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 This application claims the benefit of U.S. Provisional application No. 60/246,449 (filed Nov. 7, 2000), U.S. Provisional application No. 60/257,131 (filed Dec. 20, 2000), U.S. Provisional application No. 60/301,715 (filed Jun. 287, 2001), and U.S. Provisional application No. 60/315,565 (filed Aug. 29, 2001), the contents of which are incorporated by reference.
 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.
 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.,
 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.
 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,
 One such receptor is TACI, transmembrane activator and CAML-interactor (von Bülow and Bram,
 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.
 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.
 1. Overview
 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.,
 To investigate this possibility further, I
 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
 ZTNF4 appears to bind to virtually all mature CD19
 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.
 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 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
 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.
 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, (l) 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.
 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.
 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.
 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′)
 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 IDNO: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.
 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).
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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 Fc fragment. The present invention further includes isolated nucleic acid molecules that encode such fusion proteins.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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
 In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
 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., α-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, phosphorodiselerioate, 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.
 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′.
 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.
 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).
 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.
 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.
 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.
 “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.
 “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.
 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.,
 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.
 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.
 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.
 “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.
 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.”
 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.
 A peptide or polypeptide encoded by a non-host DNA molecule is a “heterologous” peptide or polypeptide.
 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.
 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.
 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.
 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.
 “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
 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 immtnoglobulin. 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
 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.
 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.
 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.
 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.
 An “isolated polypeptid” 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.
 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.
 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.
 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.
 As used herein, the term “immunomodulator” includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
 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
 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.
 An “antibody fragment” is a portion of an antibody such as F(ab′)
 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.
 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.
 “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.
 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.
 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.
 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.,
 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.
 As used herein, the term “antibody component” includes both an entire antibody and an antibody fragment.
 A “bispecific antibody” has binding sites for two different antigens within a single antibody molecule.
 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.
 An “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
 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.
 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.
 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.
 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.
 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.
 A “ribozyme” is a nuclcic 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.”
 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.”
 The term “variant Ztnf12 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.
 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,
 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.
 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.
 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.
 “Paralogs” are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, α-globin, β-globin, and myoglobin are paralogs of each other.
 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.
 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
 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.
 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.),
 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.,
 In order to construct a cDNA library, poly(A)
 Double-stranded cDNA molecules are synthesized from poly(A)
 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 λgt10 vector. See, for example, Huynh et al., “Constructing and Screening cDNA Libraries in λgL10 and λgt11,” in
 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.).
 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
 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.
 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).
 Alternatively, human genomic libraries can be obtained from commercial sources such as ResGen (Huntsville, Ala.) and the American Type Culture Collection (Manassas, Va.).
 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).
 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
 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 λgt11 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 λ expression libraries with antibody and protein probes,” in
 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.,
 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,
 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)).
 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
 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.
 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 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
 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 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 TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
 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.
 Different species can exhibit “preferential codon usage.” In general, see, Grantham et al.,
 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.
 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.
 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.
 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.
 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° C. lower than the thermal melting point (T
 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
 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
 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.
 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
 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
 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° C. overnight in a solution comprising 50% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution (100× Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 μg/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° 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.
 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×-2× SSC with 0.1% sodium dodecyl sulfate (SDS) at 55-65° 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×-2× SSC with 0.1% SDS at 55-65° C., including 0.5× SSC with 0.1% SDS at 55° C., or 2× SSC with 0.1% SDS at 65° C. One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.
 Typical highly stringent washing conditions include washing in a solution of 0.1×-0.2× SSC with 0.1% sodium dodecyl sulfate (SDS) at 50-65° 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. lx -0.2× SSC with 0.1% SDS at 50-65° C., including 0.1× SSC with 0.1% SDS at 50° C., or 0.2× SSC with 0.1% SDS at 65° C.
 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.
 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×-2× SSC with 0.1% SDS at 55-65° 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×-0.2× SSC with 0.1% SDS at 50-65° 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.
 Percent sequence identity is determined by conventional methods. See, for example, Altschul et al.,
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
 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,
 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.
 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.
 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,
 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
 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.
 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.),
 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
 In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al.,
 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.
 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,
 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.,
 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 (
 Variants of the disclosed Ztnfr12 nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer,
 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.
 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).
 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,
 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.
 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.,
 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.,
 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,
 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.
 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.
 Ztnfr12 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroisni, 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.
 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.
 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).
 5. Production of Ztnfr12 Polypeptides
 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.
 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.
 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.
 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-K1; ATCC CCL61; CHO DG44 (Chasin et al.,
 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.
 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.,
 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.,
 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.),
 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.
 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.,
 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 Gamier et al.,
 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
 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,
 The recombinant virus or bacmid is used to transfect host cells. Suitable insect host cells include cell lines derived from IPLB-Sf-21, a
 Established techniques for producing recombinant proteins in baculovirus systems are provided by Bailey et al., “Manipulation of Baculovirus Vectors,” in
 Fungal cells, including yeast cells, can also be used to express the genes described herein. Yeast species of particular interest in this regard include
 Transformation systems for other yeasts, including
 For example, the use of
 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
 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
 Suitable prokaryotic hosts include
 When expressing a Ztnfr12 polypeptide in bacteria such as
 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
 Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995).
 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
 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,
 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
 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.,
 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. Nos. 5,783,384, Kay, et. al., 5,747,334, and Kauffman et al., 5,723,323.
 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.
 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. Nos. 5,037,743; Holland et al., 5,143,830).
 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 α-factor (encoded by the MFα1 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
 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
 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.,
 Another form of fusion protein comprises a Ztnfr12 polypeptide and an immunoglobulin heavy chain constant region, typically an F
 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 γ4 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.
 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
 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.
 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
 The Fc fragment, or Fc domain, consists of the disulfide linked heavy chain hinge regions, C
 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 FcγRI binding and complement C1q binding. Specifically, three amino acid substitutions were introduced to reduce FcγRI 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 γ1 region). Substitutions at these positions have been shown to reduce binding to FcγRI (Duncan et al.,
 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,
 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.
 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.
 Fc7 is identical to the wild type γ1 Fc except for an amino acid substitution at EU index position 297 located in the C
 Fc8 is identical to the wild type immunoglobulin yl 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.
 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.
 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.
 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 IgGγ1 constant region (Putman,
TABLE 5 Allotypic Determinants of the Human Immunoglobulin γ1 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 —
 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 β2-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.
 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.
 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.
 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.
 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.
 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.
 The present invention further provides a variety of other polypeptide fusions. For example, part or all of a clomain(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.,
 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.
 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)).
 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.,
 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.
 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,
 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.
 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.,
 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
 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
 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.,
 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.
 The product of PEGylation by acylation is typically a polyPEGylated Ztnfr12 product, wherein the lysine ε-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.
 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
 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 ε-amino groups of the lysine residues and the α-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.
 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 α-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.
 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 α-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 α-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.
 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.
 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.,
 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.
 In addition, compositions can comprise a carrier, a Ztnfr12 polypeptide, and at least one of a TACI polypeptidc 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 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
 In another approach, increasing concentrations of
 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 1×10
 Ztnfr12 polypeptides or Ztnfr12-immunoglobulin proteins can be diluted from about 5 μg/ml to about 6 ng/ml, and incubated with the B cells for five days, pulsing overnight on day four with 1 μCi
 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.),
 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.
 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 glomernlonephritis 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
 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 x NZW)F
 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.
 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.,
 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 μg/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×, 10×, or 100× 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.
 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,
 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 μl 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 μg, given three times a week as a 200 μl 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.
 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.
 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
 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 μg 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 μg), and group C is injected with 100 μg 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 (lays 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.),
 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.
 Approximately 100 mice can be immunized with 20 μg 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 μg control Fc, group B is injected with 20 μg control Fc, group C is injected with 100 μg Ztnfr12 polypeptide or Zntfr12-immunoglobulin fusion protein, and group D is injected with 20 μg 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.
 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
 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.
 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.
 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,
 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.
 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,
 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
 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.
 With regard to the first characteristic, antibodies specifically bind if they bind to a Ztnfr12 polypeptide, peptide or epitope with a binding affinity (K
 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.
 As an illustration, potential antigenic sites in Ztnfr12 were identified using the Jameson-Wolf method, Jameson and Wolf,
 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.,
 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.
 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.
 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.
 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.
 Signal-inducing anti-Ztnfr 12 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).
 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
 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.,
 Alternatively, monoclonal anti-Ztnfr12 antibodies can be generated. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al.,
 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.
 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 enclogenous 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.,
 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
 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′)
 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.
 For example, Fv fragments comprise an association of V
 The Fv fragments may comprise V
 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 immuobilized 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
 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.,
 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
 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.,
 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.
 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.,
 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.
 As another example, bispecific F(ab′)
 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.
 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.,
 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′)
 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.
 Bispecific antibodies can be screened using standard techniques, such as a bispecific ELISA.
 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
 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
 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.
 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.
 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
 Ztnfr12 oligonucleotide probes are also useful for in vivo diagnosis. As an illustration,
 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.),
 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.
 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
 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.
 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 colorimetric assay.
 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.,
 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
 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.
 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.),
 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.
 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.
 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.
 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.
 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.,
 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.
 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.
 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.
 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.
 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.
 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.
 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
 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.
 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.
 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.
 11. Use of Anti-Ztnfr12 Antibodies to Detect Ztnfr12
 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.
 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.
 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
 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.
 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.
 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
 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.
 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. 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.
 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 polysp(cific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
 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.,
 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 for performing immunoassays are well-established. See, for example, Cook and Self, “Monoclonal Antibodies in Diagnostic Immunoassays,” in
 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
 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.
 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.
 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.
 ZTNF4 is expressed in CD8
 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.
 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.
 The present invention also provides methods for treatment of renal or urological neoplasms, multiple mylelomas, lymphomas, light chain neuropathy or amyloidosis.
 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.
 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.
 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.
 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.
 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.
 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.
 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
 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.),
 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.
 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.,
 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.,
 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.,
 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.,
 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.,
 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,
 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.,
 Polypeptides having Ztnfr12 binding activity can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al.,
 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,
 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.
 Other dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich,
 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
 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.
 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,
 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.
 Alternatively, a Ztnfr12 gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al.,
 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.,
 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).
 Moreover, adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector. Such adenoviruses are El-deleted, and in addition, contain deletions of E2A or E4 (Lusky et al.,
 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.,
 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.,
 Electroporation is another alternative mode of administration. For example, Aihara and Miyazaki,
 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.
 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. Nos. 5,496,698, McSwiggen, 5,525,468, Chowrira and McSwiggen, 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.
 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.,
 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.,
 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,
 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.
 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
 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.,
 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.
 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.
 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.
 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.,
 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.,
 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.
 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.,
 Suitable type II ribosome-inactivating proteins include polypeptides from
 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.
 Additional polypeptide cytotoxins include ribonuclease, DNase I, Staphylococcal enterotoxin-A, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,
 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.
 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, [L-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-α, -β, -γ, -ω, -ε, and -τ), the stem cell growth factor designated “S1 factor,” erythropoietin, and thrombopoietin. Illustrative immunomodulator moieties include IL-2, IL-6, IL-10, interferon-γ, TNF-α, and the like.
 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
 The present invention also includes immunocongugates that comprise a nucleic acid molecule encoding a cytotoxin. As an example of this approach, Hoganson et al.,
 Other suitable toxins are known to those of skill in the art.
 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.,
 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.,
 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 α-emitting radioisotope, a β-emitting radioisotope, a γ-emitting radioisotope, an Auger electron emitter, a neutron capturing agent that emits α-particles or a radioisotope that decays by electron capture. Suitable radioisotopes include
 A radioisotope can be attached to an antibody component directly or indirectly, via a chelating agent. For example,
 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
 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.,
 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.
 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.
 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
 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.
 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.
 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.
 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.
 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
 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
 For example, a method for producing a transgenic mouse that expresses a Ztnfr12 gene can begin with adult, fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2f1, 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.
 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,
 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.
 Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO
 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° C./5% CO
 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.
 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.
 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° C. slide warmer for a minimum of four hours.
 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.
 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.
 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° 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.
 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
 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
 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.
 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
 The COS cell transfection was performed as follows. Five microliters of pooled DNA (about 0.5-1.0 μg) and 5 μl of lipofectamine were mixed in 92 μl of serum free DMEM medium (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 μg selenium, and 5 mg fetuin in 500 ml DMEM), incubated at room temperature for 30 minutes, and then 400 μl of serum free DMEM medium were added. Five hundred microliters of this mixture were added to 1.5×10
 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
 One of the positive DNA pools, “10A11,” was identified using the method described above. The DNA of pool 10A11 was electroporated into
 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′ GCGAATTCGTCGGCACCATGAGGCCGAGGG 3′; SEQ ID NO:10) and 37549 (5′ CGCTCGAGCTGCCGGCTCCCTGCFATTGTTG 3′; SEQ ID NO:11), under the following reaction conditions: 94° C. for 2 minutes; 35 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 30 seconds; followed by 72° 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
 The blots were then washed four times with 2× SCC and 0.05% SDS at room temperature, followed by two washes in 0.1× SSC and 0.1% SDS at 50° C. One transcript size was detected at approximately 4.4 kilobases.
 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, Calif.). The methods and conditions for the dot blot analyses were the same as for the multiple tissue blots disclosed above.
 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.
 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 β-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.
 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-60D), and chronic myelogenous leukemia cells (K562).
 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 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 − − −
 A. Igγ1 Fragment Constructicon
 To prepare the Ztnfr12-]Fc4 fusion protein, the Fe region of human IgG1 (the hinge region and the CH
 The Fe 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 γ1 constant region are presented in SEQ ID Nos:17 and 18, respectively. Mutations within the Fc region were introduced by PCR to reduce FcγRI binding. The FcγRI 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 FcγR1 binding (see, for example, Duncan et al.,
 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,
 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
 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 μL final volume was added 236 ng Fc4 template, 5 μL 10× Pfu reaction buffer, 4 μL of 2.5 mM dNTPs, 1 μL 20 μM each of the primers and 1 μL Pfu polymerase (2.5 units, Stratagene). The amplification thermal profile consisted of 94° C. for 2 minutes, 5 cycles at 94° C. for 15 seconds, 42° C. for 20 seconds, 72° C. for 45 seconds, 20 cycles at 94° C. for 15 seconds, 72° C. for one minute 20 seconds, followed by a seven minute extension at 72° 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
 A protein encoding expression cassette for Ztnfr12-tcs-FcS was generated by overlap PCR (Horton et al.,
 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.
 First Round PCR Products 1, 2, 3, and 4 were separately generated using different oligonucleotide primers and DNA templates. To a 25 μl final volume each was added approximately 2 ng template DNA, 2.5 μl 10× Pfu Polymerase Reaction Buffer (Stratagene), 2 μl of 2.5 mM dNTPs, 2.5 μl Rediload (ReGen; Huntsville, Ala.), 20 pmole each 5′ oligonucleotide and 3′ oligonucleotide primers (see below), and 0.5 μl Pfu polymerase (2.5 units, Stratagene). The reaction to generate First Round PCR Product 4 also included the addition of 2.5 μl GC-Melt Reagent (Clontech). Information on the templates and primers used in the PCR amplifications is provided in Tables 7 and 8.
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 seq- ZC38, 989 ZC38, 987 uence cDNA 2 Ztnfr12 cDNA ZC38, 986 ZC38, 990 3 Ztnfr12 cDNA ZC39, 428 ZC39, 425 4 Fc5 DNA fragment ZC39, 027 ZC38, 874
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′ GGAACCACGCGGAACCAGCGCCGCCTCGCCGGCCCC 38 C 3′ ZC39,027 5′ CTGGTTCCGCGTGGTTCCGAGCCCAAATCTTCAGAC 39 3′ ZC38,874 5′ GGCGCGCCTCTAGATTATTTACCCGGAGACA 3′ 40
 The amplification thermal profile consisted of 94° C. for 3 minutes, 30 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, followed by a 4 minute extension at 72° 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.
 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 μl final volume was added 1 μl of each First Round PCR Products 1, 2, 3, and 4, 5 μl 10× Pfu Polymerase Reaction Buffer (Stratagene), 4 μl of 2.5 mM dNTPs, 5 μl Rediload (ResGen), 5 μl GC-Melt Reagent (Clontech), approximately 40 pmoles each ZC38,989, ZC38,874 and 0.5 μl Pfu Polymerase (2.5 units, Stratagene). The amplification thermal profile consisted of 94° C. for 3 minutes, 35 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 3 minutes, followed by a 6 minute extension at 72° 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.
 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.
 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.
 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.
 CHO DG44 cells were passaged in PFCHO media (JRH Biosciences, Lenexa, Kans), 4 mM L-Glutamine (JRH Biosciences), and 1× hypothanxine-thymidine supplement (Life Technologies), and the cells were incubated at 37° C. and 5% CO
 The CHO DG44 cells were prepared while the DNA pellet was drying by centrifuging 106 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 μl 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 μl of PFCHO growth media and added to the cells in the cuvette so that the total volume did not exceed 800 μl 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.
 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° C. and 5% CO
 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 μm 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.
 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
 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
 Due to the protease resistance of the Fc domain, no glycosylation modifications could be observed for that part of the protein. The single predicted N-linked carbohydrate is in the Fc domain at Asn
 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
 Ztnfr12-Fc5 was immobilized to a plate coated with goat anti-human IgG Fc, 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.
 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
 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 μl volume reaction containing 10% DMSO: 1 cycle at 94° C. for 2 minutes; 35 cycles of 94° C. for 15 seconds, 50° C. for 30 seconds, and 72° C. for 45 seconds; 1 cycle at 72° C. for 5 min; followed by 4° 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 μl of water. The cDNA was digested in a 35 μl volume using BspeI and XbaI (New England Biolabs, Beverly, Mass.) in appropriate buffer conditions for 1 hr at 37° 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 μl of water. The purified, digested sTNFR12cee PCR product was ligated into the MCS of a previously prepared and restriction enzyme digested (BspeI and XbaI) vector pZBV37L.
 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° C. in a 20 μl volume in appropriate buffer conditions. Five microliters of the ligation mix were transformed into 50 μl of ELECTOMAX DH12S cells (Life Technologies) by electroporation at 400 0 hms, 2V and 25μF in a 2 mm gap electroporation cuvette (BTX). The transformed cells were diluted in 350 μl of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl
 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 μl DH10BAC MAX EFFICIENCY competent cells (GIBCO-BRL) by heat shock for 45 seconds in a 42° C. heat block. The transformed DH10BAC cells were diluted in 900 μl of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl
 B. Transfection of Sf9 Cells
 Sf9 cells were seeded at 1×10
 C. Amplification
 Sf9 cells were seeded at 1×10
 A second round of amplification can proceed as follows: Sf9 cells are seeded at 1×10
 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 1×10
 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.
 A large viral stock is generated by the following method: Sf9 cells are grown in IL Sf-900 II SFM in a 2800 ml shake flask to an approximate density of 1×10
 Larger scale infections can be completed to provide material for downstream purification.
 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.