
a recombinant host, and to isolate the produced Ztnfr12. As
described below, particular Ztnfr12 fusion proteins also have uses
in diagnosis and therapy. One type of fusion protein comprises a
peptide that guides a Ztnfr12 polypeptide from a recombinant host
cell. To direct a Ztnfr12 polypeptide into the secretory pathway of
a eukaryotic host cell, a secretory signal sequence (also known as
a signal peptide, a leader sequence, prepro sequence or pre
sequence) is provided in the Ztnfr12 expression vector. While the
secretory signal sequence may be derived from Ztnfr12, a suitable
signal sequence may also be derived from another secreted protein
or synthesized de novo. The secretory signal sequence is operably
linked to a Ztnfr12-encoding sequence such that the two sequences
are joined in the correct reading frame and positioned to direct
the newly synthesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the nucleotide sequence encoding the polypeptide of interest,
although certain secretory signal sequences may be positioned
elsewhere in the nucleotide sequence of interest (see, e.g., Welch
et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0202] Although the secretory signal sequence of Ztnfr12 or another
protein produced by mammalian cells (e.g., tissue-type plasminogen
activator signal sequence, as described, for example, in U.S. Pat.
No. 5,641,655) is useful for expression of Ztnfr12 in recombinant
mammalian hosts, a yeast signal sequence is preferred for
expression in yeast cells. Examples of suitable yeast signal
sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0203] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, Ztnfr12 can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferease fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a Ztnfr12 fusion protein comprising a
maltose binding protein polypeptide can be isolated with an amylose
resin column, while a fusion protein comprising the C-terminal end
of a truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wis.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0204] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0205] Another form of fusion protein comprises a Ztnfr12
polypeptide and an immunoglobulin heavy chain constant region,
typically an F.sub.c fragment, which contains two or three constant
region domains and a hinge region but lacks the variable region.
Fusion proteins comprising a Ztnfr12 moiety and an Fc moiety can be
used, for example, as an in vitro assay tool. For example, the
presence of a Ztnfr12 ligand in a biological sample can be detected
using a Ztnfr12-immunoglobulin fusion protein, in which the Ztnfr12
moiety is used to bind the ligand, and a macromolecule, such as
Protein A or anti-Fc antibody, is used to bind the fusion protein
to a solid support. Such systems can be used to identify agonists
and antagonists that interfere with the binding of a Ztnfr12 ligand
to its receptor.
[0206] As an illustration, Chang et al., U.S. Pat. No. 5,723,125,
describe a fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:4). In this fusion protein, an
illustrative Fc moiety is a human .gamma.4 chain, which is stable
in solution and has little or no complement activating activity.
Accordingly, the present invention contemplates a Ztnfr12 fusion
protein that comprises a Ztnfr12 moiety and a human Fc fragment,
wherein the C-terminus of the Ztnfr12 moiety is attached to the
N-terminus of the Fc fragment via a peptide linker, such as a
peptide consisting of the amino acid sequence of SEQ ID NO:4. The
Ztnfr12 moiety can be a Ztnfr12 molecule or a fragment thereof. For
example, a fusion protein can comprise an Fc fragment (e.g., a
human Fc fragment), and amino acid residues 1 to about 69 of SEQ ID
NO:2, or amino acid residues 1 to 79 of SEQ ID NO:2.
[0207] In another variation, a Ztnfr12 fusion protein comprises an
IgG sequence, a Ztnfr12 moiety covalently joined to the
aminoterminal end of the IgG sequence, and a signal peptide that is
covalently joined to the aminoterminal of the Ztnfr12 moiety,
wherein the IgG sequence consists of the following elements in the
following order: a hinge region, a CH.sub.2 domain, and a CH.sub.3
domain. Accordingly, the IgG sequence lacks a CH.sub.1 domain. The
Ztnfr12 moiety displays a Ztnfr12 activity, as described herein,
such as the ability to bind with a Ztnfr12 ligand (e.g., ZTNF4).
This general approach to producing fusion proteins that comprise
both antibody and nonantibody portions has been described by
LaRochelle et al., EP 742830 (WO 95/21258).
[0208] Example 4 describes the construction of a Ztnfr12 fusion
protein, in which the immunoglobulin moiety, derived from IgG,
contains certain mutations. Five classes of immunoglobulin, IgG,
IgA, IgM, IgD, and IgE, have been identified in higher vertebrates.
IgG, IgD, and IgE proteins are characteristically disulfide linked
heterotetramers consisting of two identical heavy chains and two
identical light chains. Typically, IgM is found as a pentamer of a
tetramer, whereas IgA occurs as a dimer of a tetramer.
[0209] IgG comprises the major class as it normally exists as the
second most abundant protein found in plasma. In humans, IgG
consists of four subclasses, designated IgG1, IgG2, IgG3, and IgG4.
As shown in FIG. 1, each immunoglobulin heavy chain possesses a
constant region that consists of constant region protein domains
(C.sub.H1, hinge, C.sub.H2, and C.sub.H3) that are invariant for a
given subclass. The heavy chain constant regions of the IgG class
are identified with the Greek symbol .quadrature.. For example,
immunoglobulins of the IgG1 subclass contain a .quadrature.1 heavy
chain constant region.
[0210] The Fc fragment, or Fc domain, consists of the disulfide
linked heavy chain hinge regions, C.sub.H2, and C.sub.H3 domains.
In immunoglobulin fusion proteins, Fc domains of the IgG1 subclass
are often used as the immunoglobulin moiety, because IgG1 has the
longest serum half-life of any of the serum proteins. Lengthy serum
half-life can be a desirable protein characteristic for animal
studies and potential human therapeutic use. In addition, the IgG1
subclass possesses the strongest ability to carry out antibody
mediated effector functions. The primary effector function that may
be most useful in an immunoglobulin fusion protein is the ability
for an IgG1 antibody to mediate antibody dependent cellular
cytotoxicity. On the other hand, this could be an undesirable
function for a fusion protein whose primary function is as an
antagonist. Several of the specific amino acid residues that are
important for antibody constant region mediated activity in the
IgG1 subclass have been identified. Inclusion or exclusion of these
specific amino acids therefore allows for inclusion or exclusion of
specific immunoglobulin constant region mediated activity.
[0211] Example 4 describes two versions of a modified human IgG1 Fc
that were generated for creating Ztnfr12-Fc fusion protein. Fc4 and
Fc5 contain mutations to reduce effector functions mediated by the
Fc by reducing Fc.quadrature.R1 binding and complement C1q binding.
Specifically, three amino acid substitutions were introduced to
reduce Fc.quadrature.R1 binding. These are the substitutions at EU
index positions 234, 235, and 237 (amino acid residues 38, 39, and
41 of SEQ ID NO:17, which is a sequence of a wild type
immunoglobulin .quadrature.1 region). Substitutions at these
positions have been shown to reduce binding to Fc.quadrature.R1
(Duncan et al., Nature 332:563 (1988)). These amino acid
substitutions may also reduce Fc.quadrature.RIIa binding, as well
as Fc.quadrature.RIII binding (Sondermann et al., Nature 406:267
(2000); Wines et al., J. Immunol. 164:5313 (2000)).
[0212] Several groups have described the relevance of EU index
positions 330 and 331 (amino acid residues 134 and 135 of SEQ ID
NO:17) in complement C1q binding and subsequent complement fixation
(Canfield and Morrison, J. Exp. Med. 173:1483 (1991); Tao et al.,
J. Exp. Med. 178:661 (1993)). Amino acid substitutions at these
positions were introduced in Fc4 to reduce complement fixation. The
C.sub.H3 domain of Fc4 is identical to that found in the
corresponding wild-type polypeptide, except for the stop codon,
which was changed from TGA to TAA to eliminate a potential dam
methylation site when the cloned DNA is grown in dam plus strains
of E. coli.
[0213] In Fc5, the Arginine residue at EU index position 218 was
mutated back to a lysine, because the BglII cloning scheme was not
used in fusion proteins containing this particular Fc. The
remainder of the Fc5 sequence matches the above description for
Fc4.
[0214] Other useful Fc variants include Fc6, Fc7, and Fc8. Fc6 is
identical to Fc5 except that the carboxyl terminal lysine codon has
been eliminated. The C-terminal lysine of mature immunoglobulins is
often removed from mature immunoglobulins post-translationally
prior to secretion from B-cells, or removed during serum
circulation. Consequently, the C-terminal lysine residue is
typically not found on circulating antibodies. As in Fc4 and Fc5
above, the stop codon in the Fc6 sequence was changed to TAA.
[0215] Fc7 is identical to the wild type .quadrature.1 Fe except
for an amino acid substitution at EU index position 297 located in
the C.sub.H2 domain. EU index position Asn-297 (amino acid residue
101 of SEQ ID NO:17) is a site of N-linked carbohydrate attachment.
N-linked carbohydrate introduces a potential source of variability
in a recombinantly expressed protein due to potential
batch-to-batch variations in the carbohydrate structure. In an
attempt to eliminate this potential variability, Asn-297 was
mutated to a glutamine residue to prevent the attachment of
N-linked carbohydrate at that residue position. The carbohydrate at
residue 297 is also involved in Fc binding to the
Fc.quadrature.RIII (Sondermann et al., Nature 406:267 (2000)).
Therefore, removal of the carbohydrate should decrease binding of
recombinant Fc7 containing fusion proteins to the Fc.quadrature.Rs
in general. As above, the stop codon in the Fc7 sequence was
mutated to TAA.
[0216] Fc8 is identical to the wild type immunoglobulin
.quadrature.1 region shown in SEQ ID NO:17, except that the
cysteine residue at EU index position 220 (amino acid residue 24 of
SEQ ID NO:17) was replaced with a serine residue. This mutation
eliminated the cysteine residue that normally disulfide bonds with
the immunoglobulin light chain constant region.
[0217] The present invention contemplates Ztnfr12-immunoglobulin
fusion proteins that comprise a Ztnfr12 receptor moiety consisting
of amino acid residues 7 to 69 of SEQ ID NO:2, amino acid residues
7 to 79 of SEQ ID NO:2, amino acid residues 7 to 39 of SEQ ID NO:2,
amino acid residues 19 to 35 of SEQ ID NO:2, amino acid residues 1
to 69 of SEQ ID NO:2, amino acid residues 1 to 79 of SEQ ID NO:2,
amino acid residues 1 to 71 of SEQ ID NO:2, amino acid residues 7
to 71 of SEQ ID NO:2, or amino acid residues 1 to 39 of SEQ ID
NO:2. More generally, the present invention includes
Ztnfr12-immunoglobulin fusion proteins, wherein the Ztnfr12
receptor moiety consists of a fragment of amino acid residues 1 to
69 of SEQ ID NO:2, amino acid residues 1 to 71 of SEQ ID NO:2,
amino acid residues 7 to 71 of SEQ ID NO:2, or amino acid residues
1 to 79 of SEQ ID NO:2, and wherein the Ztnfr12 receptor moiety
binds at ZTNF4.
[0218] The immunoglobulin moiety of a fusion protein described
herein comprises at least one constant region of an immunoglobulin.
Preferably, the immunoglobulin moiety represents a segment of a
human immunoglobulin. The human immunoglobulin sequence can be a
wild-type amino acid sequence, or a modified wild-type amino acid
sequence, which has at least one of the amino acid mutations
discussed above.
[0219] The human immunoglobulin amino acid sequence can also vary
from wild-type by having one or more mutations characteristic of a
known allotypic determinant. Table 5 shows the allotypic
determinants of the human IgG.gamma.1 constant region (Putman, The
Plasma Proteins, Vol. V, pages 49 to 140 (Academic Press, Inc.
1987)). EU index positions 214, 356, 358, and 431 define the known
IgG.gamma.1 allotypes. Position 214 is in the C.sub.H1 domain of
the IgG.gamma.1 constant region, and, therefore, does not reside
within the Fc sequence. The wild type Fc sequence of SEQ ID NO:17
includes the Glm(1) and Glm(2-) allotypes. However, the Fc moiety
of a TACI-Fc protein can be modified to reflect any combination of
these allotypes. TABLE-US-00005 TABLE 5 Allotypic Determinants of
the Human Immunoglobulin .gamma.1 Constant Region Amino Acid Amino
Acid Position Allotype Residue EU Index SEQ ID NO: 17 Glm(1) Asp,
Leu 356, 358 160, 162 Glm(1-) Glu, Met 356, 358 160, 162 Glm(2) Gly
431 235 Glm(2-) Ala 431 235 Glm(3) Arg 214 -- Glm(3-) Lys 214
--
[0220] The examples of Ztnfr12-Fc proteins disclosed herein
comprise human IgG1 constant regions. However, suitable
immunoglobulin moieties also include polypeptides comprising at
least one constant region, such as a heavy chain constant region
from any of the following immunoglobulins: IgG2, IgG3, IgG4, IgA1,
IgA2, IgD, IgE, and IgM. The present invention also contemplates
fusion proteins that comprise a Ztnfr12 receptor moiety, as
described above, and either albumin or .beta.2-macroglobulin, and
the like, to produce Ztnfr12 dimers and multimers. Additional
protein moieties suitable to produce Ztnfr12 fusion protein dimers
and multimers are known to those of skill in the art.
[0221] In the treatment of certain conditions, it may be
advantageous to combine a Ztnfr12-immunoglobulin fusion protein
with at least one of a TACI-immunoglobulin fusion protein and
BCMA-immunoglobulin fusion protein. This combination therapy can be
achieved by administering various types of immunoglobulin fusion
proteins, for example as dimers, or by administering heterodimers
of Ztnfr12-immunoglobulin, TACI-immunoglobulin and
BCMA-immunoglobulin fusion proteins.
[0222] The fusion proteins of the present invention can have the
form of single chain polypeptides, dimers, trimers, or multiples of
dimers or trimers. Dimers can be homodimers or heterodimers, and
trimers can be homotrimers or heterotrimers. Examples of
heterodimers include a Ztnfr12-immunoglobulin polypeptide with a
BCMA-immunoglobulin polypeptide, a Ztnfr12-immunoglobulin
polypeptide with a TACI-immunoglobulin polypeptide, and a
BCMA-immunoglobulin polypeptide with a TACI-immunoglobulin
polypeptide. Examples of heterotrimers include a
Ztnfr12-immunoglobulin polypeptide with two BCMA-immunoglobulin
polypeptides, a Ztnfr12-immunoglobulin polypeptide with two
TACI-immunoglobulin polypeptides, a BCMA-immunoglobulin polypeptide
with two Ztnfr12-immunoglobulin polypeptides, two
TACI-immunoglobulin polypeptides with a BCMA-immunoglobulin
polypeptide, one TACI-immunoglobulin polypeptide with two
Ztnfr12-immunoglobulin polypeptides, two BCMA-immunoglobulin
polypeptides with a TACI-immunoglobulin polypeptide, and a trimer
of a TACI-immunoglobulin polypeptide, a BCMA-immunoglobulin
polypeptide, and a Ztnfr12-immunoglobulin polypeptide.
[0223] In such fusion proteins, the TACI receptor moiety can
comprise at least one of the following amino acid sequences of SEQ
ID NO:8: amino acid residues 30 to 154, amino acid residues 34 to
66, amino acid residues 71 to 104, amino acid residues 47 to 62,
and amino acid residues 86 to 100. The BCMA receptor moiety can
comprise at least one of the following amino acid sequences of SEQ
ID NO:7: amino acid residues 1 to 48, amino acid residues 8 to 41,
and amino acid residues 21 to 37. The Ztnfr12 receptor moiety can
comprise at least one of the following amino acid sequences of SEQ
ID NO:2: amino acid residues 7 to 69, amino acid residues 7 to 79,
amino acid residues 7 to 39, amino acid residues 19 to 35, amino
acid residues 1 to 69, amino acid residues 1 to 79 of SEQ ID NO:2,
amino acid residues 1 to 71 of SEQ ID NO:2, amino acid residues 7
to 71 of SEQ ID NO:2, or amino acid residues 1 to 39.
[0224] Immunoglobulin fusion proteins can be produced using
standard methods. As an illustration, Example 4 describes the use
of PCR methods used to construct the illustrative Ztnfr12-Fc5
fusion protein.
[0225] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a Ztnfr12
fragment that contains a Ztnfr12 extracellular domain. Such
molecules can be used to target particular tissues for the benefit
of Ztnfr12 binding activity.
[0226] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between Ztnfr12 of
the present invention with the functionally equivalent domain(s)
from another member of the tumor necrosis factor receptor family.
Polypeptide fusions can be expressed in recombinant host cells to
produce a variety of Ztnfr12 fusion analogs. A Ztnfr12 polypeptide
can be fused to two or more moieties or domains, such as an
affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See, for example, Tuan et al., Connective Tissue
Research 34:1 (1996).
[0227] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0228] Ztnfr12 may bind ligands other than ZTNF4. Ztnfr12
polypeptides can be used to identify and to isolate such
additional, potential Ztnfr12 ligands. For example, proteins and
peptides of the present invention can be immobilized on a column
and used to bind ligands from a biological sample that is run over
the column (Hermanson et al. (eds.), Immobilized Affinity Ligand
Techniques, pages 195-202 (Academic Press 1992)).
[0229] The activity of a Ztnfr12 polypeptide can be observed by a
silicon-based biosensor microphysiometer, which measures the
extracellular acidification rate or proton excretion associated
with receptor binding and subsequent physiologic cellular
responses. An exemplary device is the CYTOSENSOR Microphysiometer
manufactured by Molecular Devices, Sunnyvale, Calif. A variety of
cellular responses, such as cell proliferation, ion transport,
energy production, inflammatory response, regulatory and receptor
activation, and the like, can be measured by this method (see, for
example, McConnell et al., Science 257:1906 (1992), Pitchford et
al., Meth. Enzymol. 228:84 (1997), Arimilli et al., J. Immunol.
Meth. 212:49 (1998), Van Liefde et al., Eur. J. Pharmacol. 346:87
(1998)). The microphysiometer can be used for assaying eukaryotic,
prokaryotic, adherent, or non-adherent cells. By measuring
extracellular acidification changes in cell media over time, the
microphysiometer directly measures cellular responses to various
stimuli, including agonists, ligands, or antagonists of
Ztnfr12.
[0230] For example, the microphysiometer is used to measure
responses of a Ztnfr12-expressing eukaryotic cell, compared to a
control eukaryotic cell that does not express Ztnfr12 polypeptide.
Suitable cells responsive to Ztnfr12-modulating stimuli include
recombinant host cells comprising a Ztnfr12 expression vector, and
cells that naturally express Ztnfr12. Extracellular acidification
provides one measure for a Ztnfr12-modulated cellular response. In
addition, this approach can be used to identify ligands, agonists,
and antagonists of Ztnfr12 ligands. For example, a molecule can be
identified as an agonist of Ztnfr12 ligand by providing cells that
express a Ztnfr12 polypeptide, culturing a first portion of the
cells in the absence of the test compound, culturing a second
portion of the cells in the presence of the test compound, and
determining whether the second portion exhibits a cellular
response, in comparison with the first portion.
[0231] Alternatively, a solid phase system can be used to identify
a new Ztnfr12 ligand, or an agonist or antagonist of ZTNF4. For
example, a Ztnfr12 polypeptide or Ztnfr12 fusion protein can be
immobilized onto the surface of a receptor chip of a commercially
available biosensor instrument (BIACORE, Biacore AB; Uppsala,
Sweden). The use of this instrument is disclosed, for example, by
Karlsson, Immunol. Methods 145:229 (1991), and Cunningham and
Wells, J. Mol. Biol. 234:554 (1993).
[0232] In brief, a Ztnfr12 polypeptide or fusion protein is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within a flow cell. A
test sample is then passed through the cell. If a ligand is present
in the sample, it will bind to the immobilized polypeptide or
fusion protein, causing a change in the refractive index of the
medium, which is detected as a change in surface plasmon resonance
of the gold film. This system allows the determination of on- and
off-rates, from which binding affinity can be calculated, and
assessment of stoichiometry of binding. This system can also be
used to examine antibody-antigen interactions, and the interactions
of other complement/anti-complement pairs.
[0233] Ztnfr12 binding domains can be further characterized by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids of Ztnfr12 ligand agonists. See, for
example, de Vos et al., Science 255:306 (1992), Smith et al., J.
Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59
(1992).
[0234] The present invention also contemplates chemically modified
Ztnfr12 compositions, in which a Ztnfr12 polypeptide is linked with
a polymer. Illustrative Ztnfr12 polypeptides are soluble
polypeptides that lack a functional transmembrane domain, such as a
polypeptide consisting of amino acid residues 1 to about 69 of SEQ
ID NO:2, or a polypeptide consisting of amino acid residues 1 to
about 79 of SEQ ID NO:2. Typically, the polymer is water-soluble so
that the Ztnfr12 conjugate does not precipitate in an aqueous
environment, such as a physiological environment. An example of a
suitable polymer is one that has been modified to have a single
reactive group, such as an active ester for acylation, or an
aldehyde for alkylation, In this way, the degree of polymerization
can be controlled. An example of a reactive aldehyde is
polyethylene glycol propionaldehyde, or mono-(C.sub.1-C.sub.10)
alkoxy, or aryloxy derivatives thereof (see, for example, Harris,
et al., U.S. Pat. No. 5,252,714). The polymer may be branched or
unbranched. Moreover, a mixture of polymers can be used to produce
Ztnfr12 conjugates.
[0235] Ztnfr12 conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
Suitable water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C.sub.1-C.sub.10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A Ztnfr12
conjugate can also comprise a mixture of such water-soluble
polymers.
[0236] One example of a Ztnfr12 conjugate comprises a Ztnfr12
moiety and a polyalkyl oxide moiety attached to the N-terminus of
the Ztnfr12 moiety. PEG is one suitable polyalkyl oxide. As an
illustration, Ztnfr12 can be modified with PEG, a process known as
"PEGylation." PEGylation of Ztnfr12 can be carried out by any of
the PEGylation reactions known in the art (see, for example, EP 0
154 316, Delgado et al., Critical Reviews in Therapeutic Drug
Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin.
Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol
68:1 (1998)). For example, PEGylation can be performed by an
acylation reaction or by an alkylation reaction with a reactive
polyethylene glycol molecule. In an alternative approach, Ztnfr12
conjugates are formed by condensing activated PEG, in which a
terminal hydroxy or amino group of PEG has been replaced by an
activated linker (see, for example, Karasiewicz et al., U.S. Pat.
No. 5,382,657).
[0237] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a Ztnfr12 polypeptide. An
example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between Ztnfr12 and a water-soluble
polymer: amide, carbamate, urethane, and the like. Methods for
preparing PEGylated Ztnfr12 by acylation will typically comprise
the steps of (a) reacting a Ztnfr12 polypeptide with PEG (such as a
reactive ester of an aldehyde derivative of PEG) under conditions
whereby one or more PEG groups attach to Ztnfr12, and (b) obtaining
the reaction product(s). Generally, the optimal reaction conditions
for acylation reactions will be determined based upon known
parameters and desired results. For example, the larger the ratio
of PEG:Ztnfr12, the greater the percentage of polyPEGylated Ztnfr12
product.
[0238] The product of PEGylation by acylation is typically a
polyPEGylated Ztnfr12 product, wherein the lysine .epsilon.-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting Ztnfr12
will be at least 95% mono-, di-, or tri-pegylated, although some
species with higher degrees of PEGylation may be formed depending
upon the reaction conditions. PEGylated species can be separated
from unconjugated Ztnfr12 polypeptides using standard purification
methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
[0239] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with Ztnfr12 in the presence of
a reducing agent. PEG groups can be attached to the polypeptide via
a --CH.sub.2--NH group.
[0240] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and the .alpha.-amino
group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups. The
present invention provides a substantially homogenous preparation
of Ztnfr12 monopolymer conjugates.
[0241] Reductive alkylation to produce a substantially homogenous
population of monopolymer Ztnfr12 conjugate molecule can comprise
the steps of: (a) reacting a Ztnfr12 polypeptide with a reactive
PEG under reductive alkylation conditions at a pH suitable to
permit selective modification of the .alpha.-amino group at the
amino terminus of the Ztnfr12, and (b) obtaining the reaction
product(s). The reducing agent used for reductive alkylation should
be stable in aqueous solution and able to reduce only the Schiff
base formed in the initial process of reductive alkylation.
Illustrative reducing agents include sodium borohydride, sodium
cyanoborohydride, dimethylamine borane, trimethylamine borane, and
pyridine borane.
[0242] For a substantially homogenous population of monopolymer
Ztnfr12 conjugates, the reductive alkylation reaction conditions
are those which permit the selective attachment of the water
soluble polymer moiety to the N-terminus of Ztnfr12. Such reaction
conditions generally provide for pKa differences between the lysine
amino groups and the .alpha.-amino group at the N-terminus. The pH
also affects the ratio of polymer to protein to be used. In
general, if the pH is lower, a larger excess of polymer to protein
will be desired because the less reactive the N-terminal
.alpha.-group, the more polymer is needed to achieve optimal
conditions. If the pH is higher, the polymer:Ztnfr12 need not be as
large because more reactive groups are available. Typically, the pH
will fall within the range of 3 to 9, or 3 to 6.
[0243] Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of
water-soluble polymer to Ztnfr12 will generally be in the range of
1:1 to 100:1. Typically, the molar ratio of water-soluble polymer
to Ztnfr12 will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1
for monoPEGylation.
[0244] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)).
[0245] The present invention contemplates compositions comprising a
peptide or polypeptide described herein. Such compositions can
further comprise a carrier. The carrier can be a conventional
organic or inorganic carrier. Examples of carriers include water,
buffer solution, alcohol, propylene glycol, macrogol, sesame oil,
corn oil, and the like.
[0246] In addition, compositions can comprise a carrier, a Ztnfr12
polypeptide, and at least one of a TACI polypeptide or a BCMA
polypeptide. Certain compositions can comprise soluble forms of
these receptors. Examples of such compositions include compositions
comprising carrier, a Ztnfr12 polypeptide comprising amino acid
residues 7 to 69 of SEQ ID NO:2 (e.g., a polypeptide consisting of
amino acid residues 1 to 79, 1 to 69, 7 to 79, 1 to 71, or 7 to 71,
of SEQ ID NO:2), and (1) a BCMA polypeptide comprising amino acid
residues 1 to 51 of SEQ ID NO:7, (2) a TACI polypeptide comprising
amino acid residues 1 to 166 of SEQ ID NO:8, or (3) a BCMA
polypeptide comprising amino acid residues 1 to 51 of SEQ ID NO:7,
and a TACI polypeptide comprising amino acid residues 1 to 166 of
SEQ ID NO:8.
7. Assays for Ztnfr12 Polypeptides and Fusion Proteins
[0247] The function of Ztnfr12 polypeptides and
Ztnfr12-immunoglobulin fusion proteins can be examined using a
variety of approaches to assess the ability of the fusion proteins
to bind ZTNF4. For example, one approach measures the ability of
Ztnfr12 polypeptides or Ztnfr12-immunoglobulin fusion protein to
compete with Ztnfr12-coated plates for binding of .sup.125I-labeled
ZTNF4. As an illustration, 50 .mu.g of the ZTNF4 can be labeled
with 4 mCi of .sup.125I using a single IODO-BEAD (Pierce; Rockford,
Ill.). The reaction is quenched with a 0.25% solution of bovine
serum albumin, and the free .sup.125I is removed by gel filtration
using a PD-10 column (Pierce). The specific radioactivity of
.sup.125I-ZTNF4 preparations is determined by trichloroacetic acid
precipitation before and after the desalting step. An N-terminal
fragment of the Ztnfr12 receptor, designated as "Ztnfr12-N," is
added to 96-well plates (e.g., 100 .mu.l at 0.1 .mu.g/ml), and
incubated overnight at 4.degree. C. The plates are washed, blocked
with SUPERBLOCK (Pierce), and washed again. The Ztnfr12
polypeptides or Ztnfr12-Fc constructs, at various concentrations
ranging from 0 to about 12 ng/ml, are mixed with a fixed
concentration of .sup.125I-ZTNF4, and incubated for about two hours
at 37.degree. C. on the plate coated with Ztnfr12-N. Controls
contain either Ztnfr12-N in solution, or lack Ztnfr12 polypeptides
or Ztnfr12-Fc constructs. After incubation, the plates are washed
and counted.
[0248] In another approach, increasing concentrations of .sup.125I
labeled ZTNF4 are incubated with Ztnfr12 polypeptides or Ztnfr12-Fc
constructs, and the radioactivity associated with precipitated
ZTNF4-Ztnfr12 polypeptide complexes, or ZTNF4-Ztnfr12-Fc complexes,
is determined. As an illustration, about 0.05 nM of Ztnfr12
polypeptides or Ztnfr12-Fc construct can be incubated with about
0.4 pM to about 1.5 nM .sup.125I-ZTNF4 for 30 minutes at room
temperature in a total volume of 0.25 ml/tube. A Pansorbin (Staph
A) suspension is added to each tube, and after 15 minutes, the
samples are centrifuged, washed twice, and the pellets counted.
Nonspecific binding is determined by the addition of 130 nM
unlabeled ZTNF4 to the .sup.125I-ZTNF4/Ztnfr12 polypeptide mix, or
to the .sup.125I-ZTNF4/Ztnfr12-Fc mix. Specific binding is
calculated by subtracting the cpm bound in the presence of
unlabeled ZTNF4 from the total cpm bound at each concentration of
.sup.125I-ZTNF4.
[0249] Alternatively, Ztnfr12 polypeptides and
Ztnfr12-immunoglobulin fusion proteins can be characterized by the
ability to inhibit the stimulation of human B cells by soluble
ZTNF4, as described by Gross et al., international publication No.
WO00/40716. Briefly, human B cells are isolated from peripheral
blood mononuclear cells using CD19 magnetic beads and the VarioMacs
magnetic separation system (Miltenyi Biotec; Auburn, Calif.)
according to the manufacturer's instructions. Purified B cells are
mixed with soluble ZTNF4 (25 ng/ml) and recombinant human IL-4 (10
ng/ml Pharmingen), and the cells are plated onto round bottom 96
well plates at 1.times.10.sup.5 cells per well.
[0250] Ztnfr12 polypeptides or Ztnfr12-immunoglobulin proteins can
be diluted from about 5 .mu.g/ml to about 6 ng/ml, and incubated
with the B cells for five days, pulsing overnight on day four with
1 .mu.Ci .sup.3H-thymidine per well. As a control, Ztnfr12
polypeptides or Ztnfr12-immunoglobulin protein can also be
incubated with B cells and IL-4 without ZTNF4. Plates are harvested
using Packard plate harvester, and counted using the Packard
reader.
[0251] Well-established animal models are available to test in vivo
efficacy of Ztnfr12 polypeptides or Ztnfr12-immunoglobulin proteins
in certain disease states. For example, Ztnfr12 polypeptides or
Ztnfr12-immunoglobulin proteins can be tested in a number of animal
models of autoimmune disease, such as MRL-lpr/lpr or NZB x NZW F1
congenic mouse strains, which serve as a model of SLE (systemic
lupus erythematosus). Such animal models are known in the art (see,
for example, Cohen and Miller (Eds.), Autoimmune Disease Models: A
Guidebook (Academic Press, Inc. 1994).
[0252] Offspring of a cross between New Zealand Black (NZB) and New
Zealand White (NZW) mice develop a spontaneous form of SLE that
closely resembles SLE in humans. The offspring mice, known as NZBW
begin to develop IgM autoantibodies against T-cells at one month of
age, and by five to seven months of age, anti-DNA autoantibodies
are the dominant immunoglobulin. Polyclonal B-cell hyperactivity
leads to overproduction of autoantibodies. The deposition of these
autoantibodies, particularly those directed against single stranded
DNA, is associated with the development of glomerulonephritis,
which manifests clinically as proteinuria, azotemia, and death from
renal failure.
[0253] Kidney failure is the leading cause of death in mice
affected with spontaneous SLE, and in the NZBW strain, this process
is chronic and obliterative. The disease is more rapid and severe
in females than males, with mean survival of only 245 days as
compared to 406 days for the males. While many of the female mice
will be symptomatic (proteinuria) by seven to nine months of age,
some can be much younger or older when they develop symptoms. The
fatal immune nephritis seen in the NZBW mice is very similar to the
glomerulonephritis seen in human SLE, making this spontaneous
murine model very attractive for testing of potential SLE
therapeutics (Putterman and Naparstek, "Murine Models of
Spontaneous Systemic Lupus Erythematosus," in Autoimmune Disease
Models: A Guidebook, pages 217-234 (Academic Press, Inc., 1994);
Mohan et al., J. Immunol. 154:1470 (1995); and Daikh et al., J.
Immunol. 159:3104 (1997)).
[0254] As described by Gross et al., international publication No.
WO00/40716, TACI-immunoglobulin proteins, which bind ZTNF4, can be
administered to NZBW mice to monitor its suppressive effect on B
cells over the five-week period when, on average, B-cell
autoantibody production is believed to be at high levels in NZBW
mice. This method can be applied to determine efficacy of a Ztnfr12
polypeptide of Ztnfr12-immunoglobulin fusion protein. Briefly, one
hundred 8-week old female (NZB.times.NZW)F.sub.1 mice can be
divided into six groups of 15 mice. Prior to treatment, the mice
are monitored once a month for urine protein, and blood is drawn
for CBC and serum banking. Serum can be screened for the presence
of autoantibodies. Because proteinuria is the hallmark sign of
glomerulonephritis, urine protein levels are monitored by dipstick
at regular intervals over the course of the study. Treatment can
begin when mice are approximately five months of age. Mice can
receive intraperitoneal injections of vehicle only (phosphate
buffered saline) or human immunoglobulin (control protein) or
Ztnfr12-immunoglobulin protein (e.g., 20 to 100 .mu.g test protein
per dose) three times a week for five weeks. Similar studies can be
performed with Ztnfr12 polypeptides.
[0255] Blood is collected twice during treatment, and will be
collected at least twice following treatment. Urine dipstick values
for proteinuria and body weights are determined every two weeks
after treatment begins. Blood, urine dipstick value and body weight
are collected at the time of euthanasia. The spleen and thymus are
divided for fluorescent activated cell sorting analysis and
histology. Submandibular salivary glands, mesenteric lymph node
chain, liver lobe with gall bladder, cecum and large intestine,
stomach, small intestine, pancreas, right kidney, adrenal gland,
tongue with trachea and esophagus, heart and lungs are also
collected for histology.
[0256] Murine models for experimental allergic encephalomyelitis
have been used as a tool to investigate both the mechanisms of
immune-mediated disease, and methods of potential therapeutic
intervention. The model resembles human multiple sclerosis, and
produces demyelination as a result of T-cell activation to
neuroproteins such as myelin basic protein, or proteolipid protein.
Inoculation with antigen leads to induction of CD4+, class II
MHC-restricted T-cells (Th1). Changes in the protocol for
experimental allergic encephalomyelitis can produce acute,
chronic-relapsing, or passive-transfer variants of the model
(Weinberg et al., J. Immunol. 162:1818 (1999); Mijaba et al., Cell.
Immunol. 186:94 (1999); and Glabinski, Meth. Enzym. 288:182
(1997)).
[0257] Gross et al., international publication No. WO00/40716,
describe one approach to evaluating the efficacy of
TACI-immunoglobulin proteins in the amelioration of symptoms
associated with experimental allergic encephalomyelitis. Briefly,
25 female PLxSJL F1 mice (12 weeks old) are given a subcutaneous
injection of 125 .mu.g/mouse of antigen (myelin Proteolipid
Protein, PLP, residues 139-151), formulated in complete Freund's
Adjuvant. The mice are divided into five groups of five mice.
Intraperitoneal injections of pertussis toxin (400 ng) are given on
Day 0 and 2. The groups are given a 1.times., 10.times., or
100.times. dose of TACI-immunoglobulin protein, one group will
receive vehicle only, and one group will receive no treatment.
Prevention therapy begins on Day 0, intervention therapy begins on
day 7, or at onset of clinical signs. Signs of disease, weight
loss, and paralysis manifest in approximately 10 to 14 days, and
last for about one week. Animals are assessed daily by collecting
body weights and assigning a clinical score to correspond to the
extent of their symptoms. Clinical signs of experimental allergic
encephalomyelitis appear within 10 to 14 days of inoculation and
persist for approximately one week. At the end of the study, all
animals are euthanized by gas overdose, and necropsied. The brain
and spinal column are collected for histology or frozen for mRNA
analysis. Body weight and clinical score data are plotted by
individual and by group. This approach can be used to test Ztnfr12
polypeptides or Ztnfr12-immunoglobulin fusion proteins.
[0258] In the collagen-induced arthritis model, mice develop
chronic inflammatory arthritis, which closely resembles human
rheumatoid arthritis. Since collagen-induced arthritis shares
similar immunological and pathological features with rheumatoid
arthritis, this makes it an ideal model for screening potential
human anti-inflammatory compounds. Another advantage in using the
collagen-induced arthritis model is that the mechanisms of
pathogenesis are known. The T and B cell epitopes on type II
collagen have been identified, and various immunological
(delayed-type hypersensitivity and anti-collagen antibody) and
inflammatory (cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediating arthritis have been
determined, and can be used to assess test compound efficacy in the
models (Wooley, Curr. Opin. Rheum. 3:407 (1999); Williams et al.,
Immunol. 89:9784 (1992); Myers et al., Life Sci. 61:1861 (1997);
and Wang et al., Immunol. 92:8955 (1995)).
[0259] Gross et al., international publication No. WO00/40716,
describe a method for evaluating the efficacy of
TACI-immunoglobulin proteins in the amelioration of symptoms
associated with collagen-induced arthritis. In brief, eight-week
old male DBA/1J mice (Jackson Labs) are divided into groups of five
mice/group and are given two subcutaneous injections of 50 to 100
.mu.l of 1 mg/ml collagen (chick or bovine origin), at three week
intervals. One control does not receive collagen injections. The
first injection is formulated in Complete Freund's Adjuvant, and
the second injection is formulated in Incomplete Freund's Adjuvant.
TACI-immunoglobulin protein is administered prophylactically at or
before the second injection, or after the animal develops a
clinical score of two or more that persists at least 24 hours.
Animals begin to show symptoms of arthritis following the second
collagen injection, usually within two to three weeks. For example,
TACI-Fc, a control protein, human IgFc, or phosphate-buffered
saline (vehicle) can be administered prophylactically beginning
seven days before the second injection (day-7). Proteins can be
administered at 100 .mu.g, given three times a week as a 200
.quadrature.l intraperitoneal injection, and continued for four
weeks. The extent of disease is evaluated in each paw using a
caliper to measure paw thickness and assigning a clinical score to
each paw. For example, a clinical score of "0" indicates a normal
mouse, a score of "1" indicates that one or more toes are inflamed,
a score of "2" indicates mild paw inflammation, a score of "3"
indicates moderate paw inflammation, and a score of "4" indicates
severe paw inflammation. Animals are euthanized after the disease
as been established for a set period of time, usually seven days.
Paws are collected for histology or mRNA analysis, and serum is
collected for immunoglobulin and cytokine assays. The
collagen-induced arthritis model can be used to test Ztnfr12
polypeptides or Ztnfr12-immunoglobulin fusion proteins.
[0260] Myasthenia gravis is another autoimmune disease for which
murine models are available. Myasthenia gravis is a disorder of
neuromuscular transmission involving the production of
autoantibodies directed against the nicotinic acetylcholine
receptor. This disease is acquired or inherited with clinical
features including abnormal weakness and fatigue on exertion.
[0261] A murine model of myasthenia gravis has been established.
(Christadoss et al., "Establishment of a Mouse Model of Myasthenia
gravis Which Mimics Human Myasthenia gravid Pathogenesis for Immune
Intervention," in Immunobiology of Proteins and Peptides VIII,
Atassi and Bixler (Eds.), pages 195-199 (1995)). Experimental
autoimmune myasthenia gravis is an antibody mediated disease
characterized by the presence of antibodies to acetylcholine
receptor. These antibodies destroy the receptor leading to
defective neuromuscular electrical impulses, resulting in muscle
weakness. In the experimental autoimmune myasthenia gravis model,
mice are immunized with the nicotinic acetylcholine receptor.
Clinical signs of myasthenia gravis become evident weeks after the
second immunization. Experimental autoimmune myasthenia gravis is
evaluated by several methods including measuring serum levels of
acetylcholine receptor antibodies by radioimmunoassay (Christadoss
and Dauphinee, J. Immunol. 136:2437 (1986); Lindstrom et al.,
Methods Enzymol. 74:432 (1981)), measuring muscle acetylcholine
receptor, or electromyography (Coligan et al. (Eds.), Protocols in
Immunology. Vol. 3, page 15.8.1 (John Wiley & Sons, 1997)).
[0262] The effect of Ztnfr12 polypeptides or Zntfr12-immunoglobulin
fusion proteins on experimental autoimmune myasthenia gravis can be
determined by administering fusion proteins during ongoing clinical
myasthenia gravis in B6 mice. For example, 100 B6 mice are
immunized with 20 .mu.g acetylcholine receptor in complete Freund's
adjuvant on days 0 and 30. Approximately 40 to 60% of mice will
develop moderate (grade 2) to severe (grade 3) clinical myasthenia
gravis after the boost with acetylcholine receptor. Mice with grade
2 and 3 clinical disease are divided into three groups (with equal
grades of weakness) and weighed (mice with weakness also lose
weight, since they have difficulty in consuming food and water) and
bled for serum (for pre-treatment anti-acetylcholine receptor
antibody and isotype level). Group A is injected I.P with phosphate
buffered saline, group B is injected intraperitoneally with human
IgG-Fc as a control protein (100 .mu.g), and group C is injected
with 100 .mu.g of Ztnfr12 polypeptides or Zntfr12-immunoglobulin
fusion proteins three times a week for four weeks. Mice are
screened for clinical muscle weakness twice a week, and weighed and
bled for serum 15 and 30 days after the commencement of treatment.
Whole blood is collected on day 15 to determine T/B cell ratio by
fluorescence activated cell sorter analysis using markers B220 and
CD5. Surviving mice are killed 30 to 45 days after the initiation
of treatment, and their carcasses are frozen for later extraction
of muscle acetylcholine receptor to determine the loss of muscle
acetylcholine receptor, the primary pathology in myasthenia gravis
(see, for example, Coligan et al. (Eds.), Protocols in Immunology.
Vol. 3, page 15.8.1 (John Wiley & Sons, 1997)).
[0263] Serum antibodies to mouse muscle acetylcholine receptor can
be determined by an established radioimmunoassay, and
anti-acetylcholine receptor antibody isotypes (IgM, IgG1, IgG2b and
IgG2c) is measured by ELISA. Such methods are known. The effects of
Ztnfr12 polypeptides or Zntfr12-immunoglobulin fusion proteins on
ongoing clinical myasthenia gravis, anti-acetylcholine receptor
antibody and isotype level, and muscle acetylcholine receptor loss
are determined.
[0264] Approximately 100 mice can be immunized with 20 .mu.g
acetylcholine receptor in complete Freund's adjuvant on day 0 and
30. Mice with clinical myasthenia gravis are divided into four
groups. Group A is injected intraperitoneally with 100 .mu.g
control Fc, group B is injected with 20 .mu.g control Fc, group C
is injected with 100 .mu.g Ztnfr12 polypeptide or
Zntfr12-immunoglobulin fusion protein, and group D is injected with
20 .mu.g Ztnfr12 polypeptide or Zntfr12-immunoglobulin fusion
protein three times a week for four weeks. Mice are weighed and
bled for serum before, and 15 and 30 days after the start of the
treatment. Serum is tested for anti-acetylcholine receptor antibody
and isotypes as described above. Muscle acetylcholine receptor loss
can also be measured.
[0265] These in vitro and in vivo assays can also be used to
evaluate Ztnfr12 antibody components, antibody fusion proteins,
immunoconjugates, and the like. Other suitable assays of Ztnfr12
polypeptides, Zntfr12-immunoglobulin fusion proteins, or Ztnfr12
antibody components can be determined by those of skill in the
art.
8. Isolation of Ztnfr12 Polypeptides
[0266] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95% purity with respect to
contaminating macromolecules, particularly other proteins and
nucleic acids, and free of infectious and pyrogenic agents. The
polypeptides of the present invention may also be purified to a
pharmaceutically pure state, which is greater than 99.9% pure. In
certain preparations, purified polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin.
[0267] Fractionation and/or conventional purification methods can
be used to obtain preparations of Ztnfr12 purified from natural
sources (e.g., lymph node tissue), synthetic Ztnfr12 polypeptides,
and recombinant Ztnfr12 polypeptides and fusion Ztnfr12
polypeptides purified from recombinant host cells. In general,
ammonium sulfate precipitation and acid or chaotrope extraction may
be used for fractionation of samples. Exemplary purification steps
may include hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable chromatographic
media include derivatized dextrans, agarose, cellulose,
polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and
Q derivatives are suitable. Exemplary chromatographic media include
those media derivatized with phenyl, butyl, or octyl groups, such
as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate
moieties.
[0268] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0269] Additional variations in Ztnfr12 isolation and purification
can be devised by those of skill in the art. For example,
anti-Ztnfr12 antibodies, obtained as described below, can be used
to isolate large quantities of protein by immunoaffinity
purification.
[0270] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification.
[0271] Ztnfr12 polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. Ztnfr12
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; PEGylated or non-PEGylated; and may or may not
include an initial methionine amino acid residue.
9. Production of Antibodies to Ztnfr12 Proteins
[0272] Antibodies to Ztnfr12 can be obtained, for example, using
the product of a Ztnfr12 expression vector or Ztnfr12 isolated from
a natural source as an antigen. Particularly useful anti-Ztnfr12
antibodies "bind specifically" with Ztnfr12. Antibodies are
considered to be specifically binding if the antibodies exhibit at
least one of the following two properties: (1) antibodies bind to
Ztnfr12 with a threshold level of binding activity, and (2)
antibodies do not significantly cross-react with polypeptides
related to Ztnfr12.
[0273] With regard to the first characteristic, antibodies
specifically bind if they bind to a Ztnfr12 polypeptide, peptide or
epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M.sup.-1 or greater, more preferably
10.sup.8 M.sup.-1 or greater, and most preferably 10.sup.9 M.sup.-1
or greater. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).
With regard to the second characteristic, antibodies do not
significantly cross-react with related polypeptide molecules, for
example, if they detect Ztnfr12, but not presently known
polypeptides using a standard Western blot analysis. Examples of
known related polypeptides include known tumor necrosis factor
receptors. For example, certain anti-Ztnfr12 antibodies bind with
Ztnfr12, but not with TACI or BCMA.
[0274] Anti-Ztnfr12 antibodies can be produced using antigenic
Ztnfr12 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:2 or another amino acid
sequence disclosed herein. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of the
invention, containing from 30 to 50 amino acids, or any length up
to and including the entire amino acid sequence of a polypeptide of
the invention, also are useful for inducing antibodies that bind
with Ztnfr12. It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
typically avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0275] As an illustration, potential antigenic sites in Ztnfr12
were identified using the Jameson-Wolf method, Jameson and Wolf,
CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0276] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; .alpha. region threshold=103;
.beta. region threshold=105; Garnier-Robson parameters: .alpha. and
.beta. decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible.
[0277] The results of this analysis indicated that the following
amino acid sequences of SEQ ID NO:2 would provide suitable
antigenic molecules: amino acids 1 to 17, amino acids 39 to 64, 102
to 129, amino acids 135 to 142, amino acids 146 to 159, and amino
acids 174 to 182. The present invention contemplates the use of any
one of these antigenic amino acid sequences to generate antibodies
to Ztnfr12. The present invention also contemplates polypeptides
comprising at least one of these antigenic molecules.
[0278] Similarly, the results of Jameson-Wolf analysis indicated
that the following amino acid sequences of SEQ ID NO:13 would
provide suitable antigenic molecules: amino acids 10 to 26, amino
acids 45 to 69, 106 to 113, and amino acids 139 to 151. The present
invention contemplates the use of any one of these antigenic amino
acid sequences to generate antibodies to murine Ztnfr12. The
present invention also contemplates polypeptides comprising at
least one of these antigenic molecules.
[0279] Useful antibodies can also be produced using antigenic
molecules that comprise at least one Ztnfr12 exon of the human
gene. For example, such antigenic molecules can comprise
polypeptides that consist of the following amino acid sequences of
SEQ ID NO:2: amino acid residues 1 to 45, amino acid residues 47 to
122, and amino acid residues 124 to 184.
[0280] Antibodies that block signal transduction by ZTNF4 can be
useful in therapeutic applications. Blocking anti-Ztnfr12
antibodies can be identified, for example, by their inhibition of
biotin-ZTNF4 binding to Ztnfr12 on tumor cell lines. Antibodies
that bind with the Ztnfr12 intracellular domain can also be used to
block ZTNF4-induced signal transduction. Such antibodies can bind
the intracellular domain of Ztnfr12 within amino acid residues 101
to 184 of SEQ ID NO:2. In addition, a potential TRAF binding domain
resides at amino acid residues 159 to 178 of SEQ ID NO:2. Thus,
certain signal-blocking antibodies can bind the intracellular
domain of Ztnfr12 within this region. The present invention
includes antibodies that bind Ztnfr12 within amino acid residues
159 to 178 of SEQ ID NO:2. Standard methods are available to
introduce antibodies to the intracellular compartment of cells. For
example, such antibodies can be encapsulated in liposomes.
[0281] Signal-inducing anti-Ztnfr12 antibodies are also useful.
Antibodies that induce a signal by binding to a Ztnfr12 receptor
can also be identified using a suitable reporter cell line that
contains a transcriptional reporter element and Ztnfr12. As an
illustration, an engineered mammalian cell line (e.g., Jurkat),
which expresses Ztnfr12, and a transcriptional reporter gene can be
used to test anti-Ztnfr12 monoclonal antibodies for their ability
to stimulate transcription of a reporter gene (e.g.,
luciferase).
[0282] Polyclonal antibodies to recombinant Ztnfr12 protein or to
Ztnfr12 isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a Ztnfr12 polypeptide can be increased through
the use of an adjuvant, such as alum (aluminum hydroxide) or
Freund's complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
Ztnfr12 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0283] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-Ztnfr12 antibody of the
present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310
(1990).
[0284] Alternatively, monoclonal anti-Ztnfr12 antibodies can be
generated. Rodent mono-clonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0285] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a Ztnfr12 gene product,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0286] In addition, an anti-Ztnfr12 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0287] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0288] For particular uses, it may be desirable to prepare
fragments of anti-Ztnfr12 antibodies. Such antibody fragments can
be obtained, for example, by proteolytic hydrolysis of the
antibody. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. As an
illustration, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0289] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0290] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0291] The Fv fragments may comprise V.sub.H and V.sub.L chains,
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector, which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0292] As an illustration, a scFV can be obtained by exposing
lymphocytes to Ztnfr12 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled Ztnfr12 protein or peptide).
Genes encoding polypeptides having potential Ztnfr12 polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides, which interact with a
known target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic macromolecule, or
organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the
art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.
Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are
available commercially, for instance from CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the Ztnfr12 sequences disclosed
herein to identify proteins which bind to Ztnfr12.
[0293] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0294] Another useful anti-receptor antibody is a chimeric
antibody. A chimeric antibody comprises the variable domains and
complementary determining regions derived from a rodent antibody,
while the remainder of the antibody molecule is derived from a
human antibody. See, for example, Verma and Boleti, "Engineering
Antibody Molecules," in Diagnostic and Therapeutic Antibodies,
George and Urch (Eds.), pages 35-52 (Humana Press, Inc. 2000).
[0295] Alternatively, an anti-Ztnfr12 antibody may be derived from
a "humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0296] The present invention includes the use of compositions that
comprise an antibody component that binds the Ztnfr12 extracellular
region, and an antibody component that binds at least one of a TACI
extracellular region and a BCMA extracellular region. For example,
such a "multispecific antibody composition" can comprise a
heteroantibody mixture (i.e., an aggregate of at least two antibody
components, each having a different binding specificity), a
bispecific antibody (i.e., an antibody component with two different
combining sites), a single chain bispecific polypeptide, and the
like.
[0297] Bispecific antibodies can be made by a variety of
conventional methods. As an illustration, bispecific antibodies
have been prepared by oxidative cleavage of Fab' fragments
resulting from reductive cleavage of different antibodies. See, for
example, Winter et al., Nature 349:293 (1991). This can be carried
out by mixing two different F(ab').sub.2 fragments produced by
pepsin digestion of two different antibodies, reductive cleavage to
form a mixture of Fab' fragments, followed by oxidative reformation
of the disulfide linkages to produce a mixture of F(ab').sub.2
fragments including bispecific antibodies containing a Fab' potion
specific to each of the original epitopes. General techniques for
the preparation of such bispecific antibodies can be found, for
example, in Nisonhoff et al., Arch Biochem. Biophys. 93:470 (1961),
Hammerling et al., J. Exp. Med. 128:1461 (1968), and U.S. Pat. No.
4,331,647.
[0298] Alternatively, linkage can be achieved by using a
heterobifunctional linker such as maleimide-hydroxysuccinimide
ester. Reaction of the ester with an antibody or fragment will
derivatize amine groups on the antibody or fragment, and the
derivative can then be reacted with, for example, an antibody Fab
fragment having free sulfhydryl groups (or, a larger fragment or
intact antibody with sulfhydryl groups appended thereto by, for
example, Traut's Reagent). Such a linker is less likely to
crosslink groups in the same antibody and improves the selectivity
of the linkage.
[0299] As another example, bispecific F(ab').sub.2 antibodies can
be produced by linking two Fab' fragments via their hinge region SH
groups using the bifunctional crosslinker o-phenylenedimaleimide.
See, for example, Tso, "F(ab').sub.2 Fusion Proteins and Bispecific
F(ab').sub.2," in Chamow and Ashkenazi (Eds.), Antibody Fusion
Proteins, pages 127-150 (Wiley-Liss, Inc. 1999), and French, "How
to Make Bispecific Antibodies," in George and Urch (Eds.),
Diagnostic and Therapeutic Antibodies, pages 333-339 (Humana Press,
Inc. 2000).
[0300] It is advantageous to link the antibodies or fragments at
sites remote from the antigen binding sites. This can be
accomplished by, for example, linkage to cleaved interchain
sulfydryl groups, as noted above. Another method involves reacting
an antibody having an oxidized carbohydrate portion with another
antibody which has at lease one free amine function. This results
in an initial Schiff base (imine) linkage, which can be stabilized
by reduction to a secondary amine, for example, by borohydride
reduction, to form the final composite. Such site-specific linkages
are disclosed, for small molecules, in U.S. Pat. No. 4,671,958, and
for larger addends in U.S. Pat. No. 4,699,784.
[0301] Alternatively, bispecific antibodies can be produced by
fusing two hybridoma cell lines, one cell line that produces
anti-Ztnfr12 monoclonal antibody, and one cell line that produces
either anti-BCMA monoclonal antibody, or anti-TACI monoclonal
antibody. Techniques for producing tetradomas are described, for
example, by Milstein et al., Nature 305:537 (1983), and Pohl et
al., Int. J. Cancer 54:418 (1993).
[0302] Bispecific antibodies can also be produced by genetic
engineering. For example, vectors containing DNA coding for
variable domains of an anti-Ztnfr12 monoclonal antibody can be
introduced into hybridomas that secrete anti-TACI antibodies, or
anti-BCMA antibodies. The resulting transfectomas produce
bispecific antibodies that bind Ztnfr12 and either BCMA or TACI.
Alternatively, chimeric genes can be designed that encode an
anti-Ztnfr12 binding domain and at least one anti-BCMA binding
domain or anti-TACI binding domain. A variety of genetic strategies
for producing bispecifc antibodies are available to those of skill
in the art. In one approach, for example, bispecific F(ab').sub.2
are produced using leucine zippers. See, for example, Tso,
"F(ab').sub.2 Fusion Proteins and Bispecific F(ab').sub.2," in
Chamow and Ashkenazi (Eds.), Antibody Fusion Proteins, pages
127-150 (Wiley-Liss, Inc. 1999). General techniques for producing
bispecific antibodies by genetic engineering are described, for
example, by Songsivilai et al., Biochem. Biophys. Res. Commun.
164:271 (1989), Traunecker et al., EMBO J. 10:3655 (1991), and
Weiner et al., J. Immunol. 147:4035 (1991).
[0303] A bispecific molecule of the invention can also be a single
chain bispecific molecule, such as a single chain bispecific
antibody, a single chain bispecific molecule comprising one single
chain antibody and a binding determinant, or a single chain
bispecific molecule comprising two binding determinants.
[0304] Bispecific antibodies can be screened using standard
techniques, such as a bispecific ELISA.
[0305] The present invention further includes polyclonal
anti-idiotype antibodies, which can be prepared by immunizing
animals with anti-Ztnfr12 antibodies or antibody fragments, using
standard techniques. See, for example, Green et al., "Production of
Polyclonal Antisera," in Methods In Molecular Biology:
Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press
1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-Ztnfr12 antibodies or antibody fragments as immunogens with
the techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.
77:1875 (1996).
[0306] Anti-Ztnfr12 antibody components and anti-idiotype
antibodies of the present invention can be useful to neutralize the
effects of a Ztnfr12 ligand (e.g., ZTNF4) for treating pre-B or
B-cell leukemias, such as plasma cell leukemia, chronic or acute
lymphocytic leukemia, myelomas such as multiple myeloma, plasma
cell myeloma, endothelial myeloma and giant cell myeloma, and
lymphomas such as non-Hodgkins lymphoma, which are associated with
an increase in a Ztnfr12 ligand (e.g., ZTNF4). Additional examples
of B cell lymphomas that may be treated with the molecules
described herein include Burkitt's lymphoma, Non-Burkitt's
lymphoma, follicular lymphoma, acute lymphoblastic leukemia, large
cell lymphoma, marginal zone lymphoma, mantle cell lymphoma, large
cell lymphoma (e.g., immunoblastic lymphoma), small lymphocytic
lymphoma, and other B cell lymphomas.
10. Use of Ztnfr12 Nucleotide Sequences to Detect Gene Expression
and Gene Structure
[0307] Nucleic acid molecules can be used to detect the expression
of a Ztnfr12 gene in a biological sample. Suitable probe molecules
include double-stranded nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or a portion thereof, as well
as single-stranded nucleic acid molecules having the complement of
the nucleotide sequence of SEQ ID NO:1, or a portion thereof. Probe
molecules may be DNA, RNA, oligonucleotides, and the like. As used
herein, the term "portion" refers to at least eight nucleotides to
at least 20 or more nucleotides. Illustrative probes bind with
regions of the Ztnfr12 gene that have a low sequence similarity to
comparable regions in other tumor necrosis factor receptor
genes.
[0308] In a basic assay, a single-stranded probe molecule is
incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target Ztnfr12 RNA species. After
separating unbound probe from hybridized molecules, the amount of
hybrids is detected.
[0309] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35S. Alternatively, Ztnfr12 RNA can be detected
with a nonradioactive hybridization method (see, for example, Isaac
(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes
(Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by enzymatic conversion of chromogenic or chemiluminescent
substrates. Illustrative nonradioactive moieties include biotin,
fluorescein, and digoxigenin.
[0310] Ztnfr12 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nature Medicine 4:467
(1998)).
[0311] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)).
[0312] PCR primers can be designed to amplify a portion of the
Ztnfr12 gene that has a low sequence similarity to a comparable
region in other proteins, such as other tumor necrosis factor
receptor proteins.
[0313] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with Ztnfr12 primers (see, for example, Wu et
al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)).
PCR is then performed and the products are analyzed using standard
techniques.
[0314] As an illustration, RNA is isolated from biological sample
using, for example, the guanidinium-thiocyanate cell lysis
procedure described above. Alternatively, a solid-phase technique
can be used to isolate mRNA from a cell lysate. A reverse
transcription reaction can be primed with the isolated RNA using
random oligonucleotides, short homopolymers of dT, or Ztnfr12
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. Ztnfr12 sequences are amplified by the
polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0315] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled Ztnfr12 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK calorimetric assay.
[0316] Another approach for detection of Ztnfr12 expression is
cycling probe technology, in which a single-stranded DNA target
binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
Ztnfr12 sequences can utilize approaches such as nucleic acid
sequence-based amplification, cooperative amplification of
templates by cross-hybridization, and the ligase chain reaction
(see, for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997),
Dyer et al., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur.
J. Biochem. 243:358 (1997), and Chadwick et al., J. Virol. Methods
70:59 (1998)). Other standard methods are known to those of skill
in the art.
[0317] Ztnfr12 probes and primers can also be used to detect and to
localize Ztnfr12 gene expression in tissue samples. Methods for
such in situ hybridization are well-known to those of skill in the
art (see, for example, Choo (ed.), In Situ Hybridization Protocols
(Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC
Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc.
1997)). Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc.
1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc., 1996)). Suitable test samples include blood, urine,
saliva, tissue biopsy, and autopsy material.
[0318] The Ztnfr12 gene resides in chromosome 22q13.2, a region
that is associated with diseases and disorders, such as Fechtner
syndrome, Sorsby fundus dystrophy, deafness, and neutrophil
immunodeficiency syndrome. In addition, mutations of cytokine
receptors are associated with particular diseases. For example,
polymorphisms of cytokine receptors are associated with pulmonary
alveolar proteinosis, familial periodic fever, and erythroleukemia.
Thus, Ztnfr12 nucleotide sequences can be used in linkage-based
testing for various diseases, and to determine whether a subject's
chromosomes contain a mutation in the Ztnfr12 gene. Detectable
chromosomal aberrations at the Ztnfr12 gene locus include, but are
not limited to, aneuploidy, gene copy number changes, insertions,
deletions, restriction site changes and rearrangements. Of
particular interest are genetic alterations that inactivate a
Ztnfr12 gene.
[0319] Aberrations associated with the Ztnfr12 locus can be
detected using nucleic acid molecules of the present invention by
employing molecular genetic techniques, such as restriction
fragment length polymorphism analysis, short tandem repeat analysis
employing PCR techniques, amplification-refractory mutation system
analysis, single-strand conformation polymorphism detection, RNase
cleavage methods, denaturing gradient gel electrophoresis,
fluorescence-assisted mismatch analysis, and other genetic analysis
techniques known in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular
Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular
Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren
(ed.), Laboratory Protocols for Mutation Detection (Oxford
University Press 1996), Birren et al. (eds.), Genome Analysis, Vol.
2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),
Dracopoli et al. (eds.), Current Protocols in Human Genetics (John
Wiley & Sons 1998), and Richards and Ward, "Molecular
Diagnostic Testing," in Principles of Molecular Medicine, pages
83-88 (Humana Press, Inc. 1998)).
[0320] As an illustration, large deletions in a Ztnfr12 gene can be
detected using Southern hybridization analysis or PCR
amplification. Deletions in a particular Ztnfr12 exon can be
detected using PCR primers that flank the exon. Table 1 provides
the locations of Ztnfr12 exons present in the nucleotide sequences
of SEQ ID NOs:1 and 9. This information can be used to design
primers that amplify particular exons.
[0321] Mutations can also be detected by hybridizing an
oligonucleotide probe comprising a normal Ztnfr12 sequence to a
Southern blot or to membrane-bound PCR products. Discrimination is
achieved by hybridizing under conditions of high stringency, or by
washing under varying conditions of stringency. This analysis can
be targeted to a particular coding sequence. Alternatively, this
approach is used to examine splice-donor or splice-acceptor sites
in the immediate flanking intron sequences, where disease-causing
mutations are often located. Suitable oligonucleotides can be
designed by extending the sequence into an exon of choice, using
the information provided in Table 1 and SEQ ID NOs:1 and 9.
[0322] The duplication of all or part of a gene can cause a
disorder when the insertion of the duplicated material is inserted
into the reading frame of a gene and causes premature termination
of translation. Duplication and insertion can be examined directly
by analyzing a subject's genomic DNA with standard methods, such as
Southern hybridization, fluorescence in situ hybridization,
pulsed-field gel analysis, or PCR. In addition, the effect of
duplication can be detected with the protein truncation assay
described below.
[0323] A point mutation can lead to a nonconservative change
resulting in the alteration of Ztnfr12 function or a change of an
amino acid codon to a stop codon. If a point mutation occurs within
an intron, the mutation may affect the fidelity of splicing. A
point mutation can be detected using standard techniques, such as
Southern hybridization analysis, PCR analysis, sequencing, ligation
chain reaction, and other approaches. In single-strand conformation
polymorphism analysis, for example, fragments amplified by PCR are
separated into single strands and fractionated by polyacrylamide
gel electrophoresis under denaturing conditions. The rate of
migration through the gel is a function of conformation, which
depends upon the base sequence. A mutation can alter the rate of
migration of one or both single strands. In a chemical cleavage
approach, hybrid molecules are produced between test and control
DNA (e.g., DNA that encodes the amino acid sequence of SEQ ID
NO:2). Sites of base pair mismatch due to a mutation will be
mispaired, and the strands will be susceptible to chemical cleavage
at these sites.
[0324] The protein truncation test is also useful for detecting the
inactivation of a gene in which translation-terminating mutations
produce only portions of the encoded protein (see, for example,
Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to this
approach, RNA is isolated from a biological sample, and used to
synthesize cDNA. PCR is then used to amplify the Ztnfr12 target
sequence and to introduce an RNA polymerase promoter, a translation
initiation sequence, and an in-frame ATG triplet. PCR products are
transcribed using an RNA polymerase, and the transcripts are
translated in vitro with a T7-coupled reticulocyte lysate system.
The translation products are then fractionated by SDS-PAGE to
determine the lengths of the translation products. The protein
truncation test is described, for example, by Dracopoli et al.
(eds.), Current Protocols in Human Genetics, pages 9.11.1-9.11.18
(John Wiley & Sons 1998).
[0325] In an alternative approach, a mutation can be detected using
ribonuclease A, which will cleave the RNA strand of an RNA-DNA
hybrid at the site of a sequence mismatch. Briefly, a PCR-amplified
sequence of a Ztnfr12 gene or cDNA of a subject is hybridized with
in vitro transcribed labeled RNA probes prepared from the DNA of a
normal, healthy individual chosen from the general population. The
RNA-DNA hybrids are digested with ribonuclease A and analyzed using
denaturing gel electrophoresis. Sequence mismatches between the two
strands will cause cleavage of the protected fragment, and small
additional fragments will be detected in the samples derived from a
subject who has a mutated Ztnfr12 gene. The site of mutation can be
deduced from the sizes of the cleavage products.
[0326] Analysis of chromosomal DNA using the Ztnfr12 polynucleotide
sequence is useful for correlating disease with abnormalities
localized to chromosome 22q, in particular to chromosome 22q13.2.
In one embodiment, the methods of the present invention provide a
method of detecting a chromosome 22q13.2 abnormality in a sample
from an individual comprising: (a) obtaining Ztnfr12 RNA from the
sample, (b) generating Ztnfr12 cDNA by polymerase chain reaction,
and (c) comparing the nucleotide sequence of the Ztnfr12 cDNA to
the nucleic acid sequence as shown in SEQ ID NO:1. In further
embodiments, the difference between the sequence of the Ztnfr12cDNA
or Ztnfr12 gene in the sample and the Ztnfr12 sequence as shown in
SEQ ID NOs:1 or 9 is indicative of chromosome 22q13.2
abnormality.
[0327] In another embodiment, the present invention provides
methods for detecting in a sample from an individual, a chromosome
22q13.2 abnormality associated with an alteration in ZTNF4
activity, comprising the steps of: (a) contacting nucleic acid
molecules of the sample with a nucleic acid probe that hybridizes
with a nucleic acid molecule consisting of the nucleotide sequence
of SEQ ID NO:1, its complements or fragments, under stringent
conditions, and (b) detecting the presence or absence of
hybridization of the probe with nucleic acid molecules in the
sample, wherein the absence of hybridization is indicative of a
chromosome 22q13.2 abnormality, such as an abnormality that causes
a decrease in response to ZTNF4.
[0328] The present invention also provides methods of detecting in
a sample from an individual, a Ztnfr12 gene abnormality associated
with an alteration in ZTNF4 activity, comprising: (a) isolating
nucleic acid molecules that encode Ztnfr12 from the sample, and (b)
comparing the nucleotide sequence of the isolated Ztnfr12-encoding
sequence with the nucleotide sequence of SEQ ID NO:1, wherein the
difference between the sequence of the isolated Ztnfr12-encoding
sequence or a polynucleotide encoding the Ztnfr12 polypeptide
generated from the isolated Ztnfr12-encoding sequence and the
nucleotide sequence of SEQ ID NO:1 is indicative of an Ztnfr12 gene
abnormality associated with disease or susceptibility to a disease
in an individual, such as an abnormality that causes a decrease in
response to ZTNF4.
[0329] The present invention also provides methods of detecting in
a sample from a individual, an abnormality in expression of the
Ztnfr12 gene associated with disease or susceptibility to disease,
comprising: (a) obtaining Ztnfr12 RNA from the sample, (b)
generating Ztnfr12 cDNA by polymerase chain reaction from the
Ztnfr12 RNA, and (c) comparing the nucleotide sequence of the
Ztnfr12 cDNA to the nucleotide sequence of SEQ ID NO:1, wherein a
difference between the sequence of the Ztnfr12 cDNA and the
nucleotide sequence of SEQ ID NO:1 is indicative of an abnormality
in expression of the ZTNFR12 gene associated with disease or
susceptibility to disease.
[0330] In other aspects, the present invention provides methods for
detecting in a sample from an individual, a Ztnfr12 gene
abnormality, comprising: (a) contacting sample nucleic acid
molecules with a nucleic acid probe, wherein the probe hybridizes
to a nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, its complements or fragments, under stringent conditions, and
(b) detecting the presence or absence of hybridization is
indicative of a Ztnfr12 abnormality.
[0331] In situ hybridization provides another approach for
identifying Ztnfr12 gene abnormalities. According to this approach,
a Ztnfr12 probe is labeled with a detectable marker by any method
known in the art. For example, the probe can be directly labeled by
random priming, end labeling, PCR, or nick translation. Suitable
direct labels include radioactive labels such as .sup.32P, .sup.3H,
and .sup.35S and non-radioactive labels such as fluorescent markers
(e.g., fluorescein, Texas Red, AMCA blue
(7-amino-4-methyl-coumanine-3-acetate), lucifer yellow, rhodamine,
etc.), cyanin dyes, which are detectable with visible light,
enzymes, and the like. Probes labeled with an enzyme can be
detected through a colorimetric reaction by providing a substrate
for the enzyme. In the presence of various substrates, different
colors are produced by the reaction, and these colors can be
visualized to separately detect multiple probes if desired.
Suitable substrates for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. One
suitable substrate for horseradish peroxidase is
diaminobenzoate.
[0332] An illustrative method for detecting chromosomal
abnormalities with in situ hybridization is described by Wang et
al., U.S. Pat. No. 5,856,089. Following this approach, for example,
a method of performing in situ hybridization with a Ztnfr12 probe
to detect a chromosome structural abnormality in a cell from a
fixed tissue sample obtained from a subject can comprise the steps
of: (1) obtaining a fixed tissue sample from the patient, (2)
pretreating the fixed tissue sample obtained in step (1) with a
bisulfite ion composition, (3) digesting the fixed tissue sample
with proteinase, (4) performing in situ hybridization on cells
obtained from the digested fixed tissue sample of step (3) with a
probe which specifically hybridizes to the Ztnfr12 gene, wherein a
signal pattern of hybridized probes is obtained, (5) comparing the
signal pattern of the hybridized probe in step (4) to a
predetermined signal pattern of the hybridized probe obtained when
performing in situ hybridization on cells having a normal critical
chromosome region of interest, and (6) detecting a chromosome
structural abnormality in the patient's cells, by detecting a
difference between the signal pattern obtained in step (4) and the
predetermined signal pattern. Examples of Ztnfr12 gene
abnormalities include deletions, amplifications, translocations,
inversions, and the like. Such an assay may be used, for example,
to test tissue from a subject suspected of having disease or
disorder associated with altered responsiveness to ZTNF4.
[0333] The present invention contemplates kits for performing a
diagnostic assay for Ztnfr12 gene expression or to detect mutations
in the Ztnfr12 gene. Such kits comprise nucleic acid probes, such
as double-stranded nucleic acid molecules comprising the nucleotide
sequence of nucleotides 27 to 578 of SEQ ID NO:1, nucleotides 27 to
233 of SEQ ID NO:1, or a portion thereof, as well as
single-stranded nucleic acid molecules having the complement of the
nucleotide sequence of nucleotides 27 to 578 of SEQ ID NO:1,
nucleotides 27 to 233 of SEQ ID NO:1, or a portion thereof. Probe
molecules may be DNA, RNA, oligonucleotides, and the like. Kits may
comprise nucleic acid primers for performing PCR.
[0334] Such kits can contain all the necessary elements to perform
a nucleic acid diagnostic assay described above. A kit will
comprise at least one container comprising a Ztnfr12 probe or
primer. The kit may also comprise a second container comprising one
or more reagents capable of indicating the presence of Ztnfr12
sequences. Examples of such indicator reagents include detectable
labels such as radioactive labels, fluorochromes, chemiluminescent
agents, and the like. A kit may also comprise a means for conveying
to the user that the Ztnfr12 probes and primers are used to detect
Ztnfr12 gene expression. For example, written instructions may
state that the enclosed nucleic acid molecules can be used to
detect either a nucleic acid molecule that encodes Ztnfr12, or a
nucleic acid molecule having a nucleotide sequence that is
complementary to a Ztnfr12-encoding nucleotide sequence. The
written material can be applied directly to a container, or the
written material can be provided in the form of a packaging
insert.
[0335] 11. Use of Anti-Ztnfr12 Antibodies to Detect Ztnfr12
[0336] The present invention contemplates the use of anti-Ztnfr12
antibodies to screen biological samples in vitro for the presence
of Ztnfr12. In one type of in vitro assay, anti-Ztnfr12 antibodies
are used in liquid phase. For example, the presence of Ztnfr12 in a
biological sample can be tested by mixing the biological sample
with a trace amount of labeled Ztnfr12 and an anti-Ztnfr12 antibody
under conditions that promote binding between Ztnfr12 and its
antibody. Complexes of Ztnfr12 and anti-Ztnfr12 in the sample can
be separated from the reaction mixture by contacting the complex
with an immobilized protein which binds with the antibody, such as
an Fc antibody or Staphylococcus protein A. The concentration of
Ztnfr12 in the biological sample will be inversely proportional to
the amount of labeled Ztnfr12 bound to the antibody and directly
related to the amount of free-labeled Ztnfr12. Illustrative
biological samples include blood, urine, saliva, tissue biopsy, and
autopsy material.
[0337] Alternatively, in vitro assays can be performed in which
anti-Ztnfr12 antibody is bound to a solid-phase carrier. For
example, antibody can be attached to a polymer, such as
aminodextran, in order to link the antibody to an insoluble support
such as a polymer-coated bead, a plate or a tube. Other suitable in
vitro assays will be readily apparent to those of skill in the
art.
[0338] In another approach, anti-Ztnfr12 antibodies can be used to
detect Ztnfr12 in tissue sections prepared from a biopsy specimen.
Such immunochemical detection can be used to determine the relative
abundance of Ztnfr12 and to determine the distribution of Ztnfr12
in the examined tissue. General immunochemistry techniques are well
established (see, for example, Ponder, "Cell Marking Techniques and
Their Application," in Mammalian Development: A Practical Approach,
Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages
5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley
Interscience 1990), and Manson (ed.), Methods In Molecular Biology,
Vol. 10: Immunochemical Protocols (The Humana Press, Inc.
1992)).
[0339] Immunochemical detection can be performed by contacting a
biological sample with an anti-Ztnfr12 antibody, and then
contacting the biological sample with a detectably labeled
molecule, which binds to the antibody. For example, the detectably
labeled molecule can comprise an antibody moiety that binds to
anti-Ztnfr12 antibody. Alternatively, the anti-Ztnfr12 antibody can
be conjugated with avidin/streptavidin (or biotin) and the
detectably labeled molecule can comprise biotin (or
avidin/streptavidin). Numerous variations of this basic technique
are well-known to those of skill in the art.
[0340] Alternatively, an anti-Ztnfr12 antibody can be conjugated
with a detectable label to form an anti-Ztnfr12 immunoconjugate.
Suitable detectable labels include, for example, a radioisotope, a
fluorescent label, a chemiluminescent label, an enzyme label, a
bioluminescent label or colloidal gold. Methods of making and
detecting such detectably-labeled immunoconjugates are well-known
to those of ordinary skill in the art, and are described in more
detail below.
[0341] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0342] Anti-Ztnfr12 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0343] Alternatively, anti-Ztnfr12 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0344] Similarly, a bioluminescent compound can be used to label
anti-Ztnfr12 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0345] Alternatively, anti-Ztnfr12 immunoconjugates can be
detectably labeled by linking an anti-Ztnfr12 antibody component to
an enzyme. When the anti-Ztnfr12-enzyme conjugate is incubated in
the presence of the appropriate substrate, the enzyme moiety reacts
with the substrate to produce a chemical moiety, which can be
detected, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0346] Those of skill in the art will know of other suitable
labels, which can be employed in accordance with the present
invention. The binding of marker moieties to anti-Ztnfr12
antibodies can be accomplished using standard techniques known to
the art. Typical methodology in this regard is described by Kennedy
et al., Clin. Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim.
Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101 (1990),
Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.
[0347] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-Ztnfr12 antibodies that
have been conjugated with avidin, streptavidin, and biotin (see,
for example, Wilchek et al. (eds.), "Avidin-Biotin Technology,"
Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et
al., "Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0348] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0349] The present invention also contemplates kits for performing
an immunological diagnostic assay for Ztnfr12 gene expression. Such
kits comprise at least one container comprising an anti-Ztnfr12
antibody, or antibody fragment. A kit may also comprise a second
container comprising one or more reagents capable of indicating the
presence of Ztnfr12 antibody or antibody fragments. Examples of
such indicator reagents include detectable labels such as a
radioactive label, a fluorescent label, a chemiluminescent label,
an enzyme label, a bioluminescent label, colloidal gold, and the
like. A kit may also comprise a means for conveying to the user
that Ztnfr12 antibodies or antibody fragments are used to detect
Ztnfr12 protein. For example, written instructions may state that
the enclosed antibody or antibody fragment can be used to detect
Ztnfr12. The written material can be applied directly to a
container, or the written material can be provided in the form of a
packaging insert.
12. Therapeutic Uses of Polypeptides Having Ztnfr12 Activity
[0350] Amino acid sequences having Ztnfr12 activity can be used to
modulate the immune system by binding a Ztnfr12 ligand (e.g.,
ZTNF4), and thus, preventing the binding of the Ztnfr12 ligand with
endogenous Ztnfr12 receptor. Accordingly, the present invention
includes the use of proteins, polypeptides, and peptides having
Ztnfr12 activity (such as Ztnfr12 polypeptides, Ztnfr12 analogs
(e.g., anti-Ztnfr12 anti-idiotype antibodies), and Ztnfr12 fusion
proteins) to a subject which lacks an adequate amount of Ztnfr12
polypeptide, or which produces an excess of ZTNF4. Ztnfr12
antagonists (e.g., anti-Ztnfr12 antibodies) can be also used to
treat a subject, which produces an excess of either ZTNF4 or
Ztnfr12. These molecules can be administered to any subject in need
of treatment, and the present invention contemplates both
veterinary and human therapeutic uses. Illustrative subjects
include mammalian subjects, such as farm animals, domestic animals,
and human patients. Human or murine Ztnfr12 polypeptides can be
used for these applications.
[0351] Molecules having Ztnfr12 activity can be used for the
treatment of autoimmune diseases, B cell cancers, immunomodulation,
IBD and any antibody-mediated pathologies (e.g., ITCP, myasthenia
gravis and the like), renal diseases, indirect T cell immune
response, graft rejection, and graft versus host disease. The
polypeptides of the present invention can be targeted to
specifically regulate B cell responses during the immune response.
Additionally, the polypeptides of the present invention can be used
to modulate B cell development, development of other cells,
antibody production, and cytokine production. Polypeptides of the
present invention can also modulate T and B cell communication by
neutralizing the proliferative effects of ZTNF4.
[0352] Ztnfr12 polypeptides of the present invention can be useful
to neutralize the effects of ZTNF4 for treating pre-B or B-cell
leukemias, such as plasma cell leukemia, chronic or acute
lymphocytic leukemia, myelomas such as multiple myeloma, plasma
cell myeloma, endothelial myeloma and giant cell myeloma, and
lymphomas such as non-Hodgkins lymphoma, for which an increase in
ZTNF4 polypeptides is associated. Additional examples of B cell
lymphomas that may be treated with the molecules described herein
include Burkitt's lymphoma, Non-Burkitt's lymphoma, follicular
lymphoma, acute lymphoblastic leukemia, large cell lymphoma,
marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma
(e.g., immunoblastic lymphoma), small lymphocytic lymphoma, and
other B cell lymphomas.
[0353] ZTNF4 is expressed in CD8.sup.+ cells, monocytes, dendritic
cells, activated monocytes, which indicates that, in certain
autoimmune disorders, cytotoxic T-cells might stimulate B-cell
production through excess production of ZTNF4. Immunosuppressant
proteins that selectively block the action of B-lymphocytes would
be of use in treating disease. Autoantibody production is common to
several autoimmune diseases and contributes to tissue destruction
and exacerbation of disease. Autoantibodies can also lead to the
occurrence of immune complex deposition complications and lead to
many symptoms of systemic lupus erythomatosis, including kidney
failure, neuralgic symptoms and death. Modulating antibody
production independent of cellular response would also be
beneficial in many disease states. B cells have also been shown to
play a role in the secretion of arthritogenic immunoglobulins in
rheumatoid arthritis. As such, inhibition of ZTNF4-stimulated
antibody production would be beneficial in treatment of autoimmune
diseases such as myasthenia gravis and rheumatoid arthritis.
Immunosuppressant therapeutics such as soluble Ztnfr12 that
selectively block or neutralize the action of B-lymphocytes would
be useful for such purposes.
[0354] The invention provides methods employing Ztnfr12
polypeptides, fusions, antibodies, agonists or antagonists for
selectively blocking or neutralizing the actions of B-cells in
association with end stage renal diseases, which may or may not be
associated with autoimmune diseases. Such methods would also be
useful for treating immunologic renal diseases. Such methods would
be would be useful for treating glomerulonephritis associated with
diseases such as membranous nephropathy, IgA nephropathy or
Berger's Disease, IgM nephropathy, Goodpasture's Disease,
post-infectious glomerulonephritis, mesangioproliferative disease,
chronic lymphoid leukemia, minimal-change nephrotic syndrome. Such
methods would also serve as therapeutic applications for treating
secondary glomerulonephritis or vasculitis associated with such
diseases as lupus, polyarteritis, Henoch-Schonlein, Scleroderma,
HIV-related diseases, amyloidosis or hemolytic uremic syndrome. The
methods of the present invention would also be useful as part of a
therapeutic application for treating interstitial nephritis or
pyelonephritis associated with chronic pyelonephritis, analgesic
abuse, nephrocalcinosis, nephropathy caused by other agents,
nephrolithiasis, or chronic or acute interstitial nephritis.
[0355] The methods of the present invention also include use of
Ztnfr12 polypeptides, fusions, antibodies, agonists or antagonists
in the treatment of hypertensive or large vessel diseases,
including renal artery stenosis or occlusion and cholesterol emboli
or renal emboli.
[0356] The present invention also provides methods for treatment of
renal or urological neoplasms, multiple mylelomas, lymphomas, light
chain neuropathy or amyloidosis.
[0357] The invention also provides methods for blocking or
inhibiting activated B cells using Ztnfr12 polypeptides, fusions,
antibodies, agonists or antagonists for the treatment of asthma and
other chronic airway diseases such as bronchitis and emphysema.
[0358] Also provided are methods for inhibiting or neutralizing an
effector T cell response using Ztnfr12 polypeptides, fusions,
antibodies, agonists or antagonists for use in immunosuppression,
in particular for such therapeutic use as for graft-versus-host
disease and graft rejection. Moreover, Ztnfr12 polypeptides,
fusions, antibodies, agonists or antagonists would be useful in
therapeutic protocols for treatment of such autoimmune diseases as
insulin dependent diabetes mellitus (IDDM) and Crohn's Disease.
Methods of the present invention would have additional therapeutic
value for treating chronic inflammatory diseases, in particular to
lessen joint pain, swelling, anemia and other associated symptoms
as well as treating septic shock.
[0359] Compounds identified as Ztnfr12 agonists are also useful to
boost the humoral immune response. B cell responses are important
in fighting infectious diseases including bacterial, viral,
protozoan and parasitic infections. Antibodies against infectious
microorganisms can immobilize the pathogen by binding to antigen
followed by complement mediated lysis or cell mediated attack. A
Ztnfr12 agonist would serve to boost the humoral response and would
be a useful therapeutic for individuals at risk for an infectious
disease, an immunocompromised state, or as a supplement to
vaccination.
[0360] Well established animal models are available to test in vivo
efficacy of soluble Ztnfr12 polypeptides of the present invention
in certain disease states. In particular, soluble Ztnfr12
polypeptides and polypeptide fragments can be tested in vivo in a
number of animal models of autoimmune disease, such as MRL-lpr/lpr
or NZB x NZW F1 congenic mouse strains which serve as a model of
SLE (systemic lupus erythematosus). Such animal models are known in
the art, and illustrative models are described above, including
NZBW mice that develop a spontaneous form of SLE, murine models for
experimental allergic encephalomyelitis, the collagen-induced
arthritis murine model, murine experimental autoimmune myasthenia
gravis, and the like.
[0361] Generally, the dosage of administered Ztnfr12 (or Ztnfr12
analog or fusion protein) will vary depending upon such factors as
the subject's age, weight, height, sex, general medical condition
and previous medical history. Typically, it is desirable to provide
the recipient with a dosage of Ztnfr12 polypeptide, which is in the
range of from about 1 pg/kg to 10 mg/kg (amount of agent/body
weight of subject), although a lower or higher dosage also may be
administered as circumstances dictate.
[0362] Administration of a Ztnfr12 polypeptide to a subject can be
intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, intrapleural, intrathecal, by perfusion through a
regional catheter, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
[0363] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising Ztnfr12 can be prepared and inhaled with the aid of
dry-powder dispersers, liquid aerosol generators, or nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al.,
Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated
by the AERX diabetes management system, which is a hand-held
electronic inhaler that delivers aerosolized insulin into the
lungs. Studies have shown that proteins as large as 48,000 kDa have
been delivered across skin at therapeutic concentrations with the
aid of low-frequency ultrasound, which illustrates the feasibility
of trascutaneous administration (Mitragotri et al., Science 269:850
(1995)). Transdermal delivery using electroporation provides
another means to administer a molecule having Ztnfr12 binding
activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).
[0364] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having Ztnfr12 binding activity can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing
Company 1995).
[0365] For purposes of therapy, molecules having Ztnfr12 binding
activity and a pharmaceutically acceptable carrier are administered
to a patient in a therapeutically effective amount. A combination
of a protein, polypeptide, or peptide having Ztnfr12 binding
activity and a pharmaceutically acceptable carrier is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient. For example, an
agent used to treat inflammation is physiologically significant if
its presence alleviates the inflammatory response. As another
example, an agent used to inhibit the growth of tumor cells is
physiologically significant if the administration of the agent
results in a decrease in the number of tumor cells, decreased
metastasis, a decrease in the size of a solid tumor, or increased
necrosis of a tumor.
[0366] A pharmaceutical composition comprising Ztnfr12 (or Ztnfr12
analog or fusion protein) can be furnished in liquid form, in an
aerosol, or in solid form. Liquid forms, are illustrated by
injectable solutions and oral suspensions. Exemplary solid forms
include capsules, tablets, and controlled-release forms. The latter
form is illustrated by miniosmotic pumps and implants (Bremer et
al., Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery
with Infusion Pumps," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);
Yewey et al., "Delivery of Proteins from a Controlled Release
Injectable Implant," in Protein Delivery: Physical Systems, Sanders
and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0367] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0368] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0369] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0370] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull.
20:881 (1997)).
[0371] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol. Pharm. Bull.20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver. In addition, anti-Ztnfr12
antibody components can be used to direct liposomes to
Ztnfr12-expressing cells, such as tumor cells of B cell origin.
[0372] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0373] Polypeptides having Ztnfr12 binding activity can be
encapsulated within liposomes using standard techniques of protein
microencapsulation (see, for example, Anderson et al., Infect.
Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et
al. "Preparation and Use of Liposomes in Immunological Studies," in
Liposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page
317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124
(1987)). As noted above, therapeutically useful liposomes may
contain a variety of components. For example, liposomes may
comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,
Biochim. Biophys. Acta 1150:9 (1993)).
[0374] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0375] The present invention also contemplates chemically modified
polypeptides having binding Ztnfr12 activity and Ztnfr12
antagonists, in which a polypeptide is linked with a polymer, as
discussed above. In addition, the present invention contemplates
compositions, such as pharmaceutical compositions, comprising a
carrier, a Ztnfr12 polypeptide, and at least one of a BCMA
polypeptide and a TACI polypeptide, as discussed above.
[0376] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0377] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a Ztnfr12 extracellular domain or a Ztnfr12
antagonist (e.g., an antibody or antibody fragment that binds a
Ztnfr12 polypeptide). Therapeutic polypeptides can be provided in
the form of an injectable solution for single or multiple doses, or
as a sterile powder that will be reconstituted before injection.
Alternatively, such a kit can include a dry-powder disperser,
liquid aerosol generator, or nebulizer for administration of a
therapeutic polypeptide. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition. Moreover, such information may include a statement
that the Ztnfr12 composition is contraindicated in patients with
known hypersensitivity to Ztnfr12.
13. Therapeutic Uses of Ztnfr12 Nucleotide Sequences
[0378] The present invention includes the use of Ztnfr12 nucleotide
sequences to provide Ztnfr12 to a subject in need of such
treatment. An enhancement in Ztnfr12 activity can be useful as part
of a treatment of immunosuppressive diseases. In addition, a
therapeutic expression vector can be provided that inhibits Ztnfr12
gene expression, such as an anti-sense molecule, a ribozyme, or an
external guide sequence molecule. Inhibition of ZTNF4 activity can
be achieved by introducing an expression vector that encodes a form
of the Ztnfr12 receptor that either does not bind ZTNF4, or does
not produce a signal following binding with ZTNF4 (e.g., due to a
mutation in the Ztnfr12 intracellular domain). For veterinary
therapeutic use or human therapeutic use, such nucleic acid
molecules can be administered to a subject having a disorder or
disease, as discussed above. As one example discussed earlier,
nucleic acid molecules encoding a Ztnfr12-immunoglobulin fusion
protein can be used for long-term treatment of systemic lupus
erythematosus.
[0379] There are numerous approaches to introduce a Ztnfr12 gene to
a subject, including the use of recombinant host cells that express
Ztnfr12, delivery of naked nucleic acid encoding Ztnfr12, use of a
cationic lipid carrier with a nucleic acid molecule that encodes
Ztnfr12, and the use of viruses that express Ztnfr12, such as
recombinant retroviruses, recombinant adeno-associated viruses,
recombinant adenoviruses, and recombinant Herpes simplex viruses
(see, for example, Mulligan, Science 260:926 (1993), Rosenberg et
al., Science 242:1575 (1988), LaSalle et al., Science 259:988
(1993), Wolff et al., Science 247:1465 (1990), Breakfield and
Deluca, The New Biologist 3:203 (1991)). In an ex vivo approach,
for example, cells are isolated from a subject, transfected with a
vector that expresses a Ztnfr12 gene, and then transplanted into
the subject.
[0380] In order to effect expression of a Ztnfr12 gene, an
expression vector is constructed in which a nucleotide sequence
encoding a Ztnfr12 gene is operably linked to a core promoter, and
optionally a regulatory element, to control gene transcription. The
general requirements of an expression vector are described
above.
[0381] Alternatively, a Ztnfr12 gene can be delivered using
recombinant viral vectors, including for example, adenoviral
vectors (e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA
90:11498 (1993), Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215
(1994), Li et al., Hum. Gene Ther. 4:403 (1993), Vincent et al.,
Nat. Genet. 5:130 (1993), and Zabner et al., Cell 75:207 (1993)),
adenovirus-associated viral vectors (Flotte et al., Proc. Nat'l
Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki
Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,
Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat.
Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus
vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993),
Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)),
pox viruses, such as canary pox virus or vaccinia virus
(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), and
Flexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and
retroviruses (e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et
al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res
33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and
Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Pat.
No. 5,399,346). Within various embodiments, either the viral vector
itself, or a viral particle which contains the viral vector may be
utilized in the methods and compositions described below.
[0382] As an illustration of one system, adenovirus, a
double-stranded DNA virus, is a well-characterized gene transfer
vector for delivery of a heterologous nucleic acid molecule (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994); Douglas
and Curiel, Science & Medicine 4:44 (1997)). The adenovirus
system offers several advantages including: (i) the ability to
accommodate relatively large DNA inserts, (ii) the ability to be
grown to high-titer, (iii) the ability to infect a broad range of
mammalian cell types, and (iv) the ability to be used with many
different promoters including ubiquitous, tissue specific, and
regulatable promoters. In addition, adenoviruses can be
administered by intravenous injection, because the viruses are
stable in the bloodstream.
[0383] Using adenovirus vectors where portions of the adenovirus
genome are deleted, inserts are incorporated into the viral DNA by
direct ligation or by homologous recombination with a
co-transfected plasmid. In an exemplary system, the essential E1
gene is deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. Although an adenoviral delivery system with an
E1 gene deletion cannot replicate in the host cells, the host's
tissue will express and process an encoded heterologous protein.
Host cells will also secrete the heterologous protein if the
corresponding gene includes a secretory signal sequence. Secreted
proteins will enter the circulation from tissue that expresses the
heterologous gene (e.g., the highly vascularized liver).
[0384] Moreover, adenoviral vectors containing various deletions of
viral genes can be used to reduce or eliminate immune responses to
the vector. Such adenoviruses are E1-deleted, and in addition,
contain deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022
(1998); Raper et al., Human Gene Therapy 9:671 (1998)). The
deletion of E2b has also been reported to reduce immune responses
(Amalfitano et al., J. Virol. 72:926 (1998)). By deleting the
entire adenovirus genome, very large inserts of heterologous DNA
can be accommodated. The generation of so called "gutless"
adenoviruses, where all viral genes are deleted, is particularly
advantageous for insertion of large inserts of heterologous DNA
(for a review, see Yeh. and Perricaudet, FASEB J. 11:615
(1997)).
[0385] High titer stocks of recombinant viruses capable of
expressing a therapeutic gene can be obtained from infected
mammalian cells using standard methods. For example, recombinant
herpes simplex virus can be prepared in Vero cells, as described by
Brandt et al., J. Gen. Virol. 72:2043 (1991), Herold et al., J.
Gen. Virol. 75:1211 (1994), Visalli and Brandt, Virology 185:419
(1991), Grau et al., Invest. Ophthalmol. Vis. Sci. 30:2474 (1989),
Brandt et al., J. Virol. Meth. 36:209 (1992), and by Brown and
MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
[0386] Alternatively, an expression vector comprising a Ztnfr12
gene can be introduced into a subject's cells by lipofection in
vivo using liposomes. Synthetic cationic lipids can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker (Felgner et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987);
Mackey et al., Proc. Nat'l Acad. Sci. USA 85:8027 (1988)). The use
of lipofection to introduce exogenous genes into specific organs in
vivo has certain practical advantages. Liposomes can be used to
direct transfection to particular cell types, which is particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides (e.g., hormones or neurotransmitters), proteins such as
antibodies, or non-peptide molecules can be coupled to liposomes
chemically.
[0387] Electroporation is another alternative mode of
administration. For example, Aihara and Miyazaki, Nature
Biotechnology 16:867 (1998), have demonstrated the use of in vivo
electroporation for gene transfer into muscle.
[0388] In an alternative approach to gene therapy, a therapeutic
gene may encode a Ztnfr12 anti-sense RNA that inhibits the
expression of Ztnfr12. Suitable sequences for anti-sense molecules
can be derived from the nucleotide sequences of Ztnfr12 disclosed
herein.
[0389] Alternatively, an expression vector can be constructed in
which a regulatory element is operably linked to a nucleotide
sequence that encodes a ribozyme. Ribozymes can be designed to
express endonuclease activity that is directed to a certain target
sequence in an mRNA molecule (see, for example, Draper and Macejak,
U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,
Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson and
Goldberg, U.S. Pat. No. 5,225,337). In the context of the present
invention, ribozymes include nucleotide sequences that bind with
Ztnfr12 mRNA.
[0390] In another approach, expression vectors can be constructed
in which a regulatory element directs the production of RNA
transcripts capable of promoting RNase P-mediated cleavage of mRNA
molecules that encode a Ztnfr12 gene. According to this approach,
an external guide sequence can be constructed for directing the
endogenous ribozyme, RNase P, to a particular species of
intracellular mRNA, which is subsequently cleaved by the cellular
ribozyme (see, for example, Altman et al., U.S. Pat. No. 5,168,053,
Yuan et al., Science 263:1269 (1994), Pace et al., international
publication No. WO 96/18733, George et al., international
publication No. WO 96/21731, and Werner et al., international
publication No. WO 97/33991). For example, the external guide
sequence can comprise a ten to fifteen nucleotide sequence
complementary to Ztnfr12 mRNA, and a 3'-NCCA nucleotide sequence,
wherein N is preferably a purine. The external guide sequence
transcripts bind to the targeted mRNA species by the formation of
base pairs between the mRNA and the complementary external guide
sequences, thus promoting cleavage of mRNA by RNase P at the
nucleotide located at the 5'-side of the base-paired region.
[0391] In general, the dosage of a composition comprising a
therapeutic vector having a Ztnfr12 nucleotide sequence, such as a
recombinant virus, will vary depending upon such factors as the
subject's age, weight, height, sex, general medical condition and
previous medical history. Suitable routes of administration of
therapeutic vectors include intravenous injection, intraarterial
injection, intraperitoneal injection, intramuscular injection,
intratumoral injection, and injection into a cavity that contains a
tumor. As an illustration, Horton et al., Proc. Nat'l Acad. Sci.
USA 96:1553 (1999), demonstrated that intramuscular injection of
plasmid DNA encoding interferon-.alpha. produces potent antitumor
effects on primary and metastatic tumors in a murine model.
[0392] A composition comprising viral vectors, non-viral vectors,
or a combination of viral and non-viral vectors of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby vectors or viruses
are combined in a mixture with a pharmaceutically acceptable
carrier. As noted above, a composition, such as phosphate-buffered
saline is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient subject. Other
suitable carriers are well-known to those in the art (see, for
example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and Gilman's the Pharmacological Basis of
Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
[0393] For purposes of therapy, a therapeutic gene expression
vector, or a recombinant virus comprising such a vector, and a
pharmaceutically acceptable carrier are administered to a subject
in a therapeutically effective amount. A combination of an
expression vector (or virus) and a pharmaceutically acceptable
carrier is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient subject. For
example, an agent used to treat inflammation is physiologically
significant if its presence alleviates the inflammatory response.
As another example, an agent used to inhibit the growth of tumor
cells is physiologically significant if the administration of the
agent results in a decrease in the number of tumor cells, decreased
metastasis, a decrease in the size of a solid tumor, or increased
necrosis of a tumor.
[0394] When the subject treated with a therapeutic gene expression
vector or a recombinant virus is a human, then the therapy is
preferably somatic cell gene therapy. That is, the preferred
treatment of a human with a therapeutic gene expression vector or a
recombinant virus does not entail introducing into cells a nucleic
acid molecule that can form part of a human germ line and be passed
onto successive generations (i.e., human germ line gene
therapy).
14. Therapeutically Useful Immunoconjugates
[0395] The present invention contemplates the use of naked
anti-Ztnfr12 antibodies (or naked antibody fragments thereof), as
well as the use of immunoconjugates to effect treatment of various
disorders, including B-cell malignancies, as discussed above.
Immunoconjugates can be prepared using standard techniques. For
example, immunoconjugates can be produced by indirectly conjugating
a therapeutic agent to an antibody component (see, for example,
Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J.
Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No.
5,057,313). Briefly, one standard approach involves reacting an
antibody component having an oxidized carbohydrate portion with a
carrier polymer that has at least one free amine function and that
is loaded with a plurality of drug, toxin, chelator, boron addends,
or other therapeutic agent. This reaction results in an initial
Schiff base (imine) linkage, which can be stabilized by reduction
to a secondary amine to form the final conjugate.
[0396] The carrier polymer can be an aminodextran or polypeptide of
at least 50 amino acid residues, although other substantially
equivalent polymer carriers can also be used. Preferably, the final
immunoconjugate is soluble in an aqueous solution, such as
mammalian serum, for ease of administration and effective targeting
for use in therapy. Thus, solubilizing functions on the carrier
polymer will enhance the serum solubility of the final
immunoconjugate.
[0397] In an alternative approach for producing immunoconjugates
comprising a polypeptide therapeutic agent, the therapeutic agent
is coupled to aminodextran by glutaraldehyde condensation or by
reaction of activated carboxyl groups on the polypeptide with
amines on the aminodextran. Chelators can be attached to an
antibody component to prepare immunoconjugates comprising
radiometals or magnetic resonance enhancers. Illustrative chelators
include derivatives of ethylenediaminetetraacetic acid and
diethylenetriaminepentaacetic acid. Boron addends, such as
carboranes, can be attached to antibody components by conventional
methods.
[0398] Immunoconjugates can also be prepared by directly
conjugating an antibody component with a therapeutic agent. The
general procedure is analogous to the indirect method of
conjugation except that a therapeutic agent is directly attached to
an oxidized antibody component.
[0399] As a further illustration, a therapeutic agent can be
attached at the hinge region of a reduced antibody component via
disulfide bond formation. For example, the tetanus toxoid peptides
can be constructed with a single cysteine residue that is used to
attach the peptide to an antibody component. As an alternative,
such peptides can be attached to the antibody component using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)proprionate. Yu et al., Int. J. Cancer 56:244
(1994). General techniques for such conjugation are well-known in
the art. See, for example, Wong, Chemistry Of Protein Conjugation
And Cross-Linking (CRC Press 1991); Upeslacis et al., "Modification
of Antibodies by Chemical Methods," in Monoclonal Antibodies:
Principles And Applications, Birch et al. (eds.), pages 187-230
(Wiley-Liss, Inc. 1995); Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering And Clinical Application, Ritter et al.
(eds.), pages 60-84 (Cambridge University Press 1995).
[0400] As described above, carbohydrate moieties in the Fc region
of an antibody can be used to conjugate a therapeutic agent.
However, the Fc region is absent if an antibody fragment is used as
the antibody component of the immunoconjugate. Nevertheless, it is
possible to introduce a carbohydrate moiety into the light chain
variable region of an antibody or antibody fragment. See, for
example, Leung et al., J. Immunol. 154:5919 (1995); Hansen et al.,
U.S. Pat. No. 5,443,953 (1995). The engineered carbohydrate moiety
is then used to attach a therapeutic agent.
[0401] In addition, those of skill in the art will recognize
numerous possible variations of the conjugation methods. For
example, the carbohydrate moiety can be used to attach
polyethyleneglycol in order to extend the half-life of an intact
antibody, or antigen-binding fragment thereof, in blood, lymph, or
other extracellular fluids. Moreover, it is possible to construct a
divalent immunoconjugate by attaching therapeutic agents to a
carbohydrate moiety and to a free sulfhydryl group. Such a free
sulfhydryl group may be located in the hinge region of the antibody
component.
[0402] One type of immunoconjugate comprises an antibody component
and a polypeptide cytotoxin. An example of a suitable polypeptide
cytotoxin is a ribosome-inactivating protein. Type I
ribosome-inactivating proteins are single-chain proteins, while
type II ribosome-inactivating proteins consist of two nonidentical
subunits (A and B chains) joined by a disulfide bond (for a review,
see Soria et al., Targeted Diagn. Ther. 7:193 (1992)). Useful type
I ribosome-inactivating proteins include polypeptides from
Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3,
saporin-6), Momordica charantia (e.g, momordin), Byronia dioica
(e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g.,
trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin),
Phytolacca americana(e.g., pokeweed antiviral protein, pokeweed
antiviral protein-II, pokeweed antiviral protein-S), Phytolacca
dodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and
the like. Ribosome-inactivating proteins are described, for
example, by Walsh et al., U.S. Pat. No. 5,635,384.
[0403] Suitable type II ribosome-inactivating proteins include
polypeptides from Ricinus communis (e.g., ricin), Abrus precatorius
(e.g., abrin), Adenia digitata (e.g., modeccin), and the like.
Since type II ribosome-inactiving proteins include a B chain that
binds galactosides and a toxic A chain that depurinates adensoine,
type II ribosome-inactivating protein conjugates should include the
A chain. Additional useful ribosome-inactivating proteins include
bouganin, clavin, maize ribosome-inactivating proteins, Vaccaria
pyramidata ribosome-inactivating proteins, nigrine b, basic nigrine
1, ebuline, racemosine b, luffin-a, luffin-b, luffin-S, and other
ribosome-inactivating proteins known to those of skill in the art.
See, for example, Bolognesi and Stirpe, international publication
No. WO98/55623, Colnaghi et al., international publication No.
WO97/49726, Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and
Stirpe, international publication No. WO95/07297, Arias et al.,
international publication No. WO94/20540, Watanabe et al., J.
Biochem. 106:6 977 (1989); Islam et al., Agric. Biol. Chem. 55:229
(1991), and Gao et al., FEBS Lett. 347:257 (1994).
[0404] Analogs and variants of naturally-occurring
ribosome-inactivating proteins are also suitable for the targeting
compositions described herein, and such proteins are known to those
of skill in the art. Ribosome-inactivating proteins can be produced
using publicly available amino acid and nucleotide sequences. As an
illustration, a nucleotide sequence encoding saporin-6 is disclosed
by Lorenzetti et al., U.S. Pat. No. 5,529,932, while Walsh et al.,
U.S. Pat. No. 5,635,384, describe maize and barley
ribosome-inactivating protein nucleotide and amino acid sequences.
Moreover, ribosome-inactivating proteins are also commercially
available.
[0405] Additional polypeptide cytotoxins include ribonuclease,
DNase I, Staphylococcal enterotoxin-A, diphtheria toxin,
Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,
Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.--A Cancer
Journal for Clinicians 44:43 (1994).
[0406] Another general type of useful cytotoxin is a tyrosine
kinase inhibitor. Since the activation of proliferation by tyrosine
kinases has been suggested to play a role in the development and
progression of tumors, this activation can be inhibited by
anti-Ztnfr12 antibody components that deliver tyrosine kinase
inhibitors. Suitable tyrosine kinase inhibitors include
isoflavones, such as genistein (5, 7, 4'-trihydroxyisoflavone),
daidzein (7,4'-dihydroxyisoflavone), and biochanin A
(4-methoxygenistein), and the like. Methods of conjugating tyrosine
inhibitors to a growth factor are described, for example, by Uckun,
U.S. Pat. No. 5,911,995.
[0407] Another group of useful polypeptide cytotoxins includes
immunomodulators. As used herein, the term "immunomodulator"
includes cytokines, stem cell growth factors, lymphotoxins,
co-stimulatory molecules, hematopoietic factors, and the like, as
well as synthetic analogs of these molecules. Examples of
immunomodulators include tumor necrosis factor, interleukins (e.g.,
interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, and IL-21), colony stimulating factors (e.g.,
granulocyte-colony stimulating factor and granulocyte
macrophage-colony stimulating factor), interferons (e.g.,
interferons-.alpha., -.beta., -.gamma., -.omega., -.epsilon., and
-.tau.), the stem cell growth factor designated "S1 factor,"
erythropoietin, and thrombopoietin. Illustrative immunomodulator
moieties include IL-2, IL-6, IL-10, interferon-.quadrature.,
TNF-.quadrature., and the like.
[0408] Immunoconjugates that include an immunomodulator provide a
means to deliver an immunomodulator to a target cell, and are
particularly useful against tumor cells. The cytotoxic effects of
immunomodulators are well known to those of skill in the art. See,
for example, Klegerman et al., "Lymphokines and Monokines," in
Biotechnology And Pharmacy, Pessuto et al. (eds.), pages 53-70
(Chapman & Hall 1993). As an illustration, interferons can
inhibit cell proliferation by inducing increased expression of
class I histocompatibility antigens on the surface of various cells
and thus, enhance the rate of destruction of cells by cytotoxic T
lymphocytes. Furthermore, tumor necrosis factors, such as tumor
necrosis factor-.alpha., are believed to produce cytotoxic effects
by inducing DNA fragmentation.
[0409] The present invention also includes immunocongugates that
comprise a nucleic acid molecule encoding a cytotoxin. As an
example of this approach, Hoganson et al., Human Gene Ther. 9:2565
(1998), describe FGF-2 mediated delivery of a saporin gene by
producing an FGF-2-polylysine conjugate which was condensed with an
expression vector comprising a saporin gene.
[0410] Other suitable toxins are known to those of skill in the
art.
[0411] Conjugates of cytotoxic polypeptides and antibody components
can be prepared using standard techniques for conjugating
polypeptides. For example, Lam and Kelleher, U.S. Pat. No.
5,055,291, describe the production of antibodies conjugated with
either diphtheria toxin fragment A or ricin toxin. The general
approach is also illustrated by methods of conjugating fibroblast
growth factor with saporin, as described by Lappi et al., Biochem.
Biophys. Res. Commun. 160:917 (1989), Soria et al., Targeted Diagn.
Ther. 7:193 (1992), Buechler et al., Eur. J. Biochem. 234:706
(1995), Behar-Cohen et al., Invest. Ophthalmol. Vis. Sci. 36:2434
(1995), Lappi and Baird, U.S. Pat. No. 5,191,067, Calabresi et al.,
U.S. Pat. No. 5,478,804, and Lappi and Baird, U.S. Pat. No.
5,576,288. Also see, Ghetie and Vitteta, "Chemical Construction of
Immunotoxins," in Drug Targeting: Strategies, Principles, and
Applications, Francis and Delgado (Eds.), pages 1-26 (Humana Press,
Inc. 2000), Hall (Ed.), Immunotoxin Methods and Protocols (Humana
Press, Inc. 2000), and Newton and Rybak, "Construction of
Ribonuclease-Antibody Conjugates for Selective Cytotoxicity," in
Drug Targeting: Strategies, Principles, and Applications, Francis
and Delgado (Eds.), pages 27-35 (Humana Press, Inc. 2000).
[0412] Alternatively, fusion proteins comprising an antibody
component and a cytotoxic polypeptide can be produced using
standard methods. Methods of preparing fusion proteins comprising a
cytotoxic polypeptide moiety are well-known in the art of
antibody-toxin fusion protein production. For example, antibody
fusion proteins comprising an interleukin-2 moiety are described by
Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer
Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci. USA
93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and
Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al.,
Hum. Antibodies Hybridomas 6:129 (1995), describe a fusion protein
that includes an F(ab').sub.2 fragment and a tumor necrosis factor
alpha moiety. Antibody-Pseudomonas exotoxin A fusion proteins have
been described by Chaudhary et al., Nature 339:394 (1989),
Brinkmann et al., Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra
et al., Proc. Nat'l Acad. Sci. USA 89:5867 (1992), Friedman et al.,
J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can. 60:137
(1995), Fominaya et al., J. Biol. Chem. 271:10560 (1996), Kuan et
al., Biochemistry 35:2872 (1996), and Schmidt et al., Int. J. Can.
65:538 (1996). Antibody-toxin fusion proteins containing a
diphtheria toxin moiety have been described by Kreitman et al.,
Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302
(1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), and
Vallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor
Targeting 1:177 (1995), have described an antibody-toxin fusion
protein having an RNase moiety, while Linardou et al., Cell
Biophys. 24-25:243 (1994), produced an antibody-toxin fusion
protein comprising a DNase I component. Gelonin was used as the
toxin moiety in the antibody-toxin fusion protein of Better et al.,
J. Biol. Chem. 270:14951 (1995). As a further example, Dohlsten et
al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported an
antibody-toxin fusion protein comprising Staphylococcal
enterotoxin-A. Also see, Newton and Rybak, "Preparation of
Recombinant RNase Single-Chain Antibody Fusion Proteins," in Drug
Targeting: Strategies, Principles, and Applications, Francis and
Delgado (Eds.), pages 77-95 (Humana Press, Inc. 2000).
[0413] As an alternative to a polypeptide cytotoxin,
immunoconjugates can comprise a radioisotope as the cytotoxic
moiety. For example, an immunoconjugate can comprise an
anti-Ztnfr12 antibody component and an .alpha.-emitting
radioisotope, a .beta.-emitting radioisotope, .gamma.-emitting
radioisotope, an Auger electron emitter, a neutron capturing agent
that emits .alpha.-particles or a radioisotope that decays by
electron capture. Suitable radioisotopes include .sup.198Au,
.sup.199Au, .sup.32P, .sup.33P, .sup.125I, .sup.131I, .sup.123I,
.sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu, .sup.211At, .sup.47Sc,
.sup.103Pb, .sup.109Pd, .sup.212Pb, .sup.71Ge, .sup.77As,
.sup.105Rh .sup.113Ag, .sup.119Sb, .sup.121Sn .sup.131Cs,
.sup.143Pr, .sup.161Tb, .sup.177Lu, .sup.191Os, .sup.193MPt,
.sup.197Hg, and the like.
[0414] A radioisotope can be attached to an antibody component
directly or indirectly, via a chelating agent. For example,
.sup.67Cu, which provides .beta.-particles and .gamma.-rays, can be
conjugated to an antibody component using the chelating agent,
p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid. Chase and
Shapiro, "Medical Applications of Radioisotopes," in Gennaro (Ed.),
Remington: The Science and Practice of Pharmacy, 19th Edition,
pages 843-865 (Mack Publishing Company 1995). As an alternative,
.sup.90Y, which emits an energetic .beta.-particle, can be coupled
to an antibody component using diethylenetriaminepentaacetic acid.
Moreover, an exemplary suitable method for the direct radiolabeling
of an antibody component with .sup.131I is described by Stein et
al., Antibody Immunoconj. Radiopharm. 4:703 (1991). Alternatively,
boron addends such as carboranes can be attached to antibody
components, using standard techniques.
[0415] Another type of suitable cytotoxin for the preparation of
immunoconjugates is a chemotherapeutic drug. Illustrative
chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs,
purine analogs, antibiotics, epipodophyllotoxins, platinum
coordination complexes, and the like. Specific examples of
chemotherapeutic drugs include methotrexate, doxorubicin,
daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin,
bleomycin, melphalan, chlorambucil, maytansinoids, calicheamicin,
taxol, and the like. Suitable chemotherapeutic agents are described
in Remington: The Science and Practice of Pharmacy, 19th Edition
(Mack Publishing Co. 1995), and in Goodman And Gilman's The
Pharmacological Basis Of Therapeutics, 9th Ed. (MacMillan
Publishing Co. 1995). Other suitable chemotherapeutic agents are
known to those of skill in the art.
[0416] In another approach, immunoconjugates are prepared by
conjugating photoactive agents or dyes to an antibody component.
Fluorescent and other chromogens, or dyes, such as porphyrins
sensitive to visible light, have been used to detect and to treat
lesions by directing the suitable light to the lesion. This type of
"photoradiation," "phototherapy," or "photodynamic" therapy is
described, for example, by Mew et al., J. Immunol. 130:1473 (1983),
Jori et al. (eds.), Photodynamic Therapy Of Tumors And Other
Diseases (Libreria Progetto 1985), Oseroff et al., Proc. Natl.
Acad. Sci. USA 83:8744 (1986), van den Bergh, Chem. Britain 22:430
(1986), Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989),
Tatsuta et al., Lasers Surg. Med. 9:422 (1989), and Pelegrin et
al., Cancer 67:2529 (1991).
[0417] Immunoconjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties. Methods
for attaching such polymers are known to those of skill in the art,
and have been described previously.
[0418] The approaches described above can also be used to prepare
multispecific antibody compositions that comprise an
immunoconjugate. Polypeptide cytotoxins can also be conjugated with
a soluble polymer using the above methods either before or after
conjugation to an antibody component. Soluble polymers can also be
conjugated with antibody fusion proteins.
[0419] In general, anti-Ztnfr12 immunoconjugates can be
administered as discussed previously with regard to the therapeutic
uses of Ztnfr12 polypeptides. Naked anti-Ztnfr12 antibodies, or
antibody fragments, can be supplemented with immunoconjugate or
antibody fusion protein administration. In one variation, naked
anti-Ztnfr12 antibodies (or naked antibody fragments) are
administered with low-dose radiolabeled anti-Ztnfr12 antibodies or
antibody fragments. As a second alternative, naked anti-Ztnfr12
antibodies (or antibody fragments) are administered with low-dose
radiolabeled anti-Ztnfr12 antibody-cytokine immunoconjugates. As a
third alternative, naked anti-Ztnfr12 antibodies (or antibody
fragments) are administered with anti-Ztnfr12-cytokine
immunoconjugates that are not radiolabeled. With regard to "low
doses" of .sup.131I-labeled immunoconjugates, a preferable dosage
is in the range of 15 to 40 mCi, while the most preferable range is
20 to 30 mCi. In contrast, a preferred dosage of .sup.90Y-labeled
immunoconjugates is in the range from 10 to 30 mCi, while the most
preferable range is 10 to 20 mCi. Similarly, bispecific antibody
components can be supplemented with immunoconjugate or antibody
fusion protein administration.
[0420] Immunoconjugates having a boron addend-loaded carrier for
thermal neutron activation therapy will normally be effected in
similar ways. However, it will be advantageous to wait until
non-targeted immunoconjugate clears before neutron irradiation is
performed. Clearance can be accelerated using an antibody that
binds to the immunoconjugate. See U.S. Pat. No. 4,624,846 for a
description of this general principle.
[0421] The present invention also contemplates a method of
treatment in which immunomodulators are administered to prevent,
mitigate or reverse radiation-induced or drug-induced toxicity of
normal cells, and especially hematopoietic cells. Adjunct
immunomodulator therapy allows the administration of higher doses
of cytotoxic agents due to increased tolerance of the recipient
mammal. Moreover, adjunct immunomodulator therapy can prevent,
palliate, or reverse dose-limiting marrow toxicity. Examples of
suitable immunomodulators for adjunct therapy include
granulocyte-colony stimulating factor, granulocyte
macrophage-colony stimulating factor, thrombopoietin, IL-1, IL-3,
IL-12, and the like. The method of adjunct immunomodulator therapy
is disclosed by Goldenberg, U.S. Pat. No. 5,120,525.
[0422] Anti-Ztnfr12 antibodies and immunoconjugates can be tested
using the in vitro approaches and animal models described above for
the evaluation of Ztnfr12 polypeptides and Ztnfr12 fusion
proteins.
[0423] The efficacy of anti-Ztnfr12 antibody therapy can be
enhanced by supplementing naked antibody components with
immunoconjugates and other forms of supplemental therapy described
herein. In such multimodal regimens, the supplemental therapeutic
compositions can be administered before, concurrently or after
administration of naked anti-Ztnfr12 antibodies. Multimodal
therapies of the present invention further include immunotherapy
with naked anti-Ztnfr12 antibody components supplemented with
administration of anti-Ztnfr12 immunoconjugates. In another form of
multimodal therapy, subjects receive naked anti-Ztnfr12 antibodies
and standard cancer chemotherapy.
[0424] Pharmaceutical compositions may be supplied as a kit
comprising a container that comprises anti-Ztnfr12 antibody
components, or bispecific antibody components. Therapeutic
molecules can be provided in the form of an injectable solution for
single or multiple doses, or as a sterile powder that will be
reconstituted before injection. Alternatively, such a kit can
include a dry-powder disperser, liquid aerosol generator, or
nebulizer for administration of an anti-Ztnfr12 antibody component.
Such a kit may further comprise written information on indications
and usage of the pharmaceutical composition. Moreover, such
information may include a statement that the composition is
contraindicated in patients with known hypersensitivity to
exogenous antibodies.
15. Production of Transgenic Mice
[0425] Transgenic mice can be engineered to over-express the
Ztnfr12 gene in all tissues or under the control of a
tissue-specific or tissue-preferred regulatory element. These
over-producers of Ztnfr12 can be used to characterize the phenotype
that results from over-expression, and the transgenic animals can
serve as models for human disease caused by excess Ztnfr12.
Transgenic mice that over-express Ztnfr12 also provide model
bioreactors for production of Ztnfr12, such as soluble Ztnfr12, in
the milk or blood of larger animals. Methods for producing
transgenic mice are well-known to those of skill in the art (see,
for example, Jacob, "Expression and Knockout of Interferons in
Transgenic Mice," in Overexpression and Knockout of Cytokines in
Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd.
1994), Monastersky and Robl (eds.), Strategies in Transgenic Animal
Science (ASM Press 1995), and Abbud and Nilson, "Recombinant
Protein Expression in Transgenic Mice," in Gene Expression Systems:
Using Nature for the Art of Expression, Fernandez and Hoeffler
(eds.), pages 367-397 (Academic Press, Inc. 1999)).
[0426] For example, a method for producing a transgenic mouse that
expresses a Ztnfr12 gene can begin with adult, fertile males
(studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown,
N.Y.)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic
Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks,
(Taconic Farms)) and adult fertile females (recipients) (B6D2fl,
2-4 months, (Taconic Farms)). The donors are acclimated for one
week and then injected with approximately 8 IU/mouse of Pregnant
Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, Mo.)
I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic
Gonadotropin (hCG (Sigma)) I.P. to induce superovulation. Donors
are mated with studs subsequent to hormone injections. Ovulation
generally occurs within 13 hours of hCG injection. Copulation is
confirmed by the presence of a vaginal plug the morning following
mating.
[0427] Fertilized eggs are collected under a surgical scope. The
oviducts are collected and eggs are released into urinanalysis
slides containing hyaluronidase (Sigma). Eggs are washed once in
hyaluronidase, and twice in Whitten's W640 medium (described, for
example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and
Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are then stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
[0428] Ten to twenty micrograms of plasmid DNA containing a Ztnfr12
encoding sequence is linearized, gel-purified, and resuspended in
10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final
concentration of 5-10 nanograms per microliter for microinjection.
For example, the Ztnfr12 encoding sequences can encode a
polypeptide comprising amino acid residues 1 to 69 of SEQ ID NO:2,
comprising amino acid residues 1 to 79 of SEQ ID NO:2, or
comprising amino acid residues 1 to 69 of SEQ ID NO:13.
[0429] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0430] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pre-gassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0431] The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0432] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy two-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, a nick in the oviduct is made
with a 28 g needle close to the bursa, making sure not to tear the
ampulla or the bursa.
[0433] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs allowed to slide in. The peritoneal wall is
closed with one suture and the skin closed with a wound clip. The
mice recuperate on a 37.degree. C. slide warmer for a minimum of
four hours.
[0434] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0435] Genomic DNA is prepared from the tail snips using, for
example, a QIAGEN DNEASY kit following the manufacturer's
instructions. Genomic DNA is analyzed by PCR using primers designed
to amplify a Ztnfr12 gene or a selectable marker gene that was
introduced in the same plasmid. After animals are confirmed to be
transgenic, they are back-crossed into an inbred strain by placing
a transgenic female with a wild-type male, or a transgenic male
with one or two wild-type female(s). As pups are born and weaned,
the sexes are separated, and their tails snipped for
genotyping.
[0436] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the zyphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum and
the left lateral lobe of the liver exteriorized. Using 4-0 silk, a
tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14 ml polypropylene round bottom tube and snap frozen in liquid
nitrogen and then stored on dry ice. The surgical site is closed
with suture and wound clips, and the animal's cage placed on a
37.degree. C. heating pad for 24 hours post operatively. The animal
is checked daily post operatively and the wound clips removed 7-10
days after surgery. The expression level of Ztnfr12 mRNA is
examined for each transgenic mouse using an RNA solution
hybridization assay or polymerase chain reaction.
[0437] In addition to producing transgenic mice that over-express
Ztnfr12, it is useful to engineer transgenic mice with either
abnormally low or no expression of the gene. Such transgenic mice
provide useful models for diseases associated with a lack of
Ztnfr12. As discussed above, Ztnfr12 gene expression can be
inhibited using anti-sense genes, ribozyme genes, or external guide
sequence genes. To produce transgenic mice that under-express the
Ztnfr12 gene, such inhibitory sequences are targeted to Ztnfr12
mRNA. Methods for producing transgenic mice that have abnormally
low expression of a particular gene are known to those in the art
(see, for example, Wu et al., "Gene Underexpression in Cultured
Cells and Animals by Antisense DNA and RNA Strategies," in Methods
in Gene Biotechnology, pages 205-224 (CRC Press 1997)).
[0438] An alternative approach to producing transgenic mice that
have little or no Ztnfr12 gene expression is to generate mice
having at least one normal Ztnfr12 allele replaced by a
nonfunctional Ztnfr12 gene. One method of designing a nonfunctional
Ztnfr12 gene is to insert another gene, such as a selectable marker
gene, within a nucleic acid molecule that encodes Ztnfr12. Standard
methods for producing these so-called "knockout mice" are known to
those skilled in the art (see, for example, Jacob, "Expression and
Knockout of Interferons in Transgenic Mice," in Overexpression and
Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages
111-124 (Academic Press, Ltd. 1994), and Wu et al., "New Strategies
for Gene Knockout," in Methods in Gene Biotechnology, pages 339-365
(CRC Press 1997)).
[0439] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and is not intended to be
limiting of the present invention.
EXAMPLE 1
Isolation of a Nucleic Acid Molecule Encoding Ztnfr12
[0440] This study used a human B-lymphoid precursor cell line,
designated as "Reh" (ATCC No. CRL-8286). A cDNA library was
prepared from Reh cells, and arrayed using sixteen 96-well plates.
Each well contained about 250 E. coli colonies with each colony
containing one cDNA clone. DNA minipreps were prepared in 96-well
format using the Qiaprep96 Turbo kit (Qiagen, Inc.; Valencia,
Calif.). The DNA was then divided into 128 pools that represented
3000 clones each. These pools were transfected into COS-7 cells in
12-well plates, and the positive pools were determined by
cell-surface ZTNF4 binding.
[0441] The COS cell transfection was performed as follows. Five
microliters of pooled DNA (about 0.5-1.0 .mu.g) and 5 .quadrature.l
of lipofectamine were mixed in 92 .quadrature.l of serum free DMEM
medium (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg
transferrin, 2.5 mg insulin, 1 .mu.g selenium, and 5 mg fetuin in
500 ml DMEM), incubated at room temperature for 30 minutes, and
then 400 .mu.l of serum free DMEM medium were added. Five hundred
microliters of this mixture were added to 1.5.times.10.sup.5 COS
cells/well plated on 12-well fibrinectin-pretreated tissue culture
plates, and the cells were incubated for 5 hours at 37.degree. C.
Then, 500 [ ] of 20% FBS DMEM medium (100 ml FBS, 55 mg sodium
pyruvate, and 146 mg L-glutamine in 500 ml DMEM) were added per
well, and the cells were incubated overnight.
[0442] The cell-surface binding assay was performed using
biotinylated FLAG-tagged ZTNF4 as follows. Media were rinsed from
the cells with 1% BSA/PBS and the cells were blocked for 1 hour
with TNB (0.1 M Tris-HCL, 0.15 M NaCl, and 0.5% Blocking
Reagent--NEN Renaissance TSA-Direct Kit Cat# NEL701--in H.sub.2O).
Then, the cells were incubated for 1 hour with 3 .mu.g/ml
biotinylated FLAG-tagged ZTNF4 in TNB. Cells were washed with 1%
BSA/PBS, and then incubated for another hour with 1:300 diluted
streptavidin-HRP (NEN kit) in TNB. Cells were washed with 1%
BSA/PBS and then fixed for 15 minutes with 1.8% formaldehyde in
PBS. Next, the cells were washed with TNT (0.1 M Tris-HCL, 0.15 M
NaCl, and 0.05% Tween-20 in water). Binding was detected by
incubating cells for four to five minutes with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit). Cells were
washed with TNT, and were preserved with VECTASHIELD Mounting Media
(Vector Labs; Burlingame, Calif.) diluted 1:5 in TNT. The cells
were visualized using an FITC filter on a fluorescent
microscope.
[0443] One of the positive DNA pools, "10A11," was identified using
the method described above. The DNA of pool 10A11 was
electroporated into E. coli DH10B, and the colonies were
filter-lifted and washed. DNA isolated from E. coli was then
transfected into COS cells, and the positive pool DNA "4D" was
identified using the ZTNF4 binding assay. The colonies of pool 4D
were transferred into a 96-well, and the DNA was isolated using the
Qiaprep96 Turbo kit. The positive clone 4D2H-6 was identified using
the method mentioned above. To test the specificity of ZTNF4
binding, the 4D2H-6 clone DNA was transfected into COS cells, and
the binding of ZTNF4 and ZTNF2 was tested. Although ZTNF4 binds to
COS cells transfected with 4D2H-6 DNA, ZTNF2 did not bind with the
cells. In contrast, both ZTNF4 and ZTNF2 bound to COS cells
transfected with TACI DNA.
EXAMPLE 2
Ztnfr12 Gene Expression in Human and Murine Tissues
[0444] Northern blot analysis was performed using Human Multiple
Tissue Blots (MTN I, MTN II, and MTN III) (CLONTECH Laboratories,
Inc.; Palo Alto, Calif.), Human Immune System blot (CLONTECH),
Human normal mRNA blot (Invitrogen, San Diego, Calif.) and Human
Fetal Multiple Tissue Blots (CLONTECH). A 570 base pair human probe
was generated by PCR with oligonucleotides 37550 (5'
GCGAATTCGTCGGCACCATGAGGCGAGGG 3'; SEQ ID NO:10) and 37549
(5'CGCTCGAGCTGCCGGCTCCCTGCTATTGTTG 3'; SEQ ID NO:11), under the
following reaction conditions: 94.degree. C. for 2 minutes; 35
cycles of 94.degree. C. for 30 seconds, 60.degree. C. for 30
seconds, 72.degree. C. for 30 seconds; followed by 72.degree. C.
for 5 minutes. The PCR fragment was gel-purified using QIAQUICK gel
extraction kit (QIAGEN, Inc.; Santa Clarita, Calif.). The probe was
radioactively labeled with .sup.32P using the REDIPRIME II DNA
Labeling system (AMERSHAM, Inc.; UK) according to the
manufacturer's specifications. The probe was purified using a
NUCTRAP push column (Stratagene; La Jolla, Calif.). EXPRESSHYB
(CLONTECH) solution was used for the hybridizing solution for the
blots. Hybridization took place overnight at 65.degree. C.
[0445] The blots were then washed four times with 2.times.SCC and
0.05% SDS at room temperature, followed by two washes in
0.1.times.SSC and 0.1% SDS at 50.degree. C. One transcript size was
detected at approximately 4.4 kilobases.
[0446] Tumor Blots were also examined with human uterus tumor blot
(Invitrogen, San Diego, Calif.), human tumor panel blot 4 and 5
(Invitrogen Corporation; San Diego, Calif.), human lymphoma blot
(Invitrogen), human cancer cell line blot (CLONTECH) and a human
leukemia blot. Dot blots were also analyzed using a Human Multiple
Tissue Expression Blot (CLONTECH) and a Human Cancer Gene Screening
Blot (Biochain Institute, Inc.; Hayward, Ca). The methods and
conditions for the dot blot analyses were the same as for the
multiple tissue blots disclosed above.
[0447] Ztnfr12 gene expression was observed in spleen, lymph node,
peripheral blood lymphocytes, kidney, heart, liver, skeletal
muscle, pancreas, adrenal gland, testis, brain, uterus, stomach,
bone marrow, trachea thymus, placenta, fetal liver and Raji cells.
The strongest signals were associated with spleen tissue, lymph
node tissue, and in peripheral blood.
EXAMPLE 3
Ztnfr12 Gene Expression in Cell Lines
[0448] TAQMAN RT-PCR (Applied Biosystems; Foster City, Calif.) was
used to further examine expression of the Ztnfr12 gene, as well as
TACI and BCMA genes. In these studies, the expression of endogenous
human .quadrature.-glucuronidase or glyceraldehyde-3-phosphate
dehydrogenase were used as controls. Ztnfr12, TACI, and BCMA RNA
levels were compared against the RNA levels of these control
genes.
[0449] As shown in Table 6, the results indicated that Ztnfr12 is
primarily exclusively expressed on B lineage cells. In particular,
Ztnfr12 gene expression was observed in transformed B lymphoma cell
lines, such as cells derived from Burkitt's lymphoma (e.g., RAMOS
cells, DAUDI cells, RAJI cells, BJAB cells, and HS Sultan cells),
cells derived from Non-Hodgkin's lymphoma (RL cells), B-cell
lymphoblastic leukemia cells (IM9, SUP-B15, and REH cells), and the
B-cell lymphoma cell lines, DOHH-2, and WSU-NHL. In contrast,
Ztnfr12 gene expression was not detectable in acute T-cell lymphoma
cells (Jurkat), monocytic leukemia cells (THP-1 and U937),
promyelocytic leukemia cells (HL-60), and chronic myelogenous
leukemia cells (K562).
[0450] These results indicate that the Ztnfr12 protein could
provide a useful target in monoclonal antibody therapy against
Burkitt's lymphoma, Non-Hodgkin's lymphoma, acute lymphoblastic
leukemia, and a variety of other B-cell lymphomas. Ztnfr12
expression is also quite high in many of these cells lines compared
with the expression levels of similar receptors. For example, BCMA
seems to be primarily expressed on plasma cells. TABLE-US-00006
TABLE 6 Ztnfr12, TACI, and BCMA Gene Expression Level of Receptor
Gene Expression Cell Line Ztnfr12 TACI BCMA IM9 +++ ++ + RAMOS +++
+ - DAUDI +++ + - RAJI +++ + - HS Sultan ++ - ++ MC-116 - - + BJAB
+++ - +/- RL +++ - + SUP-B15 ++ + + DOHH-2 ++ - + WSU-NHL + + - REH
+ - - K562 - - - HL-60 - -/+ - THP-1 - - - U937 - - -
EXAMPLE 4
Construction and Expression of Ztnfr12-Fc Fusion Protein
A. Ig .quadrature..sub.1 Fragment Construction
[0451] To prepare the Ztnfr12-Fc4 fusion protein, the Fe region of
human IgG1 (the hinge region and the CH.sub.2 and CH.sub.3 domains)
was modified to remove Fc.quadrature.1 receptor (Fc.quadrature.RI)
and complement (C1q) binding functions. This modified version of
human IgG1 Fe was designated "Fc4."
[0452] The Fc region was isolated from a human fetal liver library
(Clontech) PCR using oligo primers 5' ATCAGCGGAA TTCAGATCTT
CAGACAAAAC TCACACATGC CCAC 3' (SEQ ID NO:15) and 5' GGCAGTCTCT
AGATCATTTA CCCGGAGACA GGGAG 3' (SEQ ID NO:16). The nucleotide and
amino acid sequences of a wild-type human [ ]1 constant region are
presented in SEQ ID Nos:17 and 18, respectively. Mutations within
the Fe region were introduced by PCR to reduce Fe.quadrature.RI
binding. The Fe.quadrature.RI binding site (Leu-Leu-Gly-Gly; amino
acid residues 38 to 41 of SEQ ID NO:18, which correspond to EU
index positions 234 to 237) was mutated to Ala-Glu-Gly-Ala to
reduce Fc.quadrature.R1 binding (see, for example, Duncan et al.,
Nature 332:563 (1988); Baum et al., EMBO J. 13:3992 (1994)).
Oligonucleotide primers 5' CCGTGCCCAG CACCTGAAGC CGAGGGGGCA
CCGTCAGTCT TCCTCTTCCC C3' (SEQ ID NO:19) and 5' GGATTCTAGA
TTATTTACCC GGAGACAGGG A 3' (SEQ ID NO:20) were used to introduce
the mutation. To a 50 .quadrature.l final volume was added 570 ng
of IgFc template, 5 .quadrature.l of 10.times.Pfu reaction Buffer
(Stratagene), 8 .quadrature.l of 1.25 mM dNTPs, 31 .quadrature.l of
distilled water, 2 .quadrature.l of 20 mM oligonucleotide primers.
An equal volume of mineral oil was added and the reaction was
heated to 94.degree. C. for one minute. Pfu polymerase (2.5 units,
Stratagene) was added followed by 25 cycles at 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds, 72.degree. C. for one minute
followed by a seven minute extension at 72.degree. C. The reaction
products were fractioned by electrophoresis, and the band
corresponding to the predicted size of about 676 base pairs was
detected. This band was excised from the gel and recovered using a
QIAGEN QIAQUICK Gel Extraction Kit (Qiagen) according to the
manufacturer's instructions. This fragment was designated as the
Fc.quadrature.RI binding site mutated IgFc sequence.
[0453] PCR was also used to introduce a mutation of Ala to Ser
(amino acid residue 134 of SEQ ID NO:18, which corresponds to EU
index position 330) and Pro to Ser (amino acid residue 135 of SEQ
ID NO:18, which corresponds to EU index position 331) to reduce
complement C1q binding or complement fixation (Duncan and Winter,
Nature 332:788 (1988)). Two first round reactions were performed
using the Fc.gamma.RI binding side-mutated IgFc sequence as a
template. To a 50 .quadrature.l final volume was added 1
.quadrature.l of Fc.gamma.RI binding site mutated IgFc template, 5
.quadrature.l of 10.times.Pfu Reaction Buffer (Stratagene), 8
.quadrature.l of 1.25 mM dNTPs, 31 of .quadrature.l distilled
water, 2 .quadrature.l of 20 mM 5' GGTGGCGGCT CCCAGATGGG TCCTGTCCGA
GCCCAGATCT TCAGACAAAA CTCAC 3' (SEQ ID NO:21), a 5' primer
beginning at nucleotide 36 of SEQ ID NO:17, and 2 .quadrature.l of
20 mM 5' TGGGAGGGCT TTGTTGGA 3' (SEQ ID NO:22), a 3' primer
beginning at the complement of nucleotide 405 of SEQ ID NO:17. The
second reaction contained 2 .quadrature.l each of 20 mM stocks of
oligonucleotide primers 5' TCCAACAAAG CCCTCCCATC CTCCATCGAG
AAAACCATCT CC 3' (SEQ ID NO:23), a 5' primer beginning at
nucleotide 388 of SEQ ID NO:17 and 5' GGATGGATCC ATGAAGCACC
TGTGGTTCTT CCTCCTGCTG GTGGCGGCTC CCAGATG 3' (SEQ ID NO:24), a 3'
primer, to introduce the Ala to Ser mutation, XbaI restriction site
and stop codon. An equal volume of mineral oil was added and the
reactions were heated to 94.degree. C. for one minute. Pfu
polymerase (2.5 units, Stratagene) was added followed by 25 cycles
at 94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
72.degree. C. for 2 minutes followed by a seven minute extension at
72.degree. C. The reaction products were fractionated by
electrophoresis, and bands corresponding to the predicted sizes,
about 370 and about 395 base pairs respectively, were detected. The
bands were excised from the gel and extracted using a QIAGEN
QIAQUICK Gel Extraction Kit (Qiagen) according to the
manufacturer's instructions.
[0454] A second round reaction was performed to join the above
fragments and add the 5' BamHI restriction site and a signal
sequence from the human immunoglobulin JBL 2'C.sub.L heavy chain
variable region (Cogne et al., Eur. J. Immunol. 18:1485 (1988)). To
a 50 .quadrature.l final volume was added 30 .quadrature.l of
distilled water, 8 .quadrature.l of 1.25 mM dNTPs, 5 .quadrature.l
of 10.times. Pfu polymerase reaction buffer (Stratagene) and 1
.quadrature.l each of the two first two PCR products. An equal
volume of mineral oil was added and the reaction was heated to
94.degree. C. for one minute. Pfu polymerase (2.5 units,
Stratagene) was added followed by five cycles at 94.degree. C. for
30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 2
minutes. The temperature was again brought to 94.degree. C. and 2
.mu.l each of 20 mM stocks of 5' GGATGGATCC ATGAAGCACC TGTGGTTCTT
CCTCCTGCTG GTGGCGGCTC CCAGATG 3' (SEQ ID NO:25), a 5' primer
beginning at nucleotide 1 of SEQ ID NO:17 that introduces a BamHI
restriction site, and 5' GGATTCTAGA TTATTTACCC GGAGACAGGG A 3' (SEQ
ID NO:26) were added followed by 25 cycles at 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds and 72.degree. C. for two
minutes, and a final seven minute extension at 72.degree. C. A
portion of the reaction was visualized using gel electrophoresis. A
789 base pair band corresponding the predicted size was detected.
The remainder of the mutated Fc PCR fragment was digested with the
restriction enzymes BamHI and XbaI. The digested fragment was
cloned and verified by sequence analysis. The mutated Fc was
designated as "Fc4." The nucleotide and amino acid sequences of Fc4
are provided as SEQ ID NOs:27 and 28, respectively.
[0455] The Ig fusion segment designated as "Fc5" was generated by
using PCR to amplify the Fc4 Ig fusion segment with oligonucleotide
primers 5' GAGCCCAAAT CTTCAGACAA AACTCACACA TGCCCA 3' (SEQ ID
NO:29) and 5' TAATTGGCGC GCCTCTAGAT TATTTACCCG GAGACA 3' (SEQ ID
NO:30). The conditions of the PCR amplification were as follows. To
a 50 .mu.L final volume was added 236 ng Fc4 template, 5 .mu.L
10.times. Pfu reaction buffer, 4 .mu.L of 2.5 mM dNTPs, 1 .mu.L 20
.mu.M each of the primers and 1 .mu.L Pfu polymerase (2.5 units,
Stratagene). The amplification thermal profile consisted of
94.degree. C. for 2 minutes, 5 cycles at 94.degree. C. for 15
seconds, 42.degree. C. for 20 seconds, 72.degree. C. for 45
seconds, 20 cycles at 94.degree. C. for 15 seconds, 72.degree. C.
for one minute 20 seconds, followed by a seven minute extension at
72.degree. C. The reaction product was electrophoresed on a
preparative agarose gel and the band corresponding to the predicted
size of 718 bp was detected. The band was excised from the gel and
recovered using a QIAGEN QIAQUICK Gel Extraction Kit (Qiagen)
according to the manufacturer's instructions. The mutated Fc
fragment was cloned and verified by sequence analysis. The
nucleotide and amino acid sequences of Fc5 are provided as SEQ ID
NOs:31 and 32, respectively.
B. Ztnfr12-Fc5 Expression Vector Construction
[0456] A protein encoding expression cassette for Ztnfr12-tcs-Fc5
was generated by overlap PCR (Horton et al., Gene 77:61 (1989))
using a mouse immunoglobulin heavy chain variable region (Ig VH)
signal sequence cDNA, a Ztnfr12 cDNA, and an Fc5 DNA fragment as
PCR templates. The term "tcs" indicates the presence of a thrombin
cleavage site between the Ztnfr12 segment and the Fc5 segment.
[0457] The first round PCR amplifications consisted of four
separate reactions that generated the four PCR products (designated
as First Round PCR Products 1, 2, 3, and 4) to be used in the
second round, overlap PCR.
[0458] First Round PCR Products 1, 2, 3, and 4 were separately
generated using different oligonucleotide primers and DNA
templates. To a 25 .quadrature.l final volume each was added
approximately 2 ng template DNA, 2.5 .quadrature.l 10.times. Pfu
Polymerase Reaction Buffer (Stratagene), 2 .quadrature.l of 2.5 mM
dNTPs, 2.5 .quadrature.l Rediload (ReGen; Huntsville, Ala.), 20
pmole each 5' oligonucleotide and 3' oligonucleotide primers (see
below), and 0.5 .quadrature.l Pfu polymerase (2.5 units,
Stratagene). The reaction to generate First Round PCR Product 4
also included the addition of 2.5 .quadrature.l GC-Melt Reagent
(Clontech). Information on the templates and primers used in the
PCR amplifications is provided in Tables 7 and 8. TABLE-US-00007
TABLE 7 Templates and Primers Used in the First Round of PCR
Amplification PCR Product Number Template 5' Primer 3' Primer 1
Murine Ig VH 26-10 signal ZC38,989 ZC38,987 sequence cDNA 2 Ztnfr12
cDNA ZC38,986 ZC38,990 3 Ztnfr12 cDNA ZC39,428 ZC39,425 4 Fc5 DNA
fragment ZC39,027 ZC38,874
[0459] TABLE-US-00008 TABLE 8 Oligonucleotide Sequences SEQ ID
Primer Nucleotide Sequence NO. ZC38, 989 5' GGCCGGCCACCATGGGAT 3'
33 ZC38, 987 5' TCGCCTCATAGAGAGGACACCTGCAGT 3' 34 ZC38, 986 5'
GTCCTCTCTATGAGGCGAGGGCCCCGGA 3' 35 ZC38, 990 5'
CGGCGTGCGTAGGAGCCCGCAGGCCAC 3' 36 ZC39, 428 5'
GGGCTCCTACGCACGCCGCGGCCGAAACC 3' 37 ZC39, 425 5'
GGAACCACGCGGAACCAGCGCCGCCTCGCCGGCC 38 CCC 3' ZC39, 027 5'
CTGGTTCCGCGTGGTTCCGAGCCCAAATCTTCAG 39 AC 3' ZC38, 874 5'
GGCGCGCCTCTAGATTATTTACCCGGAGACA 3' 40
[0460] The amplification thermal profile consisted of 94.degree. C.
for 3 minutes, 30 cycles at 94.degree. C. for 30 seconds,
55.degree. C. for 30 seconds, 72.degree. C. for 2 minutes, followed
by a 4 minute extension at 72.degree. C. The reaction products were
fractionated using agarose gel electrophoresis and the bands
corresponding to the predicted sizes were excised from the gel and
recovered using a QIAGEN QIAQUICK Gel Extraction Kit (Qiagen)
according to the manufacturer's instructions.
[0461] The second round PCR amplification, or overlap PCR
amplification reaction, was performed using the gel-purified
fragments from the first round PCR as DNA templates. The conditions
of the second round PCR amplification were as follows. To a 50
.mu.l final volume was added 1 .mu.l of each First Round PCR
Products 1, 2, 3, and 4, 5 .quadrature.l 10.times. Pfu Polymerase
Reaction Buffer (Stratagene), 4 .quadrature.l of 2.5 mM dNTPs, 5
.quadrature.l Rediload (ResGen), 5 .quadrature.l GC-Melt Reagent
(Clontech), approximately 40 pmoles each ZC38,989, ZC38,874 and 0.5
[ ] Pfu Polymerase (2.5 units, Stratagene). The amplification
thermal profile consisted of 94.degree. C. for 3 minutes, 35 cycles
at 94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
72.degree. C. for 3 minutes, followed by a 6 minute extension at
72.degree. C. The reaction product, designated as "Ztnfr12-tcs-Fc5
PCR," was fractionated using agarose gel electrophoresis, and the
band corresponding to the predicted size was excised from the gel
and recovered using a QIAGEN QIAQUICK Gel Extraction Kit (Qiagen)
according to the manufacturer's instructions.
[0462] The Ztnfr12-tcs-Fc5 PCR product was cloned using
Invitrogen's ZEROBLUNT TOPO PCR Cloning Kit following the
manufacturer's recommended protocol and the DNA sequence was
verified. The nucleotide and amino acid sequences of
Ztnfr12-tcs-Fc5 are provided as SEQ ID NOs:41 and 42, respectively.
In SEQ ID NO:42, the murine VH 26-10 signal sequence is represented
by amino acid residues 1 to 19, a Ztnfr12 extracellular domain is
represented by amino acid residues 20 to 90 (i.e., amino acid
residues 1 to 71 of SEQ ID NO:2), the thrombin cleavage site is
represented by amino acid residues 91 to 96, and the Fc5
immunoglobulin moiety is represented by amino acid residues 97 to
328.
[0463] The plasmid encoding the sequence-verified Ztnfr12-tcs-Fc5
was digested with FseI and AscI to release the coding segment. The
FseI-AscI fragment was ligated into a mammalian expression vector
containing a cytomegalovirus promoter (CMV) promoter, an SV40 poly
A segment, and the murine dihydrofolate reductase gene.
EXAMPLE 5
Production of Ztnfr12-Fc Proteins by Chinese Hamster Ovary
Cells
[0464] The Ztnfr12-Fc5 expression construct was used to transfect,
via electroporation, suspension-adapted Chinese hamster ovary (CHO)
DG44 cells grown in animal protein-free medium (Urlaub et al., Som.
Cell. Molec. Genet. 12:555 (1986)). CHO DG44 cells lack a
functional dihydrofolate reductase gene due to deletions at both
dihydrofolate reductase chromosomal locations. Growth of the cells
in the presence of increased concentrations of methotrexate results
in the amplification of the dihydrofolate reductase gene, and the
linked recombinant protein-encoded gene on the expression
construct.
[0465] CHO DG44 cells were passaged in PFCHO media (JRH
Biosciences, Lenexa, Kans.), 4 mM L-Glutamine (JRH Biosciences),
and 1.times. hypothanxine-thymidine supplement (Life Technologies),
and the cells were incubated at 37.degree. C. and 5% CO.sub.2 in
Corning shake flasks at 120 RPM on a rotating shaker platform. The
cells were transfected separately with linearized expression
plasmids. To ensure sterility, a single ethanol precipitation step
was performed on ice for 25 minutes by combining 200 .mu.g of
plasmid DNA in an Eppendorf tube with 20 .mu.l of sheared salmon
sperm carrier DNA (5'.fwdarw.3' Inc. Boulder, Colo., 10 mg/ml), 22
.quadrature.l of 3M NaOAc (pH 5.2), and 484 .quadrature.l of 100%
ethanol (Gold Shield Chemical Co., Hayward, Calif.). After
incubation, the tube was centrifuged at 14,000 RPM in a microfuge
placed in a 4.degree. C. cold room, the supernatant removed and the
pellet washed twice with 0.5 ml of 70% ethanol and allowed to air
dry.
[0466] The CHO DG44 cells were prepared while the DNA pellet was
drying by centrifuging 10.sup.6 total cells (16.5 ml) in a 25 ml
conical centrifuge tube at 900 RPM for five minutes. The CHO DG44
cells were resuspended into a total volume of 300 .mu.l of PFCHO
growth media, and placed in a Gene-Pulser Cuvette with a 0.4 cm
electrode gap (Bio-Rad). The DNA, after approximately 50 minutes of
drying time, was resuspended into 500 .quadrature.l of PFCHO growth
media and added to the cells in the cuvette so that the total
volume did not exceed 800 .quadrature.l and was allowed to sit at
room temperature for five minutes to decrease bubble formation. The
cuvette was placed in a BioRad Gene Pulser II unit set at 0.296 kV
(killiVolts) and 0.950 HC (high capacitance) and electroporated
immediately.
[0467] The cells were incubated five minutes at room temperature
before placement in 20 ml total volume of PFCHO media in a CoStar
T-75 flask. The flask was placed at 37.degree. C. and 5% CO.sub.2
for 48 hours when the cells were then counted by hemocytometer
utilizing trypan blue exclusion and put into PFCHO selection media
without hypothanxine-thymidine supplement and containing 200 mM
methotrexate (Cal Biochem). Upon recovery of the methotrexate
selection process, the conditioned media containing the secreted
Ztnfr12-Fc5 proteins were examined by Western Blot analysis.
[0468] In one study, fusion proteins were purified as follows. Ten
liters of conditioned media from CHO cells were clarified and
sterile-filtered via passage through a 0.22 .mu.m filter. The
filtered medium sample was then applied to a 72 ml protein A column
(Poros 50A) for the capture of Ztnfr12-Fc5 target molecule.
Flow-through material from the original application was reprocessed
twice on the protein A column to enhance maximal recovery. Analysis
with non-reducing SDS-PAGE indicated that the bound material
recovered at this step was both multimeric and dimeric. After
fractionation with reducing SDS-PAGE, only monomeric fusion protein
having a molecular weight of 36 kD was observed. The recovered
mixture of Ztnfr12-Fc5 species was then applied to a Superdex-200
size exclusion chromatography column (318 ml) to further purify and
buffer-exchange the material. This step provided resolution of the
dimeric material from the mulitmeric material.
EXAMPLE 6
Characterization of Ztnfr12-Fc Fusion Proteins
[0469] Edman degradation was performed to identify the N-terminus
of the Ztnfr12-Fc fusion protein. The results indicate that the
N-terminus was digested, and that the first amino acid was
Ser.sup.7.
[0470] Ztnfr12-Fc was digested with thrombin using standard
techniques. Briefly, thrombin digestion was performed by adding the
thrombin at a 1:25 ratio by weight to protein, and incubating at
room temperature for 30 minutes. The reaction was stopped by
immediate injection onto reverse-phase HPLC column for the LC
separation part of the analysis. The eluate from the reverse phase
column was directed into an LCQ mass spectrometer and MS and MS/MS
data were collected. Each digest was analyzed with and without
reduction and peaks observed to be differentially recovered were
identified by mass matching and sequence (MS/MS) confirmation
analysis where possible. Thrombin digestion of the protein
identified the presence of the following two cleavage sites in the
Ztnfr12 domain in addition to the engineered site:
Arg.sup.39-Thr.sup.40, and Arg.sup.54-Thr.sup.55.
[0471] Due to the protease resistance of the Fe domain, no
glycosylation modifications could be observed for that part of the
protein. The single predicted N-linked carbohydrate is in the Fe
domain at Asn.sup.159.
[0472] However, numerous heterogeneous O-glycans were observed
attached to the Ztnfr12 domain. The fully formed structure of these
O-glycans is consistent with previously characterized O-glycans
found on proteins recombinantly produced in CHO cells and is a
tetra-saccharide of the form, (N-acetyl hexosamine)-(N-acetyl
neuramic acid (i.e., sialic acid))-(hexose)-(N-acetyl neuramic
acid). The most predominant form observed was the tri-saccharide,
(N-acetyl hexosamine)-(hexose)-(N-acetyl neuramic acid). Each site
was observed to be partially and heterogeneously occupied with
multiple forms of the carbohydrate ranging from a single N-acetyl
hexosamine to the fully formed tetra-saccharide. Due to the
heterogeneity of the carbohydrates and the incomplete nature of
this analysis, a clear assignment of percent site occupancy was not
possible. The residues that were observed to be modified at some
level were Thr.sup.17, Thr.sup.40, Ser.sup.49, Ser.sup.50,
Thr.sup.55, and Ser.sup.62. These carbohydrate modifications
distinguish this fusion protein from TACI-FC, which has only a
single N-linked carbohydrate in the Fe domain.
[0473] Ztnfr12-Fc5 was immobilized to a plate coated with goat
anti-human IgG Fe, and incubated with ZTNF4-biotin. The results of
this study showed that Ztnfr12-Fc5 binds ZTNF4. Additional studies
showed that Ztnfr12-Fc5 inhibited the proliferation of human
peripheral blood cells, which had been co-activated with soluble
ZTNF4 and recombinant human IL-4, and that Ztnfr12-Fc5 inhibited
ZTNF4-biotin binding to soluble TACI receptor.
EXAMPLE 7
Baculovirus Expression of Soluble Ztnfr12
[0474] An expression vector, pZBV37L:sTNFR12cee, was designed to
express soluble Ztnfr12 polypeptide (amino acid residues 1 to 71 of
SEQ ID NO:2) with a C-terminal "EE" tag (EYMPMD; SEQ ID NO:45),
after cleavage of the signal peptide.
A. Construction of pZBV37L:sTNFR12cee
[0475] A 257 base pair sTNFR12cee fragment containing BspeI and
XbaI restriction sites on the 5' and 3' ends, respectively, was
generated by PCR amplification from a plasmid containing Ztnfr12
cDNA, using primers 5' ATGCATTCCG GAATGAGGCG AGGGCCCCGG AGCCTG 3'
(SEQ ID NO:43) and 5' ATGCATTCTA GATCAGTCCA TCGGCATGTA TTCCGCCGCC
TCGCCGGCCC CCGC 3' (SEQ ID NO:44). The PCR reaction conditions were
as follows, using the Expand High Fidelity PCR System (Boehringer
Mannheim) for a 100 .quadrature.l volume reaction containing 10%
DMSO: 1 cycle at 94.degree. C. for 2 minutes; 35 cycles of
94.degree. C. for 15 seconds, 50.degree. C. for 30 seconds, and
72.degree. C. for 45 seconds; 1 cycle at 72.degree. C. for 5 min;
followed by 4.degree. C. soak. Five microliters of the reaction mix
were visualized by gel electrophoresis (1% NuSieve agarose). The
remainder of the reaction mix was purified via Qiagen PCR
purification kit as per manufacturer's instructions and eluted in
30 .quadrature.l of water. The cDNA was digested in a 35
.quadrature.l volume using Bspel and XbaI (New England Biolabs,
Beverly, Mass.) in appropriate buffer conditions for 1 hr at
37.degree. C. The digested PCR product band was run through a 1%
agarose TAE gel, excised and extracted using a QIAQUICK Gel
Extraction Kit (Qiagen) and eluted in 30 .quadrature.l of water.
The purified, digested sTNFR12cee PCR product was ligated into the
MCS of a previously prepared and restriction enzyme digested (Bspel
and XbaI) vector pZBV37L.
[0476] The pZBV37L vector is a modification of the PFASTBAC1 (Life
Technologies) expression vector, where the polyhedron promoter has
been removed and replaced with the late activating Basic Protein
Promoter and the EGT leader signal sequence upstream of the
multiple cloning site. Five microliters of the restriction digested
sTNFR12cee and about 50 ng of the corresponding pZBV37L vector were
ligated overnight at 16.degree. C. in a 20 .quadrature.l volume in
appropriate buffer conditions. Five microliters of the ligation mix
were transformed into 50 .quadrature.l of ELECTOMAX DH12S cells
(Life Technologies) by electroporation at 400 Ohms, 2V and 25 .mu.F
in a 2 mm gap electroporation cuvette (BTX). The transformed cells
were diluted in 350 .mu.l of SOC media (2% Bacto Tryptone, 0.5%
Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl.sub.2,
10 mM MgSO.sub.4 and 20 mM glucose) incubated for one hour at
37.degree. C., and 50 .mu.l of the dilution were plated onto LB
plates containing 100 .mu.g/ml ampicillin.
[0477] Clones were analyzed by PCR and restriction digestion.
Positive clones were selected, plated and submitted for sequencing.
Once proper sequence was confirmed, 25 ng of positive clone DNA was
transformed into 100 .mu.l DH10BAC MAX EFFICIENCY competent cells
(GIBCO-BRL) by heat shock for 45 seconds in a 42.degree. C. heat
block. The transformed DH10BAC cells were diluted in 900 .mu.l of
SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M
NaCl, 1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM
glucose) incubated for 37.degree. C. for one hour, and 100 .mu.l
were plated onto Luria Agar plates containing 50 .mu.g/ml
kanamycin, 7 .mu.g/ml gentamicin, 10 .mu.g/ml tetracycline, 40
.mu.g/mL IPTG and 200 .mu.g/mL BLUO-GAL
(5-bromo-3-indolyl-.quadrature.-D-galactopyranoside). The plates
were incubated for 48 hours at 37.degree. C. A color selection was
used to identify those cells having transposed viral DNA (referred
to as a "bacmid"). Those colonies, which were white in color, were
picked. Positive white colonies (containing desired bacmid) were
selected for outgrowth and subsequent bacmid DNA purification.
Spodoptera Frugiperda (Sf9) cells were transfected after culture
outgrowth and bacmid isolation.
B. Transfection of Sf9 Cells
[0478] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate and allowed to attach for 1 hour at 27.degree. C.
About 5 .quadrature.g of bacmid DNA were diluted with 100 .mu.l
Sf-900 II SFM (Life Technologies). Twenty microliters of
LIPOFECTAMINE Reagent (Life Technologies) were diluted with 100
.mu.l of Sf-900 II SFM. The bacmid DNA and lipid solutions were
gently mixed and incubated for 45 minutes at room temperature.
Eight hundred microliters of Sf-900 II SFM were added to the
lipid-DNA mixture. The media was aspirated from the well and the 1
ml of DNA-lipid mix added to the cells. The cells were incubated at
27.degree. C. overnight. The DNA-lipid mix was aspirated and 2 ml
of Sf-900 II media were added to each plate. The plates were
incubated at 27.degree. C., 90% humidity, for about seven days, and
then the virus was harvested.
C. Amplification
[0479] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate in 2 ml of SF-900II. Five hundred microliters of
virus from the transfection plate were placed in the well, and the
plate was incubated at 27.degree. C., 90% humidity, for 96 hours
after which the virus was harvested for the primary amplification.
Additional amplification can be achieved using the following
procedure.
[0480] A second round of amplification can proceed as follows: Sf9
cells are seeded at 1.times.10.sup.6 cells per well in a 6-well
plate in 2 ml of SF-900II. One hundred microliters of virus from
the primary amplification plate are placed in the well and the
plate is incubated at 27.degree. C., 90% humidity, for 96 hours,
and then the virus was harvested to complete the secondary
amplification.
[0481] An additional round of amplification can be performed. Sf9
cells are grown in 50 ml Sf-900 II SFM in a 250 ml shake flask to
an approximate density of 1.times.10.sup.6 cells/ml. They are then
infected with 500 .mu.l of the viral stock from the above plate and
incubated at 27.degree. C. for 3 days after which time the virus is
harvested.
[0482] This viral stock is titered by a growth inhibition curve and
the titer culture that indicated a MOI of 1 is allowed to proceed
for a total of 48 hours. The supernatant is analyzed via a
non-reduced Western using a primary monoclonal antibody specific
for the GFD of zVegf4 (E3595) and a HRP conjugated goat anti-Mu
secondary antibody. Results should indicate a dimer band of about
79 kDa and additional higher molecular weight species. Supernatant
can also be used for activity analysis.
[0483] A large viral stock is generated by the following method:
Sf9 cells are grown in 1L Sf-900 II SFM in a 2800 ml shake flask to
an approximate density of 1.times.10.sup.6 cells/ml. They are then
infected with 10 ml of the viral stock from the last amplification,
and incubated at 27.degree. C. for 96 hours, after which time the
virus is harvested.
[0484] Larger scale infections can be completed to provide material
for downstream purification.
[0485] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
46 1 586 DNA Homo sapiens CDS (27)...(578) 1 gcagcttgtg cggcggcgtc
ggcacc atg agg cga ggg ccc cgg agc ctg cgg 53 Met Arg Arg Gly Pro
Arg Ser Leu Arg 1 5 ggc agg gac gcg cca gcc ccc acg ccc tgc gtc ccg
gcc gag tgc ttc 101 Gly Arg Asp Ala Pro Ala Pro Thr Pro Cys Val Pro
Ala Glu Cys Phe 10 15 20 25 gac ctg ctg gtc cgc cac tgc gtg gcc tgc
ggg ctc ctg cgc acg ccg 149 Asp Leu Leu Val Arg His Cys Val Ala Cys
Gly Leu Leu Arg Thr Pro 30 35 40 cgg ccg aaa ccg gcc ggg gcc agc
agc cct gcg ccc agg acg gcg ctg 197 Arg Pro Lys Pro Ala Gly Ala Ser
Ser Pro Ala Pro Arg Thr Ala Leu 45 50 55 cag ccg cag gag tcg gtg
ggc gcg ggg gcc ggc gag gcg gcg ctg ccc 245 Gln Pro Gln Glu Ser Val
Gly Ala Gly Ala Gly Glu Ala Ala Leu Pro 60 65 70 ctg ccc ggg ctg
ctc ttt ggc gcc ccc gcg ctg ctg ggc ctg gca ctg 293 Leu Pro Gly Leu
Leu Phe Gly Ala Pro Ala Leu Leu Gly Leu Ala Leu 75 80 85 gtc ctg
gcg ctg gtc ctg gtg ggt ctg gtg agc tgg agg cgg cga cag 341 Val Leu
Ala Leu Val Leu Val Gly Leu Val Ser Trp Arg Arg Arg Gln 90 95 100
105 cgg cgg ctt cgc ggc gcg tcc tcc gca gag gcc ccc gac gga gac aag
389 Arg Arg Leu Arg Gly Ala Ser Ser Ala Glu Ala Pro Asp Gly Asp Lys
110 115 120 gac gcc cca gag ccc ctg gac aag gtc atc att ctg tct ccg
gga atc 437 Asp Ala Pro Glu Pro Leu Asp Lys Val Ile Ile Leu Ser Pro
Gly Ile 125 130 135 tct gat gcc aca gct cct gcc tgg cct cct cct ggg
gaa gac cca gga 485 Ser Asp Ala Thr Ala Pro Ala Trp Pro Pro Pro Gly
Glu Asp Pro Gly 140 145 150 acc acc cca cct ggc cac agt gtc cct gtg
cca gcc aca gag ctg ggc 533 Thr Thr Pro Pro Gly His Ser Val Pro Val
Pro Ala Thr Glu Leu Gly 155 160 165 tcc act gaa ctg gtg acc acc aag
acg gcc ggc cct gag caa caa 578 Ser Thr Glu Leu Val Thr Thr Lys Thr
Ala Gly Pro Glu Gln Gln 170 175 180 tagcaggg 586 2 184 PRT Homo
sapiens 2 Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro
Ala Pro 1 5 10 15 Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu
Val Arg His Cys 20 25 30 Val Ala Cys Gly Leu Leu Arg Thr Pro Arg
Pro Lys Pro Ala Gly Ala 35 40 45 Ser Ser Pro Ala Pro Arg Thr Ala
Leu Gln Pro Gln Glu Ser Val Gly 50 55 60 Ala Gly Ala Gly Glu Ala
Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly 65 70 75 80 Ala Pro Ala Leu
Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu Val 85 90 95 Gly Leu
Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser 100 105 110
Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp 115
120 125 Lys Val Ile Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro
Ala 130 135 140 Trp Pro Pro Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro
Gly His Ser 145 150 155 160 Val Pro Val Pro Ala Thr Glu Leu Gly Ser
Thr Glu Leu Val Thr Thr 165 170 175 Lys Thr Ala Gly Pro Glu Gln Gln
180 3 552 DNA Artificial Sequence This degenerate nucleotide
sequence encodes the amino acid sequence of SEQ ID NO2.
misc_feature 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 39, 42, 45, 48,
51, 54, 60, 63, 66, 81, 84, 87, 90, 99, 102, 108, 111, 114, 117,
120, 123, 126, 129, 135, 138, 141, 144, 147, 150, 153, 156, 159,
162, 165, 168, 171, 177, 186, 189, 192, 195, 198 n = A,T,C or G
misc_feature 201, 204, 210, 213, 216, 219, 222, 225, 228, 231, 234,
240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276,
279, 282, 285, 288, 291, 294, 297, 300, 306, 309, 312, 318, 321,
324, 327, 330, 333, 336, 339, 342, 348, 351, 357 n = A,T,C or G
misc_feature 369, 372, 378, 381, 390, 399, 402, 405, 408, 414, 420,
423, 426, 429, 432, 438, 441, 444, 447, 456, 459, 462, 465, 468,
471, 474, 480, 483, 486, 489, 492, 495, 498, 504, 507, 510, 513,
519, 522, 525, 528, 534, 537, 540, 543 n = A,T,C or G 3 atgmgnmgng
gnccnmgnws nytnmgnggn mgngaygcnc cngcnccnac nccntgygtn 60
ccngcngart gyttygayyt nytngtnmgn caytgygtng cntgyggnyt nytnmgnacn
120 ccnmgnccna arccngcngg ngcnwsnwsn ccngcnccnm gnacngcnyt
ncarccncar 180 garwsngtng gngcnggngc nggngargcn gcnytnccny
tnccnggnyt nytnttyggn 240 gcnccngcny tnytnggnyt ngcnytngtn
ytngcnytng tnytngtngg nytngtnwsn 300 tggmgnmgnm gncarmgnmg
nytnmgnggn gcnwsnwsng cngargcncc ngayggngay 360 aargaygcnc
cngarccnyt ngayaargtn athathytnw snccnggnat hwsngaygcn 420
acngcnccng cntggccncc nccnggngar gayccnggna cnacnccncc nggncaywsn
480 gtnccngtnc cngcnacnga rytnggnwsn acngarytng tnacnacnaa
racngcnggn 540 ccngarcarc ar 552 4 16 PRT Artificial Sequence
Peptide linker. 4 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 1 5 10 15 5 285 PRT Homo sapiens 5 Met Asp Asp Ser
Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys
Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro 20 25 30
Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu 35
40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val
Val 50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala
Ser Leu Arg 65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu
Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala
Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys Ile Phe Glu Pro Pro Ala
Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120 125 Ser Arg Asn Lys Arg
Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140 Asp Cys Leu
Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys 145 150 155 160
Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165
170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly
Tyr 180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr
Tyr Ala Met 195 200 205 Gly His Leu Ile Gln Arg Lys Lys Val His Val
Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile
Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr
Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255 Asp Glu Leu Gln
Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270 Asp Gly
Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 285 6 250 PRT
Homo sapiens 6 Met Pro Ala Ser Ser Pro Phe Leu Leu Ala Pro Lys Gly
Pro Pro Gly 1 5 10 15 Asn Met Gly Gly Pro Val Arg Glu Pro Ala Leu
Ser Val Ala Leu Trp 20 25 30 Leu Ser Trp Gly Ala Ala Leu Gly Ala
Val Ala Cys Ala Met Ala Leu 35 40 45 Leu Thr Gln Gln Thr Glu Leu
Gln Ser Leu Arg Arg Glu Val Ser Arg 50 55 60 Leu Gln Gly Thr Gly
Gly Pro Ser Gln Asn Gly Glu Gly Tyr Pro Trp 65 70 75 80 Gln Ser Leu
Pro Glu Gln Ser Ser Asp Ala Leu Glu Ala Trp Glu Asn 85 90 95 Gly
Glu Arg Ser Arg Lys Arg Arg Ala Val Leu Thr Gln Lys Gln Lys 100 105
110 Lys Gln His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys
115 120 125 Asp Asp Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu
Arg Arg 130 135 140 Gly Arg Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg
Ile Gln Asp Ala 145 150 155 160 Gly Val Tyr Leu Leu Tyr Ser Gln Val
Leu Phe Gln Asp Val Thr Phe 165 170 175 Thr Met Gly Gln Val Val Ser
Arg Glu Gly Gln Gly Arg Gln Glu Thr 180 185 190 Leu Phe Arg Cys Ile
Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr 195 200 205 Asn Ser Cys
Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile 210 215 220 Leu
Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro 225 230
235 240 His Gly Thr Phe Leu Gly Phe Val Lys Leu 245 250 7 184 PRT
Homo sapiens 7 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr
Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg
Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys
Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Ile
Leu Trp Thr Cys Leu Gly Leu Ser Leu 50 55 60 Ile Ile Ser Leu Ala
Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile 65 70 75 80 Ser Ser Glu
Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu 85 90 95 Leu
Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu 100 105
110 Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys
115 120 125 Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His
Cys Phe 130 135 140 Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu
Val Thr Thr Lys 145 150 155 160 Thr Asn Asp Tyr Cys Lys Ser Leu Pro
Ala Ala Leu Ser Ala Thr Glu 165 170 175 Ile Glu Lys Ser Ile Ser Ala
Arg 180 8 293 PRT Homo sapiens 8 Met Ser Gly Leu Gly Arg Ser Arg
Arg Gly Gly Arg Ser Arg Val Asp 1 5 10 15 Gln Glu Glu Arg Phe Pro
Gln Gly Leu Trp Thr Gly Val Ala Met Arg 20 25 30 Ser Cys Pro Glu
Glu Gln Tyr Trp Asp Pro Leu Leu Gly Thr Cys Met 35 40 45 Ser Cys
Lys Thr Ile Cys Asn His Gln Ser Gln Arg Thr Cys Ala Ala 50 55 60
Phe Cys Arg Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Phe Tyr Asp 65
70 75 80 His Leu Leu Arg Asp Cys Ile Ser Cys Ala Ser Ile Cys Gly
Gln His 85 90 95 Pro Lys Gln Cys Ala Tyr Phe Cys Glu Asn Lys Leu
Arg Ser Pro Val 100 105 110 Asn Leu Pro Pro Glu Leu Arg Arg Gln Arg
Ser Gly Glu Val Glu Asn 115 120 125 Asn Ser Asp Asn Ser Gly Arg Tyr
Gln Gly Leu Glu His Arg Gly Ser 130 135 140 Glu Ala Ser Pro Ala Leu
Pro Gly Leu Lys Leu Ser Ala Asp Gln Val 145 150 155 160 Ala Leu Val
Tyr Ser Thr Leu Gly Leu Cys Leu Cys Ala Val Leu Cys 165 170 175 Cys
Phe Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg Gly Asp Pro 180 185
190 Cys Ser Cys Gln Pro Arg Ser Arg Pro Arg Gln Ser Pro Ala Lys Ser
195 200 205 Ser Gln Asp His Ala Met Glu Ala Gly Ser Pro Val Ser Thr
Ser Pro 210 215 220 Glu Pro Val Glu Thr Cys Ser Phe Cys Phe Pro Glu
Cys Arg Ala Pro 225 230 235 240 Thr Gln Glu Ser Ala Val Thr Pro Gly
Thr Pro Asp Pro Thr Cys Ala 245 250 255 Gly Arg Trp Gly Cys His Thr
Arg Thr Thr Val Leu Gln Pro Cys Pro 260 265 270 His Ile Pro Asp Ser
Gly Leu Gly Ile Val Cys Val Pro Ala Gln Glu 275 280 285 Gly Gly Pro
Gly Ala 290 9 5759 DNA Homo sapiens exon (1001)...(1136) exon
(1443)...(1673) exon (2220)...(2404) 9 taatcccagc actttgggag
gccgaggagg gcggatcacc tgaggtcagg agttcgagac 60 cagcctggcc
aaatggtgaa accccacctc tactaaaaat acaaaaatta gccgggcatg 120
gtggtgcatg cctgtaatcc cagttactcg ggaggctgag gcaggagaat cgcttgaatc
180 tgggaggcag aggttgcagt gagccgagat tgcaccactg cacagagcca
gactcttttt 240 caaaaaaaaa aaaaaaaaaa gcaggtgtct gatatcccca
accagctctg tccaggaagg 300 gcaggaagag aggaggagac aggtgggttg
ggggagatgg caggggagca ctcggggtca 360 tggagaggct ttggctagac
ggagcagagg aaccactcag ggtctgtgtc ctggctgcgg 420 ggtccatccc
cctccctacc caagaggagg ctctggtctg cccccagaca cctcccagca 480
cccagcagag ggcccaccca ggcggtgctg gttgaggggc tgaattgggg aaccacaggt
540 agaaagagag gccaggccgg gtgcggtggc tcacgcctgt aatcctagca
ctttgggagg 600 ccgaggcagg tggatcacga ggtcaggagt tcaaaaccag
tctgaccaag atggtgaaac 660 cccgtctcta ctaaaaatac aaaaattagc
cgggcgtggc agtgggcgcc tacaatctca 720 gctactcggg aggctgaggc
agagaattgt ttgaacccgg gaggcagagc ttgcagtgag 780 ccgagatagc
gccattgcac tccagcctgg gcgacagagc gagactccgt ctcaaaaaaa 840
aaaaaagaaa agaaaggggg gccccaggcg agctcggtcc cacccagcag gcgggggcgg
900 ggcagggcag agtgctcccc ccgccccccg cttcctcccc gagggccccg
gagcccagct 960 cagcctcagt ccccgcagct tgtgcggcgg cgtcggcacc
atgaggcgag ggccccggag 1020 cctgcggggc agggacgcgc cagcccccac
gccctgcgtc ccggccgagt gcttcgacct 1080 gctggtccgc cactgcgtgg
cctgcgggct cctgcgcacg ccgcggccga aaccgggtaa 1140 gggggaccca
cggggcgcgc ggcgccggca gctgcgggga gaacggggcc ccgatcgcca 1200
gggcgcaggc agagccccga cccccggggg cgccgagggc tgaaaggacc ctgtgggcag
1260 ggcctggagg ggcccgcgat caccgcgtgg ccctcaccgc cgcctctctc
cctccccttg 1320 tccaccgccc cccggctgtc cctcccctcc ccggccagcc
tcgcccccct ccgcccctcc 1380 ccgtccccgc tcctccctcc cctcggcccc
ctggcctccc tccctgtccc ctcccgaagc 1440 agccggggcc agcagccctg
cgcccaggac ggcgctgcag ccgcaggagt cggtgggcgc 1500 gggggccggc
gaggcggcgc tgcccctgcc cgggctgctc tttggcgccc ccgcgctgct 1560
gggcctggca ctggtcctgg cgctggtcct ggtgggtctg gtgagctgga ggcggcgaca
1620 gcggcggctt cgcggcgcgt cctccgcaga ggcccccgac ggagacaagg
acggtgagtt 1680 ccgtgtgtag gggaaccagt ctgagcgggg cgcaggggct
ggggtgtcgg gacggtctag 1740 ggggagatgc atggcccctg agggggagag
aagggcttta aggggaaacg gagacagagt 1800 ggaaaagagt ttgcgctgag
gagctcaggc tggggaggga gacatagtcc tgcccctgag 1860 accctggtcg
gagccaggaa cagctctgcc ctgacagacc ccagtgtgag gaagagacag 1920
gttgcaggaa gggggccccg tctgagggag gagtctgtcc tgccctttgg gagtctaacg
1980 gagcttggtt cccccataaa tccctgaaga gtttgccaga ctgagcccca
gctccccaac 2040 tccctgccct gccagctccc cactggccag gcctctggac
tcagggtcag ccaggtgcca 2100 cccctcccca ccctgcattg ggcttcattt
gacggaggac tgcccctcca gaggagtctt 2160 ctaggggagg gagggaaggc
tgtgactgag tacggagcct ctaccccctc tgcctgcagc 2220 cccagagccc
ctggacaagg tcatcattct gtctccggga atctctgatg ccacagctcc 2280
tgcctggcct cctcctgggg aagacccagg aaccacccca cctggccaca gtgtccctgt
2340 gccagccaca gagctgggct ccactgaact ggtgaccacc aagacggccg
gccctgagca 2400 acaatagcag ggagccggca ggaggtggcc cctgccctcc
ctctggaccc ccagccaggg 2460 gcttggaaat caaattcagc tcttcactcc
agcatgcaca tgccctcttt ctgggaccag 2520 gctaactctg cagaagcaca
gacactacag accacagcat tcagccccca tggagtttgg 2580 tgtgcttgcc
tttggcttca gacctcacca tctttgacag cccttgaagg tggtagccca 2640
gctcctgttc ctgtgccttc aaaaggctgg ggcactatga gtaaaagacc gcttttaaaa
2700 tggggaaggc accattaagc caaaatgaat ctgaaaaaag accaagtggg
aggatgccta 2760 atttgtagtt gggtggtttt ttgtgtttgt ttgatttttt
tttttttttt tttttttttt 2820 ttttgagacg agtcttgctc tgtcgccagg
ctggagtaca gtggcgcgat ctcgcctcac 2880 tgtaacctcc gactccttgg
ttcaagtgat tctcctgcct cagcctcccg aatagctggg 2940 attacaggca
cgcaccacca cagccagcta atttttgtat ttttagtaga gacgggattt 3000
caccatgttg gccaggatgg tctcgatctc ctgacctcgt gatccgccct gctcggcctc
3060 ccaaagtgct gggattacag gcatgagcca ccatgcccgg ccttgttttg
tgttttggtt 3120 ttttttgaga cagggtcttg ctgtatcacg caggctggag
tgcaacgatg ggtccccagc 3180 taattgcagc ctgggctcaa gcaatctttc
tgcttcagcc tcctgagtag gtgggactac 3240 aggtgctcca ccttgcccag
ctaatttttt tttttttttt ttttttgaga cagagtctcg 3300 ctctgtcgcc
taggctggag tgcagtgacg caatttcggc tcacttcaag ctccgcctcc 3360
tgggttcaca ccattctcct gcctgagcct cctgagtagc tgggactata ggcgcccgcc
3420 atcacgcccg gctaattttt tttgtatttt tagtagagac ggggtgtcac
cgtattagcc 3480 aggatggtct cgatctcctg acctcgtgat ccgcccgcct
cagcctccca aagtgctagg 3540 attacaggcg tgagccaccg tggctggcct
tttttttttt ttaaagacag ggtctcggta 3600 tgttgcccag gccagtctca
aactcctggc ctcaggcagt cctccccgaa gggctgggat 3660 tataggcagg
aggcgccggg cctggcctct atttctttaa tacctgccct cagagccgtc 3720
ctgcttttaa actgatgcaa atggcagcag tgaataatca attcatgggt tcgcttgctt
3780 tgggatttag tacaagatta ttttaagaaa taacaaagga gaaaagattg
agctggattt 3840 tgctgattct caggtatctg taccccaggg tcaaccctga
gattggccca atctgccaga 3900 acctaagatg ccattttgac cccagtggca
gcttccagag ccacaagcct gcatttggga 3960 ggaggtgcgc agcctccagg
gctggactcc aaactcgagc tgaaggaaca tgctggcagg 4020 tagctctctc
agggtccacc tggggttcca gctgctccaa gacacagcac atttagccat 4080
aaacatggaa gggctgggac ctacaggagg ctcctttgtg tagaggccag ggcaggcgtg
4140 tgaccttgaa ccaggtggac atgaggtccc ttgaactccc ttctctctct
ggcttgttcc 4200 cctgaagcac tggcccccca gtgctgggct tatttggcca
tcctgtgcca gccccctggt 4260 cacccaccgt cacagtggaa gctgctccct
cctcttacaa tggtgttcct tgctctgggg 4320 tctggggaca agaccatctc
tttccactcc tggacgacac tgtgcactca gtcagccgcc 4380 tcctacctgc
cctcctgcac agggtcagaa cctcctgacc tggacttcgc cctgggcagc 4440
ttccttggaa cttgttgcag cttagcatct ttggaaggga gcactggact aggagtcaga
4500 aggtggagtt ctgggtgctg ttttcccact cttgggccct gggcatgttc
tgttctgtgc 4560 atcagatgtt ttctgcctgc ctccccctga gctgccacca
gagccacctg tagacccctg 4620 cttctcctgc ccctcctgtc ccttgccagg
cctctggact cagtgcttcc tgtcccaagg 4680 acctgttgtg tgcccagctt
ggaagacagt ggccactcac atccaggtct ctgcctggct 4740 atttgctcca
tgaacccatc tcagatgccc ctgcccccag ggagccaggt gacagggtga 4800
ggtcaagtga caagcagtag gtgtgggaga ggtttggtgg ggtcaggccc acatgggccc
4860 agccatccac tccccttcca tctggagttg ggggagtctg agctgctcct
ggccacccca 4920 tctgccccac cccatctttt cctggccacc ccagctccca
ccccatcttc tcctggccac 4980 cccatctccc accccatctt ctcttgacac
cccatctccc accccatctt ctcctggcca 5040 ccccatctcc caccccatct
tctcctggcc accccatctt ctcctggcca ccccatctcc 5100 cccaccccat
cttctcctgg acacccccat ctcccacccc atcttttgct acagccaggg 5160
ttagctcagc aggtgaaaac cccgagggtg ggtgaaaccc ctctggggct cagacatgca
5220 aaccttgggc atctctctgt cccagctggc cccgccagcc ggtaggaagt
ttcccctgag 5280 ttctcagttt tttcttctga aaaatgaggg gttgtatgca
aggttctcct cctggcctgt 5340 ggtccccaga gaagggcagg aaggaacctt
agataattct catatgcatt taacagacga 5400 ggaaactgag acccagagcc
gtcacatcaa tacctcattt gatcttcata agagcacctg 5460 gaggaggggg
gtggggtgtt tgtgtttgtt taaatttttt tttgtgaaaa aaatgaagat 5520
aggcattttg tagacaatct ggaagttctg gaccggaatc catgatgtag tcagggaaga
5580 aatgacccgt gtccagtaac cccaggcctc gagtgtgtgg tgtatttttc
tacataattg 5640 taatcattct atacatacaa attcatgtct tgaccatcat
attaatattt ggtaagtttc 5700 tctctcttta gagactccac aataaagttt
tcaacatggt aaggttttcc acctgggca 5759 10 29 DNA Artificial Sequence
PCR primer. 10 gcgaattcgt cggcaccatg aggcgaggg 29 11 31 DNA
Artificial Sequence PCR primer. 11 cgctcgagct gccggctccc tgctattgtt
g 31 12 685 DNA Mouse CDS (36)...(560) 12 gaattcgccc ttccccagct
gcatgaggcg gcgac atg ggc gcc agg aga ctc 53 Met Gly Ala Arg Arg Leu
1 5 cgg gtc cga agc cag agg agc cgg gac agc tcg gtg ccc acc cag tgc
101 Arg Val Arg Ser Gln Arg Ser Arg Asp Ser Ser Val Pro Thr Gln Cys
10 15 20 aat cag acc gag tgc ttc gac cct ctg gtg aga aac tgc gtg
tcc tgt 149 Asn Gln Thr Glu Cys Phe Asp Pro Leu Val Arg Asn Cys Val
Ser Cys 25 30 35 gag ctc ttc cac acg ccg gac act gga cat aca agc
agc ctg gag cct 197 Glu Leu Phe His Thr Pro Asp Thr Gly His Thr Ser
Ser Leu Glu Pro 40 45 50 ggg aca gct ctg cag cct cag gag ggc tcc
gcg ctg aga ccc gac gtg 245 Gly Thr Ala Leu Gln Pro Gln Glu Gly Ser
Ala Leu Arg Pro Asp Val 55 60 65 70 gcg ctg ctc gtc ggt gcc ccc gca
ctc ctg gga ctg ata ctg gcg ctg 293 Ala Leu Leu Val Gly Ala Pro Ala
Leu Leu Gly Leu Ile Leu Ala Leu 75 80 85 acc ctg gtg ggt cta gtg
agt ctg gtg agc tgg agg tgg cgt caa cag 341 Thr Leu Val Gly Leu Val
Ser Leu Val Ser Trp Arg Trp Arg Gln Gln 90 95 100 ctc agg acg gcc
tcc cca gac act tca gaa gga gtc cag caa gag tcc 389 Leu Arg Thr Ala
Ser Pro Asp Thr Ser Glu Gly Val Gln Gln Glu Ser 105 110 115 ctg gaa
aat gtc ttt gta ccc tcc tca gaa acc cct cat gcc tca gct 437 Leu Glu
Asn Val Phe Val Pro Ser Ser Glu Thr Pro His Ala Ser Ala 120 125 130
cct acc tgg cct ccg ctc aaa gaa gat gca gac agc gcc ctg cca cgc 485
Pro Thr Trp Pro Pro Leu Lys Glu Asp Ala Asp Ser Ala Leu Pro Arg 135
140 145 150 cac agc gtc ccg gtg ccc gcc aca gaa ctg ggc tcc acc gag
ctg gtg 533 His Ser Val Pro Val Pro Ala Thr Glu Leu Gly Ser Thr Glu
Leu Val 155 160 165 acc acc aag aca gct ggc cca gag caa tagcagcagt
ggaggctgga 580 Thr Thr Lys Thr Ala Gly Pro Glu Gln 170 175
acccagggat ctctactggg cttgtggact tcacccaaca gcttgggaaa gaacttggcc
640 cttcagtgac ggagtccttt gcctgggggg cgaaagggcg aattc 685 13 175
PRT Mouse 13 Met Gly Ala Arg Arg Leu Arg Val Arg Ser Gln Arg Ser
Arg Asp Ser 1 5 10 15 Ser Val Pro Thr Gln Cys Asn Gln Thr Glu Cys
Phe Asp Pro Leu Val 20 25 30 Arg Asn Cys Val Ser Cys Glu Leu Phe
His Thr Pro Asp Thr Gly His 35 40 45 Thr Ser Ser Leu Glu Pro Gly
Thr Ala Leu Gln Pro Gln Glu Gly Ser 50 55 60 Ala Leu Arg Pro Asp
Val Ala Leu Leu Val Gly Ala Pro Ala Leu Leu 65 70 75 80 Gly Leu Ile
Leu Ala Leu Thr Leu Val Gly Leu Val Ser Leu Val Ser 85 90 95 Trp
Arg Trp Arg Gln Gln Leu Arg Thr Ala Ser Pro Asp Thr Ser Glu 100 105
110 Gly Val Gln Gln Glu Ser Leu Glu Asn Val Phe Val Pro Ser Ser Glu
115 120 125 Thr Pro His Ala Ser Ala Pro Thr Trp Pro Pro Leu Lys Glu
Asp Ala 130 135 140 Asp Ser Ala Leu Pro Arg His Ser Val Pro Val Pro
Ala Thr Glu Leu 145 150 155 160 Gly Ser Thr Glu Leu Val Thr Thr Lys
Thr Ala Gly Pro Glu Gln 165 170 175 14 525 DNA Artificial Sequence
This degenerate nucleotide sequence encodes the amino acid sequence
of SEQ ID NO13. misc_feature 6, 9, 12, 15, 18, 21, 24, 27, 30, 36,
39, 42, 48, 51, 54, 57, 60, 75, 90, 93, 96, 99, 108, 111, 120, 129,
132, 138, 141, 147, 150, 153, 156, 162, 165, 168, 171, 174, 180,
189, 192, 195, 198, 201, 204, 210, 213, 216, 219, 222, 225 n =
A,T,C or G misc_feature 228, 231, 234, 237, 240, 243, 246, 252,
255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 294,
300, 309, 312, 315, 318, 321, 324, 330, 333, 339, 342, 354, 357,
366, 372, 375, 378, 381, 387, 390, 396, 399, 402, 405, 408, 414 n =
A,T,C or G misc_feature 417, 420, 432, 438, 441, 444, 447, 450,
456, 459, 462, 465, 468, 471, 474, 480, 483, 486, 489, 495, 498,
501, 504, 510, 513, 516, 519 n = A,T,C or G 14 atgggngcnm
gnmgnytnmg ngtnmgnwsn carmgnwsnm gngaywsnws ngtnccnacn 60
cartgyaayc aracngartg yttygayccn ytngtnmgna aytgygtnws ntgygarytn
120 ttycayacnc cngayacngg ncayacnwsn wsnytngarc cnggnacngc
nytncarccn 180 cargarggnw sngcnytnmg nccngaygtn gcnytnytng
tnggngcncc ngcnytnytn 240 ggnytnathy tngcnytnac nytngtnggn
ytngtnwsny tngtnwsntg gmgntggmgn 300 carcarytnm gnacngcnws
nccngayacn wsngarggng tncarcarga rwsnytngar 360 aaygtnttyg
tnccnwsnws ngaracnccn caygcnwsng cnccnacntg gccnccnytn 420
aargargayg cngaywsngc nytnccnmgn caywsngtnc cngtnccngc nacngarytn
480 ggnwsnacng arytngtnac nacnaaracn gcnggnccng arcar 525 15 44 DNA
Artificial Sequence PCR primer. 15 atcagcggaa ttcagatctt cagacaaaac
tcacacatgc ccac 44 16 35 DNA Artificial Sequence PCR primer. 16
ggcagtctct agatcattta cccggagaca gggag 35 17 762 DNA Homo sapiens
CDS (7)...(759) 17 ggatcc atg aag cac ctg tgg ttc ttc ctc ctg ctg
gtg gcg gct ccc 48 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala
Ala Pro 1 5 10 aga tgg gtc ctg tcc gag ccc aaa tct tgt gac aaa act
cac aca tgc 96 Arg Trp Val Leu Ser Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 15 20 25 30 cca ccg tgc cca gca cct gaa ctc ctg ggg gga
ccg tca gtc ttc ctc 144 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 35 40 45 ttc ccc cca aaa ccc aag gac acc ctc
atg atc tcc cgg acc cct gag 192 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 50 55 60 gtc aca tgc gtg gtg gtg gac
gtg agc cac gaa gac cct gag gtc aag 240 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 65 70 75 ttc aac tgg tac gtg
gac ggc gtg gag gtg cat aat gcc aag aca aag 288 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 80 85 90 ccg cgg gag
gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc 336 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 95 100 105 110
acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag 384
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115
120 125 gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc
aaa 432 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 130 135 140 gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg
ccc cca tcc 480 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 145 150 155 cgg gat gag ctg acc aag aac cag gtc agc ctg
acc tgc ctg gtc aaa 528 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 160 165 170 ggc ttc tat ccc agc gac atc gcc gtg
gag tgg gag agc aat ggg cag 576 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 175 180 185 190 ccg gag aac aac tac aag
acc acg cct ccc gtg ctg gac tcc gac ggc 624 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 195 200 205 tcc ttc ttc ctc
tac agc aag ctc acc gtg gac aag agc agg tgg cag 672 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220 cag ggg
aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 720 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 225 230 235
cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 762 His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 240 245 250 18 251 PRT
Homo sapiens 18 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro 20 25 30 Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro 35 40 45 Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr 50 55 60 Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 65 70 75 80 Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90 95 Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 100 105
110 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
115 120 125 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys 130 135 140 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp 145 150 155 160 Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe 165 170 175 Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 180 185 190 Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 195 200 205 Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 210 215 220 Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 225 230
235 240 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 19 51
DNA Artificial Sequence PCR primer. 19 ccgtgcccag cacctgaagc
cgagggggca ccgtcagtct tcctcttccc c 51 20 31 DNA Artificial Sequence
PCR primer. 20 ggattctaga ttatttaccc ggagacaggg a 31 21 55 DNA
Artificial Sequence PCR primer. 21 ggtggcggct cccagatggg tcctgtccga
gcccagatct tcagacaaaa ctcac 55 22 18 DNA Artificial Sequence PCR
primer. 22 tgggagggct ttgttgga 18 23 42 DNA Artificial Sequence PCR
primer. 23 tccaacaaag ccctcccatc ctccatcgag aaaaccatct cc 42 24 47
DNA Artificial Sequence PCR primer. 24 atgaagcacc tgtggttctt
cctcctgctg gtggcggctc ccagatg 47 25 57 DNA Artificial Sequence PCR
primer. 25 ggatggatcc atgaagcacc tgtggttctt cctcctgctg gtggcggctc
ccagatg 57 26 31 DNA Artificial Sequence PCR primer. 26 ggattctaga
ttatttaccc ggagacaggg a 31 27 718 DNA Artificial Sequence Modified
immunoglobulin moiety. CDS (1)...(696) 27 gag ccc aga tct tca gac
aaa act cac aca tgc cca ccg tgc cca gca 48 Glu Pro Arg Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 cct gaa gcc gag
ggg gca ccg tca gtc ttc ctc ttc ccc cca aaa ccc 96 Pro Glu Ala Glu
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 aag gac
acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg 144 Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45
gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg 192
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50
55 60 gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag
cag 240 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 65 70 75 80 tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc
ctg cac cag 288 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln 85 90 95 gac tgg ctg aat ggc aag gag tac aag tgc aag
gtc tcc aac aaa gcc 336 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala 100 105 110 ctc cca tcc tcc atc gag aaa acc atc
tcc aaa gcc aaa ggg cag ccc 384 Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro 115 120 125 cga gaa cca cag gtg tac acc
ctg ccc cca tcc cgg gat gag ctg acc 432 Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 aag aac cag gtc agc
ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 480 Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 gac atc
gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 528 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175
aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac 576
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180
185 190 agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc
ttc 624 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe 195 200 205 tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac
acg cag aag 672 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys 210 215 220 agc ctc tcc ctg tct ccg ggt aaa taatctagag
gcgcgccaat ta 718 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 28 232
PRT Artificial Sequence Modified immunoglobulin moiety. 28 Glu Pro
Arg Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15
Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20
25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150
155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr 180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195
200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 29 36 DNA
Artificial Sequence PCR primer. 29 gagcccaaat cttcagacaa aactcacaca
tgccca 36 30 36 DNA Artificial Sequence PCR primer. 30 taattggcgc
gcctctagat tatttacccg gagaca 36 31 718 DNA Artificial Sequence
Modified immunoglobulin moiety. CDS (1)...(696) 31 gag ccc aaa tct
tca gac aaa act cac aca tgc cca ccg tgc cca gca 48 Glu Pro Lys Ser
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 cct gaa
gcc gag ggg gca ccg tca gtc ttc ctc ttc ccc cca aaa ccc 96 Pro Glu
Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30
aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg 144
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35
40 45 gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac
gtg 192 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val 50 55 60 gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg
gag gag cag 240 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln 65 70 75 80 tac aac agc acg tac cgt gtg gtc agc gtc ctc
acc gtc ctg cac cag 288 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln 85 90 95 gac tgg ctg aat ggc aag gag tac aag
tgc aag gtc tcc aac aaa gcc 336 Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 100 105 110 ctc cca tcc tcc atc gag aaa
acc atc tcc aaa gcc aaa ggg cag ccc 384 Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 cga gaa cca cag gtg
tac acc ctg ccc cca tcc cgg gat gag ctg acc 432 Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 aag aac cag
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 480 Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160
gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 528
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165
170 175 aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc
tac 576 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr 180 185 190 agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg
aac gtc ttc 624 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe 195 200 205 tca tgc tcc gtg atg cat gag gct ctg cac aac
cac tac acg cag aag 672 Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys 210 215 220 agc ctc tcc ctg tct ccg ggt aaa
taatctagag gcgcgccaat ta 718 Ser Leu Ser Leu Ser Pro Gly Lys 225
230 32 232 PRT Artificial Sequence Modified immunoglobulin moiety.
32 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15 Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130
135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu
Ser Pro Gly Lys 225 230 33 18 DNA Artificial Sequence PCR primer.
33 ggccggccac catgggat 18 34 27 DNA Artificial Sequence PCR primer.
34 tcgcctcata gagaggacac ctgcagt 27 35 28 DNA Artificial Sequence
PCR primer. 35 gtcctctcta tgaggcgagg gccccgga 28 36 27 DNA
Artificial Sequence PCR primer. 36 cggcgtgcgt aggagcccgc aggccac 27
37 29 DNA Artificial Sequence PCR primer. 37 gggctcctac gcacgccgcg
gccgaaacc 29 38 37 DNA Artificial Sequence PCR primer. 38
ggaaccacgc ggaaccagcg ccgcctcgcc ggccccc 37 39 36 DNA Artificial
Sequence PCR primer. 39 ctggttccgc gtggttccga gcccaaatct tcagac 36
40 31 DNA Artificial Sequence PCR primer. 40 ggcgcgcctc tagattattt
acccggagac a 31 41 987 DNA Artificial Sequence Ztnfr12-tcs-Fc5. CDS
(1)...(984) 41 atg gga tgg agc tgg atc ttt ctc ttt ctt ctg tca gga
act gca ggt 48 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly
Thr Ala Gly 1 5 10 15 gtc ctc tct atg agg cga ggg ccc cgg agc ctg
cgg ggc agg gac gcg 96 Val Leu Ser Met Arg Arg Gly Pro Arg Ser Leu
Arg Gly Arg Asp Ala 20 25 30 cca gcc ccc acg ccc tgc gtc ccg gcc
gag tgc ttc gac ctg ctg gtc 144 Pro Ala Pro Thr Pro Cys Val Pro Ala
Glu Cys Phe Asp Leu Leu Val 35 40 45 cgc cac tgc gtg gcc tgc ggg
ctc cta cgc acg ccg cgg ccg aaa ccg 192 Arg His Cys Val Ala Cys Gly
Leu Leu Arg Thr Pro Arg Pro Lys Pro 50 55 60 gcc ggg gcc agc agc
cct gcg ccc agg acg gcg ctg cag ccg cag gag 240 Ala Gly Ala Ser Ser
Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu 65 70 75 80 tcg gtg ggc
gcg ggg gcc ggc gag gcg gcg ctg gtt ccg cgt ggt tcc 288 Ser Val Gly
Ala Gly Ala Gly Glu Ala Ala Leu Val Pro Arg Gly Ser 85 90 95 gag
ccc aaa tct tca gac aaa act cac aca tgc cca ccg tgc cca gca 336 Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 100 105
110 cct gaa gcc gag ggg gca ccg tca gtc ttc ctc ttc ccc cca aaa ccc
384 Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
115 120 125 aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc
gtg gtg 432 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val 130 135 140 gtg gac gtg agc cac gaa gac cct gag gtc aag ttc
aac tgg tac gtg 480 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val 145 150 155 160 gac ggc gtg gag gtg cat aat gcc aag
aca aag ccg cgg gag gag cag 528 Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 165 170 175 tac aac agc acg tac cgt gtg
gtc agc gtc ctc acc gtc ctg cac cag 576 Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 180 185 190 gac tgg ctg aat ggc
aag gag tac aag tgc aag gtc tcc aac aaa gcc 624 Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 195 200 205 ctc cca tcc
tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 672 Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 210 215 220 cga
gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc 720 Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 225 230
235 240 aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc
agc 768 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser 245 250 255 gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag
aac aac tac 816 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr 260 265 270 aag acc acg cct ccc gtg ctg gac tcc gac ggc
tcc ttc ttc ctc tac 864 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr 275 280 285 agc aag ctc acc gtg gac aag agc agg
tgg cag cag ggg aac gtc ttc 912 Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe 290 295 300 tca tgc tcc gtg atg cat gag
gct ctg cac aac cac tac acg cag aag 960 Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 305 310 315 320 agc ctc tcc ctg
tct ccg ggt aaa taa 987 Ser Leu Ser Leu Ser Pro Gly Lys 325 42 328
PRT Artificial Sequence Ztnfr12-tcs-Fc5. 42 Met Gly Trp Ser Trp Ile
Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val Leu Ser Met
Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala 20 25 30 Pro Ala
Pro Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val 35 40 45
Arg His Cys Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro 50
55 60 Ala Gly Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln
Glu 65 70 75 80 Ser Val Gly Ala Gly Ala Gly Glu Ala Ala Leu Val Pro
Arg Gly Ser 85 90 95 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 100 105 110 Pro Glu Ala Glu Gly Ala Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 115 120 125 Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 130 135 140 Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 145 150 155 160 Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 165 170 175
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 180
185 190 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 195 200 205 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 210 215 220 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 225 230 235 240 Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser 245 250 255 Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 260 265 270 Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 275 280 285 Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 290 295 300
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 305
310 315 320 Ser Leu Ser Leu Ser Pro Gly Lys 325 43 36 DNA
Artificial Sequence PCR primer. 43 atgcattccg gaatgaggcg agggccccgg
agcctg 36 44 54 DNA Artificial Sequence PCR primer. 44 atgcattcta
gatcagtcca tcggcatgta ttccgccgcc tcgccggccc ccgc 54 45 6 PRT
Artificial Sequence C-terminal tag. 45 Glu Tyr Met Pro Met Asp 1 5
46 17 PRT Artificial Sequence Motif. VARIANT (2)...(2) Xaa = N, V,
P, or S. VARIANT (3)...(3) Xaa = Q, P, or E. VARIANT (4)...(4) Xaa
= T, A, E, or N. VARIANT (5)...(5) Xaa = E or Q. VARIANT (6)...(6)
Xaa = C or Y. VARIANT (7)...(7) Xaa = F or W. VARIANT (9)...(9) Xaa
= P, L, or S. VARIANT (11)...(11) Xaa = V or L. VARIANT (12)...(12)
Xaa = R, G, or H. VARIANT (13)...(13) Xaa = N, H, T, or A. VARIANT
(15)...(15) Xaa = V, M, or I. VARIANT (16)...(16) Xaa = S, A, or P.
46 Cys Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Leu Xaa Xaa Xaa Cys Xaa Xaa
1 5 10 15 Cys
* * * * *