HK1134015A - Soluble receptor br43x2 and methods of using - Google Patents

Description

Soluble receptor BR43X2 and methods of use

Background

The cellular interactions that occur during the course of an immune response are regulated by members of several cell surface receptor families, including the Tumor Necrosis Factor Receptor (TNFR) family. The TNFR family consists of a variety of integral membrane glycoprotein receptors, many of which combine with their respective ligands to regulate interactions between different hematopoietic cell lines (Smith et al, TNF Receptor Superfamily of Cellular and viral Proteins: Activation, Costimulation and Death (The TNF Receptor Superfamily of Cellular and viral Proteins: Activation, Costimation and Death), 76: 959-62, 1994; Cosman, Stem Cells (Stem Cells) 12: 440-55, 1994).

One such receptor is the transmembrane activator and CAML-interactor TACI (von Bulow and Bram, Science 228: 138-41, 1997; and WIPO publication WO 98/39361). TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudo-repeats, a transmembrane domain and a cytoplasmic domain that interacts with CAMLs (calcium regulators and cyclophilin ligands), an integral membrane protein, located on intracellular vesicles, and is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with B cells and one T cell subtype. von Bulow and Bram (supra) reported that the ligand for TACI was unknown.

It has been found that the polypeptides of the invention, i.e.the TACI isoforms with only one cysteine-rich pseudo-repeat (BR 43X 2), TACI and the related B-cell protein BCMA (Gras et al, International immunology (int. Immunol.) 17: 1093-106, 1995), can bind to ligands of the presently known neutrokine alpha (WIPO publication, WO98/18921), BlyS (Moore et al, science, 285: 260-3, 1999), BAFF (Schneider et al, J.Exp.Med.; 189: 1747-56, 1999), TALL-1(Shu et al, J.Leukoc.biol.) 65: 680-3, 1999) or THANK (Mukhoddhayay et al, J.biol.chem.;. 274: 78-81, 1999) TNF 4. Thus, BR43x2, TACI, and BCMA would be useful for modulating the activity of ztnf4, particularly for modulating B cell activation.

To this end, the present invention provides protein therapeutics, related compositions and methods for modulating the activity of ztnf4 or other BR43x2, TACI, or BCMA ligands, as well as other uses that will be apparent to those of skill in the art from this disclosure.

Summary of The Invention

In one aspect, the present invention provides a method of inhibiting ztnf4 activity in a mammal comprising administering an amount of a compound selected from the group consisting of: a) a polypeptide comprising BR34 x2 extracellular domain; b) a polypeptide comprising a TACI extracellular domain; c) a polypeptide comprising a BCMA extracellular domain; d) comprises the amino acid sequence shown in SEQ ID NO: 10; e) and SEQ ID NO: 2 or an antibody or antibody fragment that specifically binds to the polypeptide of (a); f) and SEQ ID NO: 4 or an antibody or antibody fragment to which the polypeptide of (4) specifically binds; g) and SEQ ID NO: 6 or an antibody or antibody fragment to which the polypeptide of (a); h) and SEQ ID NO: 8 or an antibody or antibody fragment that specifically binds to the polypeptide of (a); i) and SEQ ID NO: 10 or an antibody or antibody fragment to which the polypeptide of claim 10 specifically binds; k) SEQ ID NO: 4; 1) SEQ ID NO: 6 at amino acid residues 1-166; and m) SEQ ID NO: 8 from 1 to 150.

In one embodiment, the compound is a fusion protein consisting of a first portion and a second portion joined together by a peptide bond, the first portion comprising a polypeptide selected from the group consisting of: a) comprises the amino acid sequence shown in SEQ ID NO: 8; b) comprises the amino acid sequence shown in SEQ ID NO: 2 at amino acid residues 25-58; c) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 34-66; d) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 71-104; e) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 25-104; f) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8 to 37; g) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 41-88; h) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8-88; and the second portion comprises another polypeptide. In another embodiment, the first part further comprises a polypeptide selected from the group consisting of: a) SEQ ID NO: 2 amino acid residues 59 to 120; b) SEQ ID NO: 6 amino acid residues 105-166; and c) SEQ ID NO: 8, 89-150 amino acid residues. In another embodiment, the first moiety is selected from: a) a polypeptide comprising BR43x2 extracellular domain; b) a polypeptide comprising a TACI extracellular domain; and c) a polypeptide comprising a BCMA extracellular domain. In a related embodiment, the first moiety is selected from the group consisting of: a) SEQ ID NO: 4; b) SEQ ID NO: 6 at amino acid residues 1-154; and c) SEQ ID NO: 8 from position 1 to 48. In another related embodiment, the second portion is a heavy chain constant region of an immunoglobulin.

In another embodiment, the antibody or antibody fragment is selected from the group consisting of: a) a polyclonal antibody; b) a murine monoclonal antibody; c) a humanized antibody derived from b); and d) a human monoclonal antibody. In a related embodiment, the antibody fragment is selected from the group consisting of F (ab '), F (ab), Fab', Fab, Fv, scFv, and minimal recognition unit. In another embodiment, the mammal is a primate.

In another embodiment, the ztnf4 activity is associated with B lymphocytes. In another related embodiment, the ztnf4 activity is associated with activated B lymphocytes. In yet another embodiment, the ztnf4 activity is associated with resting B lymphocytes. In another embodiment, the ztnf4 activity is associated with the production of antibodies. In a related embodiment, the production of the antibody is associated with an autoimmune disease. In a related embodiment, the autoimmune disease is systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, or rheumatoid arthritis. In another embodiment, the ztnf4 activity is associated with asthma, bronchitis, or emphysema. In yet another embodiment, the ztnf4 activity is associated with end-stage renal failure. In yet another embodiment, the ztnf4 activity is associated with renal disease. In a related embodiment, the kidney disease is glomerulonephritis, vasculitis, nephritis or pyelonephritis. In yet another embodiment, the renal disease is associated with a renal tumor, multiple myeloma, lymphoma, light chain neuropathy (light chain neuropathy), or an amyloidosis. In another embodiment, the ztnf4 activity is associated with effector T cells. In a related embodiment, the ztnf4 activity is associated with modulating an immune response. In yet another embodiment, the activity is associated with immunosuppression. In yet another embodiment, the immunosuppression is associated with transplant rejection, graft-versus-host disease, or inflammation. In another embodiment, the activity is associated with an autoimmune disease. In a related embodiment, the autoimmune disease is insulin-dependent diabetes mellitus or Crohn's disease. In another embodiment, the ztnf4 activity is associated with inflammation. In a related embodiment, the inflammation is associated with joint pain, swelling, anemia, septic shock. In another aspect, the present invention provides a method for inhibiting BR34 x2, TACI, or BCMA receptor-ligand engagement (receptor-ligand engagement), comprising administering an amount of the above compound. In another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with B lymphocytes. In another related embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with activated B lymphocytes. In yet another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with resting B lymphocytes.

In another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with the production of antibodies. In a related embodiment, the production of the antibody is associated with an autoimmune disease. In a related embodiment, the autoimmune disease is systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, or rheumatoid arthritis. In another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with asthma, bronchitis, or emphysema. In yet another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with end-stage renal failure. In yet another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with renal disease. In a related embodiment, the renal disease is glomerulonephritis, vasculitis, nephritis or pyelonephritis. In yet another embodiment, the renal disease is associated with a renal tumor, multiple myeloma, lymphoma, light chain neuropathy, or an amyloidosis. In another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with effector T cells. In a related embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with modulation of an immune response. In yet another embodiment, the activity is associated with immunosuppression. In yet another embodiment, the immunosuppression is associated with transplant rejection, graft-versus-host disease, or inflammation. In another embodiment, the activity is associated with an autoimmune disease. In a related embodiment, the autoimmune disease is insulin-dependent diabetes mellitus or Crohn's disease. In another embodiment, the BR34 x2, TACI, or BCMA receptor-ligand engagement is associated with inflammation. In a related embodiment, the inflammation is associated with joint pain, swelling, anemia, septic shock.

In another aspect, the invention provides a nucleic acid sequence encoding SEQ ID NO: 2. Also provided are SEQ ID NOs: 1. In a related embodiment, an expression vector is provided comprising the following operably linked elements: a transcription promoter, a polynucleotide molecule as described above, and a transcription terminator. In another embodiment, the expression vector further comprises a secretory receptor-ligand engagement sequence operably linked to the polynucleotide molecule. The present invention also provides a cultured cell into which the above-described expression vector is introduced, wherein the cultured cell expresses the polypeptide encoded by the polynucleotide fragment. The present invention also provides a method of producing a polypeptide comprising: culturing a cell into which the expression vector has been introduced; whereby said cell expresses said polypeptide encoded by said polynucleotide molecule; and recovering the expressed polypeptide. The invention also provides a polypeptide having the sequence of SEQ ID NO: 2. In a related embodiment, the polypeptide is combined with a pharmaceutically acceptable carrier (vehicle).

Brief Description of Drawings

FIG. 1 shows a multiple amino acid sequence alignment between BR 34X 2, TACI (von Bulow and Bram, supra) (SEQ ID NO: 6), BCMA (Gras et al, supra) (SEQ ID NO: 6) and BR43X 1(SEQ ID NO: 7). The cysteine-rich pseudo-repeat and transmembrane domains are indicated in the figure.

FIG. 2 shows solubility I¹²⁵-stetchard plot analysis of the binding of ztnf4 to TACI and BCMA expressed by stable BHK transfectants.

Figure 3A shows that ztnf4 assists in activating human B lymphocytes to proliferate and secrete immunoglobulins.

Figure 3B shows measured IgM and IgG levels in supernatants obtained from B cells stimulated with soluble ztnf4 in the presence of IL4 or IL4+ IL5 after 9 days of culture.

FIG. 4 shows human peripheral blood B cells stimulated in vitro with soluble ztnf4 or a control protein (ubiquitin) for 5 days in the presence of IL-4. Purified TACI-Ig, BCMA-Ig and control Fc were tested for inhibition of ztnf 4-specific proliferation.

Figure 5A shows the results obtained from ztnf4 transgenic animals that produce SLE properties.

Fig. 5B shows lymph nodes, spleen and thymocytes from ztnf4 transgenic animals stained with antibodies against CD5, CD4 and CD 8.

Figure 5C shows total IgM, IgG and IgE levels in serum of 6-23 week old transgenic ztnf4 animals.

Figure 5D shows amyloid deposition and mesangial thickening in the glomeruli identified in kidney sections of ztnf4 transgenic animals.

Fig. 5E shows effector T cells in ztnf4 transgenic mice.

FIGS. 6A and B show elevated levels of ztnf4 obtained from the sera of ZNVFF 1 mice and MRL/lpr/lpr mice that were correlated with the development of SLE.

Figure 7 shows the percentage of NZBWF1 mice that developed proteinuria during the study.

FIG. 8 shows anti-dsDNA levels of ztnf4 transgenic mice and control littermates as determined by ELISA compared to sera of ZNVWF 1 and MRL/lpr/lpr mice.

These and other aspects of the invention will be apparent upon reference to the following detailed description.

Detailed Description

Before setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms used hereinafter.

Affinity tags: as used herein refers to a polypeptide fragment that is capable of binding to a second polypeptide to provide a site for purification or detection of the second polypeptide, or for binding of the second polypeptide to a substrate. In principle, any peptide or protein from which an antibody or other specific binding agent can be obtained can be used as an affinity tag. Affinity tags include the polyhistidine sequence, protein A (Nilsson et al, EMBO J.4: 1075, 1985; Nilsson et al, Methods (Methods Enzymol.) 198: 3, 1991), glutathione S-transferase (Smith and Johnson, Gene (Gene) 67: 31, 1988), Glu-Glu affinity tag (Grussenmeyer et al, Proc. Natl.Acad.Sci.USA) 82: 7952-4, 1985), substance P, Flag^TMPeptides (Hopp et al, Biotechnology 6: 1204-10, 1988), streptavidin binding peptides, or other antigenic epitopes or binding domains. See generally Ford et al, Protein Expression and purification (Protein Expression and purification) 2: 95-107, 1991. DNA encoding affinity tags is available from commercial suppliers (e.g., pharmacia Biotech, Piscataway, NJ).

Allelic variants: refers to a gene occupying the same chromosomal locusAny one of two or more different forms of (a). Allelic variation occurs naturally through mutation and can lead to phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. The term "allelic variant" is also used herein to refer to a protein encoded by an allelic variant of a gene. Also included are the same proteins from the same species that differ from the reference amino acid sequence due to allelic variation. Allelic variation refers to the naturally occurring difference between individuals in a gene encoding a given protein.

Amino-and carboxyl-terminal: as used herein to refer to positions in polypeptides and proteins. Where the context permits, these terms are used to indicate proximity or relative position with respect to a particular sequence or portion of a polypeptide or protein. For example, a sequence located at the carboxy terminus of a reference sequence in a protein is located close to the carboxy terminus of the reference sequence, but not necessarily at the carboxy terminus of the entire protein.

Complement/anti-complement pair (complement/anti-complement pair)(ii) a Refers to non-identical moieties that under appropriate conditions form a stable pair that is not covalently linked. For example, 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. When subsequent dissociation of the complement/anti-complement pair is desired, the complement/anti-complement pair preferably has<10^-9Binding affinity of M.

Contig: refers to a polynucleotide having a stretch of identical or complementary sequence that is contiguous with another polynucleotide. Contiguous sequences are said to "overlap" with a given polynucleotide sequence, either completely or along part of the polynucleotide sequence. For example, representative contigs of polynucleotide sequence 5'-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and 3 '-gtcgacTACCGA-5'.

Complements of polynucleotide molecules: refers to a polynucleotide molecule having complementary bases and opposite orientation as compared to a reference sequence. For example, the sequence 5 'ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

Degenerate nucleotide sequences or degenerate sequences: refers to a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (e.g., GAU and GAC triplets both encode Asp).

Expression vector: a linear or circular DNA molecule comprising a segment that is operably linked to other segments that provide conditions for its transcription and that encodes a polypeptide of interest. These additional fragments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

Isoforms: refers to different forms of a protein that can be produced from different genes or from the same gene by different splicing. In certain instances, the isoforms differ in trafficking activity, expression time during development, tissue distribution, intracellular localization, or a combination of these properties.

Isolated polynucleotides: means that the polynucleotide has been removed from its natural genetic environment and therefore does not contain other extraneous or unwanted coding sequences, and that it is present in a form suitable for use in genetically engineered protein production systems. These isolated molecules are those that are isolated from their natural environment, including cDNA and genomic clones. The isolated DNA molecules of the present invention do not contain other genes to which they are normally associated, but may include naturally occurring 5 'and 3' untranslated regions such as promoters and terminators. Identification of the regions of attachment will be apparent to one of ordinary skill in the artAnd (see, e.g., Dynan and Tijan, Nature 316: 774-78, 1985).

Isolated polypeptide or protein: is a polypeptide or protein that is present in a condition other than its native environment, such as, for example, in a condition free of blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Preferably, the polypeptide is provided in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. As used herein, the term "isolated" does not exclude the presence of another physical form of the same polypeptide, such as a dimer or other glycosylated or derivatized form.

Is operably connected to: the term "operably linked" when applied to nucleotide fragments means that the fragments are arranged such that they function in concert for their intended purposes, e.g., initiating transcription in a promoter and progressing along the coding segment to a terminator.

Orthologs (ortholog): refers to a polypeptide or protein obtained from one species that is a functional counterpart of a polypeptide or protein of another species. Sequence differences between orthologs are the result of speciation.

Polynucleotide: refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The size of a polynucleotide is expressed in base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). The latter two terms may describe single-stranded or double-stranded polynucleotides as the context permits. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood to be the same as the term "base pair". One skilled in the art will appreciate that the two strands of a double-stranded polynucleotide may differ slightly in length, and their ends may be due toThe enzyme digestion is staggered; thus not all nucleotides are paired in a double stranded polynucleotide molecule. These unpaired ends are generally no longer than 20 nt in length.

Polypeptides: are polymers of amino acid residues joined by peptide bonds, whether naturally occurring or synthetically produced. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

Promoters: refers to a portion of a gene that contains a DNA sequence that provides for the binding of RNA polymerase and initiation of transcription. Promoter sequences are typically, but not always, present in the 5' non-coding region of a gene.

Protein: are macromolecules that contain one or more polypeptide chains. The protein may also contain non-peptide components, such as sugar groups. Carbohydrates and other non-peptide substituents can be added to a protein by the cell in which the protein is produced, and these carbohydrate and other non-peptide substituents vary from cell type to cell type. Proteins are defined herein in terms of their amino acid backbone structure; in general, substituents such as glycosyl groups are not specified, but may be present.

Receptors: a cell-associated protein that binds to a biologically active molecule ("ligand") and mediates the action of the ligand on a cell, or a polypeptide subunit of the protein. Binding of a ligand to a receptor results in a change in the receptor (and in some cases, multimerization of the receptor, i.e., the joining of the same or different receptor subunits), thereby causing interaction between one or more effector domains of the receptor and one or more other molecules within the cell. These interactions in turn lead to alterations in the metabolism of the cell. Metabolic events linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increased production of cyclic AMP, cellular calcium transfer, membrane lipid transfer, cell adhesion, hydrolysis of inositol lipids, and hydrolysis of phospholipids. BR43X2 has the properties of the TNF receptor, as will be discussed in more detail herein.

Secretory signal sequence: a DNA sequence encoding a polypeptide ("secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through the secretory pathway of a cell that synthesizes the larger polypeptide. The larger polypeptide is typically cleaved during transport through the secretory pathway to remove the secretory peptide.

Soluble receptors: a receptor polypeptide that does not bind to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides lacking a transmembrane domain and a cytoplasmic domain. Soluble receptors may contain additional amino acid residues, such as affinity tags that provide conditions for purification of the polypeptide or provide sites for binding of the polypeptide to a substrate. Many cell surface receptors have a naturally occurring soluble counterpart that is proteolytically produced or is translated from another spliced form of mRNA. Receptor polypeptides are considered to be substantially free of transmembrane and intracellular polypeptide fragments when they lack sufficient fragments to provide membrane anchoring or signal transduction, respectively.

The molecular weight and length of the polymer, as determined by a crude analytical method such as gel electrophoresis, are to be understood as approximate values. When the value is expressed as "about" X or "approximately" X, the X value described is to be understood as accurate as. + -. 10%.

All documents cited herein are incorporated in their entirety by reference.

The present invention is based in part on the discovery of an isoform of the receptor TACI, a 1192bp DNA sequence (SEQ ID NO: 1) and its corresponding polypeptide sequence (SEQ ID NO: 2). This isoform was named BR43 × 2. Soluble forms of BR43x2 are disclosed in SEQ ID NO: 4, the polynucleotide encoding SEQ ID NO: 3. Polynucleotides and polypeptides of the invention encoding the BR43X2 receptor were originally identified by signal cloning (signal p cloning) using the human RPMI1788 library and the N-or C-terminal FLAG-tagged, biotin-or FITC-tagged tumor necrosis factor ligand ztnf4 currently known as neutrokine alpha (WIPOWO98/18921), Blys (Moore et al, supra), BAFF (Schneider et al, supra), TALL-1(Shu et al, supra) or ANTHK (Mukhopadhyay et al, supra), as described in more detail herein. Positive pools were identified by ligand binding and then broken down into individual clones, the cDNA isolated and sequenced. Comparison of the deduced amino acid sequence of BR43X2 (shown in SEQ ID NO: 2) with known tumor necrosis factor receptors revealed that BR 34X 2 is an isoform of TACI with only one cysteine-rich pseudo-repeat that is poorly conserved.

Structurally, this TNF receptor family is characterized by an extracellular portion consisting of several components historically referred to as "cysteine-rich pseudo-repeats". A prototype TNFR family member has 4 such pseudo-repeats, each approximately 29-43 residues in length, arranged successively to the right. A typical pseudo-repeat has 6 cysteine residues. Since although they appear to originate from a common ancestral component, they are not entirely duplicative: the #1, #2, #3, and #4 pseudo-repeats have characteristic sequence characteristics that are distinct from each other, and are therefore referred to as pseudo-repeats. The crystal structure of the p55 TNF receptor shows that each of the pseudo-repeats corresponds to a single folding domain, and that all 4 pseudo-repeats fold into the same quaternary structure and are held together internally by disulfide bonds.

TACI contains two cysteine-rich pseudo-repeats (von bulow and Bram, supra), the first of which is structurally conserved and the second of which is less conserved than the other members of the TNF receptor family. The BR43x2 isoform of the invention lacks the first cysteine-rich pseudo-repeat of TACI, and retains only the second less conserved repeat unit.

For SEQ ID NO: sequence analysis of the deduced amino acid sequence of BR43X2 as shown in FIG. 2 revealed the presence of a mature protein having an extracellular domain (residues 1-120 of SEQ ID NO: 2) containing a cysteine-rich pseudo-repeat (residues 25-58 of SEQ ID NO: 2), a transmembrane domain (residues 121-133 of SEQ ID NO: 2) and a cytoplasmic domain (residues 134-247 of SEQ ID NO: 2). The cysteine-rich pseudo-repeat of BR 34X 2 has 6 conserved cysteine residues (residues 25, 40, 43, 47, 54 and 58 of SEQ ID NO: 2), one conserved aspartic acid residue (residue 34 of SEQ ID NO: 2) and two conserved leucine residues (residues 36 and 37 of SEQ ID NO: 2), and shares 46% identity with the first cysteine-rich pseudo-repeat of TACI (SEQ ID NO: 6) and 35% identity with the cysteine-rich pseudo-repeat of BCMA (SEQ ID NO: 8) (FIG. 1). The cysteine-rich pseudo-repeat can be represented by the following motif: CX [ QEK ] [ QEKNRDH ] [ QE ] X { O-2} [ YFW ] [ YFW ] DXLLX {2} C [ IMLV ] XCX {3} CX {6-8} CX {2} [ YF ] C (SEQ ID NO: 10),

wherein C represents a cysteine residue, Q represents a glutamine residue, E represents a glutamic acid residue, K represents a lysine residue, N represents an asparagine residue, R represents an arginine residue, D represents an aspartic acid residue, H represents a histidine residue, S represents a serine residue, Y represents a tyrosine residue, F represents a phenylalanine residue, W represents a tryptophan residue, L represents a leucine residue, I represents an isoleucine residue, V represents a valine residue, and X represents any naturally occurring amino acid residue other than cysteine. Amino acid residues within brackets "[ ]" indicate allowed amino acid residue variations at that position. The number within the brackets "{ }" indicates the number of amino acid residues allowed at that position.

The invention also provides SEQ ID NO: 10 is a soluble polypeptide of length 32-40 amino acid residues.

SEQ ID NO: 4, and the soluble BR43x2 receptor contains a cysteine-rich pseudo-repeat (residues 25-58 of SEQ ID NO: 4) and lacks SEQ ID NO: transmembrane and cytoplasmic domains of BR43x2 shown in 2.

Those skilled in the art will appreciate that the boundaries of these domains are approximate, and are based on alignment with known proteins and prediction of protein folding. These properties reveal that, from SEQ ID NOs: 1 and 3 is a member of the TNF receptor family.

Northern and dot blot analyses of mRNA tissue distribution using a nucleotide probe against BR43X 1 predicted to detect the expression of BR43X2, revealed in spleen, lymph nodes, CD19⁺There was expression in the cells, and weak expression in mixed lymphocyte reaction cells, Daudi and Raji cells. Using reverse transcriptase PCR, BR43X 1 was detected only in B cells and no BR43X 1 was detected in activated T cells, as previously reported for TACI (von Bulow and Bram, supra). The BR43X2 probe, which overlaps 100% with the corresponding TACI sequence, was used in spleen, lymph nodes and small intestine, stomach, salivary glands, appendix, lung, bone marrow, fetal spleen, CD19⁺TACI and BR43 × 2 were detected in cells and Raji cells.

BCMA was detected in the small intestine, spleen, stomach, colon, appendix, lymph nodes, trachea and testis using Northern blot analysis. BCMA was also detected in adenolymphoma, non-hodgkin lymphoma and parotid gland tumors, CD8⁺、CD19⁺Weak BCMA was detected in MLR cells, Daudi, Raji and Hut78 cells.

Northern blot analysis was also performed using murine ztnf4(SEQ ID NO: 19), and as with human TACI, BCMA and BR43X2, expression of murine ztnf4 was detected mainly in the spleen and thymus. Murine ztnf4 was also expressed in the lung, and weak expression was detected in skin and heart.

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, encoding the BR43x2 polypeptides disclosed herein. It will be readily apparent to those skilled in the art that considerable sequence variation is possible in these polynucleotide molecules in view of the degeneracy of the genetic code. SEQ ID NO: 11 is a polypeptide comprising the sequence encoding SEQ ID NO: 4 soluble BR43x2 polypeptide, one degenerate DNA sequence of all DNAs. Likewise, SEQ ID NO: 12 is a polypeptide comprising the sequence encoding SEQ ID NO: a degenerate DNA sequence of all the DNAs of 2BR 43X2 polypeptides. Those skilled in the art will appreciate that by substituting T with U, SEQ ID NO: 12 degenerate sequences also provide a nucleic acid encoding SEQ ID NO: 4. Thus, a polypeptide comprising SEQ ID NO: 11, nucleotides 1-360 of SEQ ID NO: 12, and RNA equivalents thereof, are contemplated by the present invention. Table 1 lists SEQ ID NOs: the one letter codes used in 11 and 12 indicate the positions of degenerate nucleotides. "resolution" refers to the nucleotide indicated by the code letter. "complementary" refers to the code of complementary nucleotides. For example, the Y code indicates C or T, and its complement, R, indicates A or G, A being complementary to T and G being complementary to C.

TABLE 1

Table 2 lists SEQ ID NOs: 11 and 12, which includes all possible codons for a given amino acid.

TABLE 2

It will be apparent to one of ordinary skill in the art that some ambiguity is introduced in determining a degenerate codon that represents all possible codons encoding each amino acid. For example, a degenerate codon for serine (WSN) may in some cases encode Arginine (AGR), while a degenerate codon for arginine (MGN) may in some cases encode serine (AGY). A similar relationship also exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but are identified by reference to SEQ ID NOs: 2 and 4 can be readily identified by one of ordinary skill in the art. The functionality of the variant sequences can be readily tested as described herein.

It will also be appreciated by those of ordinary skill in the art that different species will exhibit "biased codon usage". See generally Grantham et al, nucleic acids research (nuc. acids Res.) 8: 1893-912, 1980; haas et al, contemporary biology (curr. biol.) 6: 315-24, 1996; Wain-Hobson et al, Gene (Gene) 13: 355-64, 1981; grosjean and filirs, gene 18: 199-209, 1982; holm, nucleic acid study 14: 3075-87, 1986; ikemura, journal of molecular biology (j.mol.biol.) 158: 573-97, 1982. The term "biased codon usage" or "biased codon" as used herein is a term of art that refers to the most frequently used codons for translation of a protein in a cell of a species, thereby creating a bias to one or several possible codons encoding each amino acid (see Table 2). For example, threonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but ACC is the most commonly used codon in mammalian cells; in other species, such as insect cells, yeast, viruses or bacteria, different Thr codons may be favored. Biased codons for a particular species can be introduced into a polynucleotide of the invention by a variety of methods known in the art. The introduction of biased codon sequences into recombinant DNA can enhance protein production, for example, by making protein translation more efficient in a particular cell type or species. Thus, SEQ ID NOs: 11 and 12 can serve as templates for optimizing expression of the polynucleotides in a variety of cell types and species commonly used in the art and disclosed herein. Sequences containing biased codons can be tested for optimal expression in various species and tested for functionality as disclosed herein.

The highly conserved amino acids in the cysteine-rich pseudo-repeats of BR43x2 can be used as a tool to identify new family members. For example, sequences encoding the extracellular ligand-binding domains described above can be amplified from RNA obtained from various tissue materials or cell lines using reverse transcription-polymerase chain reaction (RT-PCR). In particular, highly degenerate primers designed from the BR43 × 2 sequence are useful for this purpose.

In a preferred embodiment of the invention, the isolated polynucleotide will hybridize to a polynucleotide of similar size as SEQ id no: 3 region, or the complement thereof, hybridizes under stringent conditions. Generally, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) for the specific sequence under certain ionic strength and pH. The Tm is the temperature (under ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are: a pH of 7, a salt concentration of up to about 0.03M, and a temperature of at least about 60 ℃.

As mentioned previously, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. Although DNA may also be prepared using RNA from other tissues or isolated as genomic DNA, it is generally preferred to isolate RNA from RPMI1788 cells, PBMNC, resting or activated transfected B cells, or tonsil tissue. Total RNA can be prepared using guanidine hydrochloride extraction followed by CsCl gradient centrifugation (Chirgwin et al, Biochemistry 18: 52-94, 1979). Poly (A) was prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972)⁺RNA. From Poly (A) using known methods⁺RNA complementary DNA (cDNA) was prepared. Polynucleotides encoding BR43x2 polypeptides are then identified and isolated, for example, by hybridization or PCR.

Those skilled in the art will appreciate that SEQ ID NOs: the sequences disclosed in 1 and 3 represent only one allele of the human gene, and allelic variation and other species splicing are expected. SEQ ID NOs: 1 and 3, including those containing silent mutations and those in which the mutation results in an amino acid sequence change, are within the scope of the present invention, as are allelic variants of the DNA sequences shown in SEQ ID NOs: 2 and 4 are also within the scope of the invention. Allelic 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.

The invention also provides polypeptides related to SEQ ID NOs: 2 and 4 and their species orthologs are substantially homologous to isolated BR43x2 polypeptides. The term "substantially homologous" is used herein to refer to sequences identical to SEQ ID NOs: 2 and 4 or their orthologues, having a sequence identity of 50%, preferably 60%, more preferably at least 80%. These polypeptides will more preferably be identical to SEQ ID NO: 2 or an orthologue thereof is at least 90% identical, most preferably 95% or more identical. Percent sequence identity is determined by conventional methods. See, e.g., Altschul et al, bull.math.bio.48: 603-66, 1986 and Henikoff, proceedings of the national academy of sciences of the united states of america 89: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a deletion region open penalty of 10, a deletion region extension penalty of 1, and the Henikoff and Henikoff (supra) "blosum 62" scoring matrices (amino acids are represented by the standard one letter code) shown in Table 3. Percent identity is then calculated as follows:

sequence identity of polynucleotide molecules is determined by similar methods using the ratios disclosed above.

Substantially homologous proteins and polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of 1 to about 30 amino acids; and small amino-or carboxy-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of no more than about 20-25 residues, or an affinity tag. The polypeptide comprising an affinity tag may further comprise a proteolytic cleavage site between the BR43x2 polypeptide and the affinity tag. Preferably, the site includes a thrombin cleavage site and a factor Xa cleavage site.

TABLE 4

Conservative amino acid substitutions

Basic: arginine

Lysine

Histidine

Acidic: glutamic acid

Aspartic acid

Polar: glutamine

Asparagine

Hydrophobic: leucine

Isoleucine

Valine

Aromatic: phenylalanine

Tryptophan

Tyrosine

Small: glycine

Alanine

Serine

Threonine

Methionine

In addition to these 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α -methylserine) may be substituted for the amino acid residues of the BR43X2 polypeptide of the present invention. Amino acid residues of the BR43x2 polypeptide may be replaced with a limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and artificial amino acids. The proteins of the invention may also contain non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2, 4-methyleneproline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine (2-azaphenylelanine), 3-aza-phenylalanine, 4-aza-phenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system employing a chemical aminoacylation-suppressing tRNA to suppress nonsense mutations can be used. Methods for synthesizing amino acids and aminoacylating trnas are known in the art. Plasmids containing nonsense mutations were transcribed and translated in a cell-free system containing E.coli (E.coli) S30 extract and commercially available enzymes and other reagents. The protein was purified by chromatography. See, e.g., Robertson et al, journal of the american chemical association (j.am.chem.soc.) 113: 2722, 1991; ellman et al, methods in enzymology (MethodsEnzymol.) 202: 301, 1991; chung et al, science 259: 806-9, 1993; and Chung et al, Proc. Natl. Acad. Sci. USA 90: 10145-9, 1993). In the second approach, translation was performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcati et al, J.Biol.chem.) 271: 19991-8, 1996). In a third method, E.coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) but in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid substitution is incorporated into a protein in its natural counterpart. See Koide et al, biochemistry (Biochem.) 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted into non-naturally occurring species by in vitro chemical modification. Chemical modifications can be combined with site-directed mutagenesis to further extend the scope of substitution (Wynn and Richards, Protein science 2: 395-403, 1993).

The amino acid residue of BR43x2 may be replaced with a limited number of non-conserved amino acids, non-genetically encoded amino acids, non-naturally occurring amino acids, and artificial amino acids.

The essential amino acids in the BR43X2 polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, science 244: 1081-5, 1989). A single alanine mutation is introduced at each residue position of the molecule, and the resulting mutant molecules are then tested for biological activity (e.g., to provide a reduction in B cell response, inhibition or reduction of autoantibody production during an immune response) to identify amino acid residues that are critical to the activity of the molecule. See also Hilton et al, journal of biochemistry 271: 4699-708, 1996. The biological interaction site, i.e., the ligand binding moiety, such as the cysteine-rich pseudo-repeat, can also be determined by physical analysis of the structure as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, and mutation of the amino acid at the putative contact site. See, e.g., de Vos et al, science 255: 306-12, 1992; smith et al, journal of molecular biology (j.mol.biol.) 224: 899-904, 1992; wlodaver et al, FEBS Lett.309: 59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology to related TNFR family members such as TACI and BCMA.

Other amino acid substitutions may also be made in the cysteine-rich pseudo-repeat of BR43X2, as long as the conserved cysteine, aspartic acid and leucine residues are retained and the higher order structure is not disturbed. Substitutions in the cysteine-rich pseudo-repeats of BR43X2 are preferably made with reference to other cysteine-rich pseudo-repeated sequences. SEQ ID NO: 10 is a generic cysteine-rich pseudo-repeat showing amino acid substitutions that can be allowed based on this alignment. Substitutions in this domain are subject to the limitations mentioned herein.

Multiple amino acid substitutions can be made and detected using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (science 241: 53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86: 2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions of a polypeptide, screening for functional polypeptides, and then sequencing the mutagenized polypeptides to determine the profile of substitutions that can be allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al, biochemistry 30: 10832-7, 1991; Ladner et al, U.S. Pat. No. 5,223,409; Huse, WIPO publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al, Gene 46: 145, 1986; Ner et al, DNA 7: 127, 1988).

According to Stemmer, nature 370: 389-91, 1994, Stemmer, journal of the national academy of sciences USA 91: 10747-51, 1994 and WIPO publication WO 97/20078, variants of the BR43X 2DNA and polypeptide sequences disclosed herein can be prepared by DNA shuffling. Briefly, randomly introduced point mutations were obtained by random fragmentation of the parental DNA followed by reassembly using PCR, and then variant DNA was generated by in vitro homologous recombination. The technique can be adapted to introduce additional variability into the procedure by employing a family of parental DNA, e.g., allelic variants or DNA from different species. Rapid "evolution" of sequences can be achieved by selecting for desirable mutations while screening for deleterious changes, selecting or screening for desired activity, followed by further rounds of mutagenesis and analysis.

The mutagenesis methods disclosed above can be combined with a large-scale automated screening method for detecting the activity of a clonally mutagenized polypeptide in a host cell. Mutagenized DNA molecules encoding active polypeptides (e.g., providing a reduction in B cell response during an immune response, suppression or reduction in autoantibody production) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the importance of individual amino acid residues in a polypeptide of interest to be rapidly determined and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can identify and/or make a variety of sequences similar to SEQ ID NO: 2 or an allelic variant thereof, which is substantially homologous and retains the B cell inhibitory properties of the wild-type protein. These polypeptides may include additional amino acids or domains from other members of the tumor necrosis factor receptor superfamily, affinity tags, or the like. BR43x2 polypeptides or fusion constructs containing functional domains of other members of the TNFR superfamily constitute hybrid tumor necrosis factor receptors exhibiting altered B cell inhibitory ability.

The invention also provides corresponding receptors and polynucleotides from other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are the BR43X2 receptors from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate receptors. Orthologues of the human BR43X2 receptor may be cloned using the information and compositions provided herein in combination with conventional cloning techniques. For example, the cDNA can be cloned using mRNA obtained from a tissue or cell type expressing the receptor. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. Libraries are then prepared from the mRNA of positive tissues or cell lines. The cDNA encoding the receptor can then be isolated by various methods, for example, by probing with a full or partial human cDNA or one or more sets of degenerate probes based on the sequences disclosed herein. The cDNA can also be cloned using PCR and primers designed from the sequences disclosed herein. In yet another method, host cells can be transformed or transfected with the cDNA library and expression of the cDNA of interest can be detected with antibodies to the receptor. Similar techniques can also be applied to isolate genomic clones.

Receptor polypeptides of the invention, including full-length receptor polypeptides, soluble receptor polypeptides, polypeptide fragments, and fusion polypeptides, can be prepared in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and that can be grown in culture, including bacteria, fungal cells, and higher eukaryotic cells in culture. Eukaryotic cells are preferred, especially cultured cells of multicellular organisms. Techniques for manipulating cloned DNA molecules and introducing foreign DNA into various host cells are disclosed in Sambrook et al, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition, Cold spring harbor, NY, 1989; and compiled by Ausubel et al, Current protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

Generally, the DNA sequence encoding the BR43x2 polypeptide and other genetic elements necessary for its expression, including generally a transcription promoter and a terminator, are operably linked together in an expression vector. The vector will also typically contain one or more selectable markers and one or more origins of replication, although it will be appreciated by those skilled in the art that in some systems selectable markers may be provided on different vectors and that replication of the exogenous DNA may be achieved by integration into the genome of the host cell. The choice of promoter, terminator, selectable marker, vector and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available from commercial suppliers.

To direct the BR43x2 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also referred to as a signal sequence, leader sequence, prepro sequence, or pre sequence) is provided in the expression vector. The secretion signal sequence may be that of the BR43X2 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretion signal sequence is linked in correct reading frame to the BR43X 2DNA sequence and is positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretion signal sequences are typically located 5' to the DNA sequence encoding the polypeptide of interest, but certain signal sequences may be placed elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Pat. No. 5,037,743; Holland et al, U.S. Pat. No. 5,143,830).

In the present invention, cultured mammalian cells are suitable hosts. Methods for introducing foreign DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al, Cell (Cell) 14: 725, 1978; Corsaro and Pearson, somatic Genetics (somatocell Genetics) 7: 603, 1981; Graham and Van der Eb, Virology (Virology) 52: 456, 1973), electroporation (Neumann et al, EMBO J.1: 841-45, 1982), DEAE-dextran-mediated transfection (Ausubel et al, supra), and liposome-mediated transfection (Hawley-Nelson et al, Focus (Focus) 15: 73, 1993; Ciccarone et al, Focus 15: 80, 1993). Production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, in Levinson et al, U.S. patent 4,713,339; hagen et al, U.S. Pat. Nos. 4,784,950; palmiter et al, U.S. patent 4,579,821; and Ringold, us patent 4,656,134. Suitable cultured mammalian cells include COS-1(ATCC CRL1650), COS-7 (ATCCRL 1651), BHK (ATCC CRL 1632), BHK570(ATCC CRL 10314), 293 (ATCCRL 1573; Graham et al, J.Gen.Virol., 36: 59-72, 1977), Jurkat (ATCC CRL-8129), BaF3 (Interleukin-3 dependent prelymoid cell line derived from mouse bone marrow, see Palactios and Steinmetz, cell 41: 727-34, 1985; Mathey-Prevot et al, molecular cell biology (mol.cell Biol.) 6: 4133-5, 1986) and Chinese hamster ovary cell lines (e.g., CHO-K1; ATCC CCL 61). Other suitable cell lines are known in the art and are available from public collections such as the American type culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as those from SV-40 or cytomegalovirus. See, for example, U.S. patent 4,956,288. Other suitable promoters include those from the metallothionein gene (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the major late promoters of adenovirus.

Drug screening is generally used to select cultured mammalian cells that have been inserted with exogenous DNA. These cells are commonly referred to as "transfectants". Cells cultured in the presence of a selective agent and capable of transmitting a gene of interest to their progeny are referred to as "stable transfectants". Preferred selectable markers are genes encoding the antibiotic neomycin resistance. Screening is performed in the presence of neomycin-like drugs such as G-418 or analogs. Selection systems may also be used to increase the expression level of a gene of interest, a process known as "amplification". Amplification was performed as follows: transfectants are cultured in the presence of low levels of a selection agent, followed by increasing the amount of selection agent to select for cells that produce high levels of the introduced gene product. A preferred amplifiable selectable marker is dihydrofolate reductase which confers resistance to methotrexate. Other drug resistance genes (e.g., hygromycin resistance, multidrug resistance, puromycin acetyltransferase) may also be used. Other markers that introduce phenotypic changes, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC class I, placental alkaline phosphatase, can be used to separate transfected cells from untransfected cells by FACS sorting or magnetic bead separation techniques.

Other higher eukaryotic cells may also be used as hosts, including plant cells, insect cells, and avian cells. For a review of Agrobacterium rhizogenes (Agrobacterium rhizogenes) as vectors for gene expression in plant cells, see Sinkar et al, journal of bioscience (j.biosci.) (Bangalore) 11: 47-58, 1987. Transformation of insect cells and production of exogenous polypeptides therein are disclosed in Guarino et al, U.S. Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, typically derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See King and Possee, baculovirus expression system: laboratory Manual (The Baculoviral expression System: A Laboratory Guide), London, ChapmanHall o' Reilly et al, baculovirus expression vector: a Laboratory Manual (Baculoviral Expression Vectors: A Laboratory Manual), New York, Oxford University Press, 1994; and Richardson, manual for baculovirus expression, Molecular Biology methods (bactabove expression protocols in Molecular Biology), Totowa, NJ, Humana Press, 1995. A second method for preparing recombinant BR43X2 baculoviruses utilizes a transposon-based system described by Luckow (Luckow et al, J.Virol., 67: 4566-79, 1993). The system utilizes a transfer vector, in Bac-to-Bac^TMThis system is sold in kits (Life technologies, Rockville, Md.). The system uses a transfer vector pFastBacl containing Tn7 transposon^TM(Life Technologies) to move the DNA encoding the BR43X2 polypeptide into a baculovirus genome maintained in E.coli as a large plasmid called a "bacmid". See Hill-Perkins and Possee, J.Gen.Virol 71: 971-6, 1990; bonning et al, journal of common virology 75: 1551-6, 1994; and Chazenbalk and Rapoport, journal of biochemistry 270: 1543-9, 1995. In addition, the transfer vector may comprise an in-frame fusion of the expressed BR43X2 polypeptide at the C-or N-terminus with DNA encoding an epitope tag, such as a Glu-Glu tag (Grussenmeyer et al, Proc. Natl. Acad. Sci. USA 82: 7952-4, 1985). Using techniques known in the art, a transfer vector containing BR43X2 was transformed into E.coli and bacmid containing the disrupted lacZ gene indicative of recombinant baculovirus was screened. Bacmid DNA containing the recombinant baculovirus genome is isolated using conventional techniques and used to transfect Spodoptera frugiperda (Spodoptera frugiperda) cells, such as sf9 cells. Subsequently, a recombinant virus expressing BR43X2 was prepared. The recombinant virus stock solution is prepared by a method generally used in the art.

The recombinant virus is used for infecting host cells, and is typically a cell line derived from fall armyworm spodoptera frugiperda. See generally Glick and Pasternak, molecular biotechnology: principles and Applications of recombinant DNA (Molecular Biotechnology: Principles and Applications), ASMPress, Washington, D.C., 1994. Another suitable cell line is HighFiveO from Trichoplusia ni (Trichoplusia ni)^TMCell line (Invitrogen) (U.S. Pat. No. 5,300,435). Is commercially availableThe serum-free medium of (a) and (b) and maintaining the cells. For sf9 cells, a suitable medium is sf900II^TM(Life Technologies) or ESF921^TM(Expression Systems); for Trichoplusia ni cells, a suitable medium is Ex-cell0405^TM(JRH Biosciences, Lenexa, KS) or Express FiveO^TM(Life technologies). Making the cells from about 2-5X 10⁵The seeded density of individual cells was grown to 1-2X 10⁶The density of individual cells, at this time, is added to the recombinant virus stock at a multiplicity of infection (MOI) of 0.1-10, more typically close to 3. The procedures are generally described in available laboratory manuals (King and Possee, supra; O' Reilly, et al, supra; Richardson, supra). The BR43x2 polypeptide may then be purified from the supernatant using the methods described herein.

Fungal cells, including yeast cells, may also be used in the present invention. Particularly interesting yeast species in this regard include Saccharomyces cerevisiae, Pichia pastoris and Pichia methanolica. Methods for transforming saccharomyces cerevisiae cells with exogenous DNA and preparing recombinant polypeptides therefrom are disclosed in, for example, Kawasaki, U.S. patent 4,599,311; kawasaki et al, U.S. Pat. Nos. 4,931,373; brake, U.S. patent 4,870,008; welch et al, U.S. Pat. No. 5,037,743; and Murray et al, U.S. Pat. No. 4,845,075. Transformed cells are selected for a phenotype determined by a selectable marker, usually drug resistance or ability to grow in the absence of a particular nutrient (e.g., leucine). One preferred vector system for s.cerevisiae is the POT1 vector system disclosed by Kawasaki et al (U.S. Pat. No. 4,931,373), which allows selection of transformed cells by growth in a medium containing glucose. Suitable promoters and terminators for yeast include those derived from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al, U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. patent 4,990,446; 5,063,154, respectively; 5,139,936, and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha (Hansenula polymorpha), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Ustilago zea (Ustilago maydis), Pichia pastoris, Pichia methanolica, Pichia quarternary (Pichia guillermondii) and Candida maltosa, are known in the art. See, e.g., Gleeson et al, journal of general microbiology (j.gen. microbiol.) 132: 3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus (Aspergillus) cells may be used according to the methods of McKnight et al, U.S. Pat. No. 4,935,349. Suminio et al, U.S. Pat. No. 5,162,228, disclose methods for the transformation of Acremonium chrysogenum. Lambowitz, U.S. Pat. No. 4,486,533, discloses a method for transformation of Neurospora.

For example, the use of Pichia methanolica as a host for the preparation of recombinant proteins is disclosed in Raymond, us patent 5,716,808; raymond, U.S. patent 5,736,383; raymond et al, Yeast (Yeast) 14: 11-23, 1998; and International publications WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules for transformation of p.methanolica are typically prepared as double-stranded circular plasmids and are preferably linearized prior to transformation. For the production of polypeptides in p.methanolica, the promoter and terminator in the plasmid are preferably the promoter and terminator of the p.methanolica gene, e.g. the alcohol utilization gene of p.methanolica (AUG1 or AUG 2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD) and Catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred that the entire expression fragment of the plasmid is surrounded by host DNA sequences at both ends. A preferred selectable marker for Pichia methanolica is the ADE2 gene encoding 5-aminoimidazole nucleotide carboxylase (AIRC; EC4.1.1.21) which allows ADE2 host cells to grow in the absence of adenine. For large scale industrial procedures, it may be desirable to minimize the use of methanol, in which case it is preferred to employ host cells in which both methanol utilization genes (AUG1 and AUG2) have been deleted. For the preparation of secreted proteins, host cells deficient in the vacuolar protease (vacuolor protease) gene (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into p. P. methanolica cells are preferably transformed by electroporation using an exponentially decaying pulsed electric field having a field strength of 2.5-4.5kv/cm, preferably about 3.75kv/cm, and a time constant (t) of 1-40 ms, most preferably about 20 ms.

Prokaryotic host cells, including strains of Escherichia coli, Bacillus, and other genera, are also useful host cells in the present invention. Techniques for transforming such hosts and expressing the cloned exogenous DNA sequences therein are well known in the art (see, e.g., Sambrook et al, supra). When expressing the BR43x2 polypeptide in bacteria, such as e.g. e.coli, the polypeptide may be retained in the cytoplasm, typically as an insoluble particle, or may be directed into the periplasmic space by bacterial secretion sequences. In the former case, the cells are lysed and the particles are recovered and then denatured, for example with guanidinium isothiocyanate or urea. The denatured polypeptide may then be refolded and dimerized by diluting the denaturant, for example, by dialysis against a urea solution and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide may be recovered in a soluble functional form from the periplasmic space by: the cells are disrupted (e.g., by sonication or osmotic shock) to release the contents of the periplasmic space and the protein is recovered, thereby avoiding the need for denaturation and refolding.

The transformed or transfected host cells are cultured according to conventional procedures in a medium containing nutrients and other components necessary for the growth of the selected host cell. A variety of suitable media, including media of known composition and complex media, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins, and minerals. The medium may also contain components such as growth factors or serum, if desired. The growth medium is generally deficient in essential nutrients, which are compensated, for example, by drug selection, or by selectable markers on the expression vector or co-transfected into the host cell,cells containing exogenously added DNA were selected. P. methanolica cells are cultured at about 25-35 ℃ in a medium containing sufficient carbon and nitrogen sources and trace nutrients. Adequate aeration is provided to the liquid culture by conventional means, such as shaking a small shake flask or sparging (spark) fermentor. For p. methanolica, a preferred medium is YEPD (2% D-glucose, 2% Bacto)^TMPeptone (Difco Laboratories, Detroit, Mi), 1% Bacto^TMYeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

The expressed recombinant BR43x2 polypeptide (or chimeric or fused BR43x2 polypeptide) may be purified using fractionation and/or conventional purification methods and media. Preferably, the protein or polypeptide of the invention is provided in a highly pure form, i.e., greater than 95% pure, more preferably greater than 99% pure. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of the sample. Typical purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable anion exchange media include derivatives of dextran, agarose, cellulose, polyacrylamide, special grade silica gel, and the like. Derivatives of PEI, DEAE, QAE and Q are preferred, DEAE Fast-FlowSepharose (Pharmacia, Piscataway, NJ) is particularly preferred. Typical chromatography media include those derivatized with phenyl, butyl or octyl groups, such as phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650(TosoHaas, Montgomeryville, Pa.), octyl-Sepharose (Pharmacia), and the like; or polypropylene resins such as Amberchrom CG 71(Toso Haas), etc. Suitable solid supports include glass beads, silicon-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, and the like which are insoluble under the conditions of their use. These supports may be modified with reactive groups that allow attachment of proteins through amino, carboxyl, thiol, hydroxyl and/or carbohydrate moieties. Examples of coupling chemistry include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistry. These and other solid phase media are well known and widely used in the art and are available from commercial suppliers. Methods for binding receptor polypeptides to a support medium are well known in the art. The choice of a particular method is a matter of routine design, which depends in part on the nature of the support chosen. See, e.g., affinity chromatography: principles and Methods (Affinity Chromatography: principles & Methods), Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptide of the present invention can be isolated by utilizing its physical properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those containing a polyhistidine tag. Briefly, the gel was first loaded with divalent metal ions to form a chelate (Sulkowski, Biochemical Advances in Biochem. 3: 1-7, 1985). The histidine-rich protein will be adsorbed onto the matrix with different affinity depending on the metal ion used and then eluted by competitive elution, lowering the pH or using a strong chelating agent. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (methods in enzymology, Vol. 182, "Guide to protein purification", M.Deutscher (eds.), Acad.Press, San Diego, 1990, pp. 529-39). In other embodiments of the invention, fusions of a polypeptide of interest with affinity tags (e.g., maltose binding protein, FLAG-tag (Asp Tyr Lys Asp Asp AspAspAspAsp Lys (SEQ ID NO: 13)), Glu-Glu tag (Glu Glu Tyr Met Pro MetGlu (SEQ ID NO: 14)), immunoglobulin domains) can be constructed to facilitate purification.

Refolding (and optionally reoxidation) of the protein may be advantageously employed. Preferably the protein is purified to > 80% purity, more preferably > 90% purity, even more preferably > 95% purity, especially preferably the protein is in a pharmaceutically pure state, i.e. more than 99.9% purity with respect to contaminating macromolecules, especially other proteins and nucleic acids, and is free of infectious and pyrogenic agents. Preferably, the purified protein is substantially free of other proteins, especially of animal origin.

The BR43 × 2 polypeptide or fragment thereof may also be prepared by chemical synthesis. The BR43x2 polypeptide may be monomeric or multimeric; glycosylated or non-glycosylated; pegylated (pegylated) or non-pegylated; and may or may not include the initial methionine residue. Examples of BR43x2 polypeptides include amino acid sequences between 32-40 residues in length, having the following motifs: XXCX [ QEK ] [ QEKNRRDHS ] [ QE ] X {0-2} [ YFW ] [ YFW ] DXLLX {2} C [ IMLV ] XCX {3} CX {6-8} CX {2} [ YF ] CXX (SEQ ID NO: 10), and subject to the limitations described herein.

The BR43x2 polypeptide may be synthesized by total solid phase synthesis, partial solid phase methods, fragment condensation, or classical solution synthesis. Preferably by solid phase peptide synthesis, for example as described by Merrifield, journal of the american chemical association 85: 2149, 1963, and preparing the polypeptide. The synthesis was performed with an amino acid protected at the alpha-amino terminus. Trifunctional amino acids with labile side chains are also protected with suitable groups to avoid unwanted chemical reactions during assembly of the polypeptide. The alpha-amino protecting group is selectively removed to allow subsequent reactions to continue at the amino terminus. The conditions used to remove the alpha-amino protecting group do not remove the side chain protecting group.

The alpha-amino protecting groups are those known to be useful in the field of stepwise polypeptide synthesis. Including acyl-based protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aryl-based protecting groups (e.g., biotinyl), aromatic carbamate (urethane) -based protecting groups (e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc)), aliphatic carbamate protecting groups (e.g., t-butoxycarbonyl (tBoc), isopropoxycarbonyl, cyclohexyloxycarbonyl), and alkyl-based protecting groups (e.g., benzyl, trityl). Preferred protecting groups are tBoc and Fmoc.

The side chain protecting group chosen must remain intact during coupling and not be removed during deprotection of the amino-terminal protecting group or in the case of coupling. The side chain protecting groups must also be capable of being removed after synthesis is complete without altering the reaction conditions of the final polypeptide. In tBoc chemistry, the side chain protecting groups of trifunctional amino acids are mostly benzyl based. In Fmoc chemistry, they are mostly based on t-butyl or trityl groups.

In tBoc chemistry, the preferred side chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and threonine, benzyloxymethyl (and dinitrophenyl) for histidine, 2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and 2-bromobenzyl for tyrosine. In Fmoc chemistry, preferred side chain protecting groups are 2,2, 5,7, 8-pentamethylbenzodihydropyran-6-sulfonyl (Pmc) or 2,2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, trityl for asparagine, cysteine, glutamine and histidine, t-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, and tBoc for lysine and tryptophan.

For phosphopeptide synthesis, phosphate groups can be incorporated directly or after assembly. In the direct incorporation strategy, the phosphate group on serine, threonine or tyrosine can be protected by methyl, benzyl or tert-butyl in Fmoc chemistry and by methyl, benzyl or phenyl in tBoc chemistry. In Fmoc chemistry, direct incorporation of phosphotyrosine without protection from phosphate can also be used. In the post-assembly incorporation strategy, the unprotected hydroxyl group of serine, threonine or tyrosine is derivatized with di-tert-butyl-, dibenzyl-, or dimethyl-N, N' -diisopropyl-phosphoramidite on a solid phase, followed by oxidation by tert-butyl hydroperoxide.

Solid phase synthesis is typically performed starting from the carboxyl terminus by coupling an alpha-amino protected (side chain protected) amino acid to a suitable solid support. When attached to a chloromethyl, chlorotrityl or hydroxymethyl resin, ester linkages are formed and the resulting polypeptide will have a free carboxyl group at the C-terminus. Alternatively, when amide resins such as benzhydrylamine or p-benzhydrylamine resins (for tBoc chemistry) and Rink amide or PAL resins (for Fmoc chemistry) are employed, amide bonds are formed and the resulting polypeptide will have a carbamoyl group at the C-terminus. These resins, whether polystyrene-or polyamide-based or polyethylene glycol-grafted, with or without a handle or linker, and with or without a first attached amino acid, are commercially available and their preparation is described in Stewart et al, "Solid Phase Peptide Synthesis" (second edition), (Pierce Chemical company, Rockford, IL, 1984); and Bayer and Rapp, chem.pept.prot.3: 3,1986, respectively; and Atherton et al, solid phase peptide synthesis: practical methods (Solid Phase Peptide Synthesis: active Approach), IRL Press, Oxford, 1989.

The C-terminal amino acid (side chain and alpha-amino group are protected if necessary) is attached to the hydroxymethyl resin using various activators including Dicyclohexylcarbodiimide (DCC), N' -diisopropylcarbodiimide (DIPCDI) and Carbonyldiimidazole (CDI). The C-terminal amino acid may be in the form of its cesium tetramethylammonium salt (cecum tetramethylammonium salt), or directly bound to chloromethyl or chlorotrityl resin in the presence of Triethylamine (TEA) or Diisopropylethylamine (DIEA). The binding of the first amino acid to the amide resin is the same as the formation of an amide bond during the coupling reaction.

After attachment to the resin support, the alpha-amino protecting group is removed using various reagents according to protective chemistry (e.g., tBoc, Fmoc). The degree of Fmoc removal can be monitored at 300-320nm or by conductivity cells. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise according to the desired sequence to obtain the desired sequence.

A variety of activating reagents can be used in the coupling reaction, including DCC, DIPCDI, 2-chloro-1, 3-dimethylanilinium hexafluorophosphate (CIP),Benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphine hexafluorophosphate (BOP) and its pyrrolidine analogue (PyBOP), bromo-trispyrrolidine-phosphine hexafluorophosphate (PyBrOP), O- (benzotriazol-1-yl) -1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HBTU) and its tetrafluoroborate analogue (TBTU) or its pyrrolidine analogue (HBPyU), O- (7-azabenzotriazol-1-yl) -1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HATU) and its Tetrafluoroborate Analogue (TATU) or its pyrrolidine analogue (HAPyU). The most common catalytic additives used in coupling reactions include 4-Dimethylaminopyridine (DMAP), 3-hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazine (HODhbt), N-hydroxybenzotriazole (HOBt), and 1-hydroxy-7-azabenzotriazole (HOAt). Each protected amino acid is used in excess (>2.0 equivalents) and the coupling is often in N-methylpyrrolidone (NMP) or in DMF, CH₂Cl₂Or mixtures thereof. The completion of the coupling reaction can be monitored at each stage by the ninhydrin reaction as described, for example, by Kaiser et al (analytical biochemistry (anal. biochem.) 34: 595, 1970).

After complete assembly of the desired peptide is completed, the peptide-resin is cleaved with reagents with appropriate scavengers. Fmoc peptides are typically used with scavengers (e.g., H)₂O, ethanedithiol (ethanedithiol), phenol and thiophenylmethane) in TFA. tBoc peptides are typically cleaved and deprotected with liquid HF at-5 ℃ to 0 ℃ for 1-2 hours, which cleaves the polypeptide from the resin and removes most of the side chain protecting groups. Scavengers, such as anisole, dimethyl sulfide, and p-toluene thiophenol, are commonly used with liquid HF to prevent cations formed during the cleavage process from alkylating and acylating amino acid residues in the polypeptide. The formyl group of tryptophan and the dinitrophenyl group of histidine need to be removed by piperidine and phenylthio groups, respectively, in DMF prior to HF cleavage. The acetamidomethyl group of cysteine can be removed by mercury (II) acetate or by iodine, thallium (III) trifluoroacetate or silver tetrafluoroborate and cysteine is simultaneously oxidized to cystine. Other strong acids for cleavage and deprotection of tBoc peptide include trifluoromethanesulfonic acidAcid (TFMSA) and trimethylsilane trifluoroacetate (TMSOTf).

The invention also provides various other polypeptide fusions and related multimeric proteins containing one or more polypeptide fusions. Soluble BR43x2, TACI or BCMA polypeptides can be expressed as fusions with an immunoglobulin heavy chain constant region (typically an Fc fragment) containing two constant region domains but lacking a variable region. Methods for making these fusions are disclosed in U.S. patents 5,155,027 and 5,567,584. These fusions are typically secreted as multimeric molecules in which the Fc portions form disulfide bonds with each other, while two non-Ig polypeptides are arranged next to each other. immunoglobulin-BR 43x 2(TACI or BCMA) polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric BR43x2 analogs. The accessory domain can be fused to a BR43x 2(TACI or BCMA) polypeptide to target a particular cell, tissue, or macromolecule. Fusions can also be made using toxins as discussed herein. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. The BR43x2 polypeptide may be fused to two or more moieties, such as affinity tags and targeting domains for purification. Polypeptide fusions may also contain one or more cleavage sites, particularly between domains. See, Tuan et al, connection.Tiss.Res.34: 1-9, 1996. Fusions of this type may also be used, for example, to affinity purify the relevant ligand from solution, as an in vitro analytical tool, to block signal by specific titration of the ligand in vitro, to bind to a cell surface ligand, or as an in vivo antagonist of BR43x2 and to block the stimulatory effect of the ligand by administration. For use in analytical assays, the fusion protein may be bound to a support via an Fc region and used in an ELISA.

The invention also provides soluble BR43X2 receptors and polypeptide fragments useful for forming fusion proteins with affinity tags or labels. Soluble BR43x 2-affinity tag fusion proteins can be used, for example, to identify BR43x2 ligands, as well as agonists and antagonists of natural ligands. Cells expressing the ligand, agonist or antagonist can be identified by fluorescence immunocytometry or immunohistochemistry using labeled soluble BR43x 2. The soluble fusion proteins are useful in studying the distribution of the ligand on tissues or specific cell lines, and in providing an understanding of receptor/ligand biology.

To purify a ligand, agonist or antagonist, the BR43X 2-Ig fusion protein is added to a sample containing the ligand, agonist or antagonist under conditions favorable to receptor/ligand binding (typically near physiological temperature, pH and ionic strength). The receptor/ligand complex is then isolated by mixing with protein A immobilized on a solid support (e.g., insoluble resin beads). The ligand, agonist, antagonist is then eluted using conventional chemical techniques, such as salt or pH gradient. In another alternative method, the fusion protein itself is bound to a solid support and the binding and elution are carried out as described above. Methods for immobilizing receptor polypeptides on solid supports, such as agarose, cross-linked agarose, glass, cellulose resins, silicon-based resins, polystyrene, cross-linked polyacrylamide, or particles of similar materials that are stable under the conditions of use, are known in the art. Methods for attachment of polypeptides to solid supports are known in the art and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium is typically formed into a column, and then a liquid containing the ligand is flowed through the column one or more times to allow binding of the ligand to the receptor polypeptide. Then using a salt concentration, chaotropic agent (MnCl)₂) Or a change in pH disrupts ligand-receptor binding to elute the ligand.

To direct the soluble receptor out of the host cell, the soluble receptor DNA is ligated with a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide. To facilitate purification of the secretory receptor domain, an N-or C-terminal overhang such as an affinity tag, or another polypeptide or protein from which an antibody or other specific binding agent can be obtained, can be fused to the receptor polypeptide.

Cells expressing the functional soluble and membrane-bound receptors of the invention can be used in screening assays. A variety of suitable analytical tests are known in the art. These assays are based on detecting biological responses in target cells. A change in metabolism compared to the control value indicates whether the test compound modulates BR43x2 mediated metabolism. One such assay is a cell proliferation assay. Cells are cultured with or without test compounds and then proliferation of the cells is detected, for example, by measuring incorporation of tritiated thymidine, or by colorimetric assays based on metabolic degradation of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazole bromide (MTT) (Mosman, J.Immunol. meth.) 65: 55-63, 1983). An alternative assay protocol employs cells that are further engineered to express a reporter gene. The reporter gene is ligated to a promoter element responsive to the receptor-associated pathway, and the assay then detects transcriptional activation of the reporter gene. A number of reporter genes which are readily assayed in Cell extracts are known in the art, such as lacZ, Chloramphenicol Acetyltransferase (CAT) and Serum Response Element (SRE) of E.coli (see, e.g., Shaw et al, Cell (Cell) 56: 563-72, 1989). A preferred such reporter gene is the luciferase gene (de Wet et al, molecular cell Biol. 7: 725, 1987). Expression of the luciferase gene can be detected by fluorescence using methods known in the art (e.g., Baumgartner et al, J.Biol.chem.) 269: 29094-101, 1994; Schenborn and Goiffin, Promega Notes 41: 11, 1993). Luciferase activity assay kits are commercially available, for example from Promega, Madison, WI. This type of target cell line can be used to screen chemical libraries, cell conditioned media, fungal cultures, soil samples, water samples, and the like. For example, a series of samples of cell conditioned media can be analyzed on target cells to identify cells that produce the ligand. Positive cells are then used to prepare a cDNA library in a mammalian expression vector, which is divided into pools (pool), transfected into host cells and expressed. Media samples from transfected cells were then analyzed, and the pools were again partitioned, re-transfected, subcultured, and positive cells were again analyzed to isolate cloned cDNA encoding the ligand.

It may also be advantageous to use a receptor (or antibody, a member of a complement/anti-complement pair) or binding fragment thereof that binds a ligand, and commercially available biosensor devices (BIAcore)^TMPharmacia Biosensor, Piscataway, NJ). The receptor, antibody, member or fragment of a complement/anti-complement pair is immobilized on the surface of the receptor chip. The application of this device is disclosed in Karlsson, journal of immunization methods 145: 229-40, 1991 and Cunningham and Wells, journal of molecular biology (J.mol.biol.) 234: 554-63, 1993. For example, BR43x2 polypeptide, fragment, antibody, or member of a complement/anti-complement pair is covalently linked to gold foil-bound dextran fibers in a flow cell using amine or thiol chemistry. The test sample is flowed through the absorption cell. If a ligand, epitope, or member of a relative complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody, or member, respectively, causing a change in the refractive index of the medium, which can be detected as a change in gold foil surface plasmon resonance (surfactam resonance). The system allows determination of the rate of binding and dissociation (on-and off-rates), from which binding affinity can be calculated and an estimate of the stoichiometry of binding can be made. Receptor polypeptides that bind a ligand may also be used in other assay systems known in the art. These systems include Scatchard analysis (see Scatchard, ann. ny acad. sci.51: 660-72, 1949) and calorimetric assays (Cunningham et al, science 253: 545-48, 1991; Cunningham et al, science 245: 821-25, 1991) for determining binding affinity.

FIG. 2 shows solubility I¹²⁵Scatchard plot analysis of the binding of ztnf4 to TACI and BCMA, and comparison of binding constants to other members of the TNFR family in table 7.

TABLE 7

Ligands	Kd M	Cell source	Reference to the literature
				TNFa high	7.14E-11	HL-60	a
TNFa Low	3.26E-10	HEP-2	a
TNFa high	2.00E-10	HL-60	b
CD27L	3.70E-10	MP-1	c
				CD27L	8.30E-09	MP-1	c
				CD40L	5.00E-10	EL40.5	d
CD40L	1.00E-09	EBNA	d
				(125I-CD40)
				4-1BBL	1.16E-09	Biocore	e
Anti-41 BBmab	4.14E-10	Biocore	e
Soluble ztnf4	1.11E-09	TACI-BHK
				Soluble ztnf4	1.25E-09	BCMA-BHK

a Hohmann et al, journal of biochemistry 264: 14927-34, 1989

b Manna and Aggarwal, journal of biochemistry 273: 33333-41, 1998

c Goodwin et al, cell 73: 447-56, 1993

d Armitage et al, Nature 357: 80-82, 1992

e Shuford et al, j.exp.med.186: 47-55, 1997

As a receptor, activation of BR43 × 2 polypeptides can be determined by measurement of extracellular acidification rate or proton efflux associated with receptor binding and subsequent physiological cellular responses by silicon-based biosensor microphysiometers. One exemplary device is a Cytosensor manufactured by Molecular Devices (Sunnyvale, Calif.)^TMMicrophysiometer. Various cellular responses, such as cell proliferation, ion transport, energy production, inflammatory responses, modulation, and receptor activation, etc., can be measured by this method. See, e.g., McConnell et al, science 257: 1906-12, 1992; pitchford et al, methods in enzymology 228: 84-108, 1997; arigilli et al, journal of immunological methods 212: 49-59, 1998; vanliende et al, european journal of pharmacology (eur.j. pharmacol.) 346: 87-95, 1998. The micro-physiological function measuring instrument can be used for analyzing adherent or non-adherent eukaryotic or prokaryotic cells. The microphysiological function meter directly measures the response of cells to various stimuli, including agonists, ligands, or antagonists of the BR43x2 polypeptide, by measuring extracellular acidification changes in the cell culture medium over a period of time. The micro-physiological function measuring instrument is preferably used for measuring the physiological function of a human body without expressing BControl eukaryotic cells expressing R43x2 polypeptides were compared to measure responses in eukaryotic cells expressing BR43x 2. Eukaryotic cells expressing BR43x 02 include cells that respond to stimuli that modulate BR43x 22 by transfecting BR43x 12 as described herein; or a cell naturally expressing BR43X2, such as BR43X2 derived from spleen tissue. Differences measured by extracellular acidification changes, such as an increase or decrease in response in cells expressing BR43x2 relative to controls, are a direct measure of cellular responses mediated by BR43x 2. Furthermore, these BR43X2 mediated responses can be assayed under a variety of stimuli. The method for identifying the agonists and antagonists of the BR43X2 polypeptide by adopting the micro physiological function tester is also provided, and comprises the following steps: providing a cell expressing a BR43x2 polypeptide, culturing a first portion of the cell in the absence of a test compound, culturing a second portion of the cell in the presence of the test compound, and detecting a change, e.g., an increase or decrease, in a cellular response of the second portion of the cell relative to the first portion of the cell. Changes in cellular responses are manifested as measurable changes in extracellular acidification rates. The method can be used for rapidly identifying antagonists and agonists of the BR43X2 polypeptide.

Soluble BR43x2 is useful in studying the distribution of ligands on tissues or specific cell lines, and in providing an understanding of receptor/ligand biology. The specificity of the TNF receptor for its ligand can also be used as a mechanism to destroy target cells bearing the ligand. For example, toxic compounds may be conjugated to BR43x2 soluble receptors or BR43x2 fusions. Examples of toxic compounds include radiopharmaceuticals that inactivate target cells; chemotherapeutic agents such as doxorubicin, daunorubicin, methotrexate, and cyclophosphamide; toxins such as ricin, diphtheria toxin, pseudomonas exotoxin a, and abrin; and antibodies to cytotoxic T cell surface molecules.

It has been found that ztnf4(5ng/ml) can bind to BR43X 2(SEQ ID NO: 2), TACI (SEQ ID NO: 6), BCMA (SEQ ID NO: 8), and BR43X 1(SEQ ID NO: 9) by FACS analysis (Flow Cytometry and Sorting, compiled by Melamed et al, Wiley-Liss, 1990; and Immunofluorescence and Cell Sorting, compiled by Current Immunology Protocols in Immunology, Vol.1, compiled by Coligan et al, John Wiley & Son, 1997). FITC-labeled soluble ztnf4 also showed specific binding, using FACS analysis, especially to PBMNC, B lymphocytes in tonsil cells, and to B cell lymphoma cell lines (Raji cells, burkitt's human lymphoma, ATCC CCL86), Ramos (burkitt's lymphoma cell line, ATCC CRL-1596), Daudi (burkitt's human lymphoma, ATCC CCL213), and RPMI1788 (a B lymphocyte cell line, ATCC CCL-156). No binding was observed to HL-60(ATCC a promyelocyte, ATCC CCL-240). Binding specificity to B cells from PBMNC and tonsil cells was verified by co-staining with antibodies against B cell specific molecules including CD19, IgD, IgM, and CD 20. The similarity of ztnf4 to CD40L suggests that there is a broader tissue distribution than observed. For example, affinity of ztnf4 was detected on monocytes, dendritic cells and purified T cells using cytokine proliferation and T cell proliferation assays, while binding or any other biological effect of ztnf4 could not be detected on any other type of cell detected. Thus, the specificity for B cells caused by the ligand and receptor suggests that the ligand and receptor are useful for the study and treatment of autoimmunity, B cell cancer, immunomodulation, IBD and any antibody-mediated pathology such as ITCP, myasthenia gravis, renal disease, indirect T cell immune response, transplant rejection, graft versus host disease.

In vitro, ztnf4 exhibits activation of B cells, resulting in B cell proliferation, antibody production, and upregulation of activation markers (see, e.g., below). These effects may require co-stimulation by IL-4 or other cytokines, or stimulation via B cell antigen receptors or other cell surface receptors that activate B cells, such as CD 40. Other tumor necrosis factor ligands, such as gp39 and TNF β, also stimulate B cell proliferation. Thus, the polypeptides of the invention can be directed to specifically modulate B cell responses during an immune response, suppressing activated B cells without affecting other cell populations, which is advantageous in the treatment of disease. In addition, the polypeptides of the invention may also be used to modulate B cell development, development of other cells, production of antibodies, and production of cytokines. The BR43x2 polypeptide may also be used to induce apoptosis and/or anergy in cells. The polypeptides of the invention may also modulate T and B cell communication by neutralizing the proliferative effect of ztnf 4. Bioassays and ELISAs can be performed to measure the response of cells to ztnf4 in the presence of soluble BR43 × 2, TACI, and/or BCMA. Other assays include those that measure changes in cytokine production as a measure of cellular response (see, e.g., Current Protocols in immunology, eds. John e. coligan et al, NIH, 1996), as well as assays that measure other cellular responses, including antibody isotype, monocyte activation, NK cell formation, function of antigen presenting cells, apoptosis.

The BR43x2 polypeptide of the invention may be used to treat pre-B cell leukemia or B cell leukemia such as plasma cell leukemia, chronic or acute lymphocytic leukemia by neutralizing the effect of ztnf 4; myelomas such as multiple myeloma, plasma cell myeloma, endothelial cell myeloma, and giant cell myeloma; and lymphomas such as non-Hodgkins lymphomas associated with an increase in ztnf4 polypeptide. Soluble BR43x2 would be a useful ingredient in a therapeutic regimen for inhibiting tumor development and survival.

Northern blot analysis showed CD8⁺Ztnf4 is expressed in cells, monocytes, tree cells (dendritic), activated monocytes. This suggests that in certain autoimmune diseases, cytotoxic T cells may stimulate B cell production by overproducing ztnf 4. Immunosuppressive proteins that selectively block the action of B lymphocytes would be useful in treating diseases. The production of autoantibodies (autoantibodies) is common for several autoimmune diseases, which promote tissue destruction and disease progression. Autoantibodies can also lead to complications of immune complex deposition and cause many systemic lupus erythematosus symptoms including renal failure, neuropathic pain symptoms and death. Modulation of antibody production independent of cellular response is also beneficial in many disease states. B cells have also been shown to play a role in the secretion of arthritis-causing (arthritis) immunoglobulins in rheumatoid arthritis (Korganow et al, Immunity 10: 451-61, 1999). Thus, the inhibition of antibody production by ztnf4 would be beneficial in the treatment of autoimmune diseases such as myasthenia gravis and rheumatoid arthritis. Immunosuppressive therapies, such as soluble BR43x2, which selectively block or neutralize B lymphocyte action, would be useful for these purposes. To test these capabilities of the BR43x2 soluble receptor polypeptides of the invention, these BR43x2 polypeptides were evaluated using assays known in the art and described herein.

The present invention provides methods of selectively blocking or neutralizing the effects of B cells associated with end stage renal disease (which may or may not be associated with autoimmune disease) using BR43x2, TACI, or BCMA polypeptides, fusions, antibodies, agonists, or antagonists. These methods are useful for treating immune kidney disease. These methods would also be useful for treating glomerulonephritis associated with diseases such as membranous nephropathy, IgA nephropathy or Berger's disease, IgM nephropathy, Goodpasture's disease, post-infection glomerulonephritis, mesangial proliferative disease (mesangial proliferative disease), minimal change nephrotic syndrome. The methods may also find therapeutic application in the treatment of secondary glomerulonephritis or vasculitis associated with diseases such as lupus, polyarteritis, Henoch-Schonlein disease, scleroderma, HIV-associated diseases, amyloidosis or hemolytic uremic syndrome. The methods of the invention may also be used as part of a therapeutic application for the treatment of interstitial nephritis or pyelonephritis associated with chronic pyelonephritis, analgesic abuse, calcinosis of the kidney, renal calculus, or chronic or acute interstitial nephritis caused by other agents.

The methods of the invention also include the use of BR43x2, TACI, or BCMA polypeptides, fusions, antibodies, agonists, or antagonists to treat hypertensive disorders or macrovascular (large vessel) disorders, including renal artery stenosis or occlusion and cholesterol or renal emboli.

The invention also provides methods for diagnosing and treating a renal or urinary tract tumor (urologic neoplasms), multiple myeloma, lymphoma, light chain neuropathy, or amyloidosis.

The invention also provides methods of using BR43x2, TACI, or BCMA polypeptides, fusions, antibodies, agonists, or antagonists to block or inhibit activated B cells for the treatment of asthma and other chronic respiratory diseases such as bronchitis and emphysema.

The invention also provides methods of using the BR43X2, TACI or BCMA polypeptides, fusions, antibodies, agonists or antagonists to inhibit or counteract effector T cell responses for immunosuppression, particularly for treatment of graft-versus-host disease, transplant rejection and the like. Other uses may also be found in the modulation of immune responses, particularly the activation and modulation of lymphocytes. BR43x2, TACI, or BCMA polypeptides, fusions, antibodies, agonists, or antagonists would be useful in therapies for treating immunodeficiency. BR43x2, TACI or BCMA polypeptides, fusions, antibodies, agonists or antagonists would be useful in therapeutic regimens for the treatment of autoimmune diseases such as Insulin Dependent Diabetes Mellitus (IDDM) and Crohn's disease. The methods of the invention will also have therapeutic value for the treatment of chronic inflammatory diseases, particularly the alleviation of joint pain, swelling, anemia and other related conditions, as well as for the treatment of septic shock.

The effect of soluble BR43x2, TACI or BCMA polypeptides and fusion proteins on the immune response can be determined by: animals immunized with antigen and subsequently injected with ztnf4 were administered the polypeptides of the invention and the production of antibody isotypes, B and T cell responses including delayed hypersensitivity reactions and in vitro proliferation, and cytokine production were measured according to methods known in the art.

Accordingly, the present invention provides a method of inhibiting ztnf4 activity in a mammal, comprising administering to said animal an amount of a compound selected from the group consisting of: a) SEQ ID NO: 4; b) SEQ ID NO: 8; c) a fusion protein; d) SEQ ID NO: 6 from amino acid residue 1 to 166; e) SEQ ID NO: 8 from position 1 to 150; f) and SEQ ID NO: 4 or an antibody or antibody fragment to which the polypeptide of (4) specifically binds; and g) to SEQ ID NO: 10 or an antibody or antibody fragment to which the polypeptide of claim 10 specifically binds. Examples of fusion proteins include fusions of soluble BR43X 2(SEQ ID NO: 4), TACI (amino acid residues 1-166 of SEQ ID NO: 6) or BCMA (amino acid residues 1-150 of SEQ ID NO: 8) with another polypeptide, preferably an Fc fragment of an immunoglobulin heavy chain constant region. Likewise, the present invention provides methods of inhibiting BR43x2, TACI, or BCMA receptor-ligand engagement.

These methods would be particularly useful where ztnf4 activity is associated with activated B lymphocytes, and for pre-treatment B cell or B cell cancers. These methods would also be useful where ztnf4 activity is associated with antibody production, particularly in connection with autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, or rheumatoid arthritis.

The invention also provides BR43x2 agonists and antagonists. Compounds identified as BR43x2 agonists are useful for modulating proliferation and development of target cells in vitro and in vivo. For example, agonist compounds, alone or in combination with other cytokines and hormones, may be used as components of a cell culture medium of known composition. Thus, agonists are useful in specifically modulating the growth and/or development of B lymphocytes carrying BR43x2 in culture. Agonists and antagonists may also be used to study effector functions of B lymphocytes, particularly the activation and differentiation of B lymphocytes. Antagonists may be used as research reagents to study the nature of ligand-receptor interactions.

Compounds identified as BR43x2 antagonists may also be useful for boosting humoral immune responses. B cell responses are important in combating infectious diseases, including bacterial, viral, protozoal and parasitic infections. Antibodies against infectious microorganisms are capable of immobilizing pathogens by binding to antigens followed by complement-mediated lysis or cell-mediated attack. Antagonists of BR43x2 will act to enhance humoral responses and will be a useful therapeutic agent for individuals at risk of infectious disease, or may be used as a supplement to vaccination.

The invention also provides antagonists which bind to a ligand which binds to either a BR43x2 polypeptide or a BR43x2 polypeptide, thereby inhibiting or abolishing the function of BR4 x 2. These BR43x2 antagonists will include antibodies; an oligonucleotide that binds to a BR43x2 polypeptide or a ligand thereof; a natural or synthetic BR43x2 ligand analog that retains the ability to bind to the receptor but does not result in signaling of the ligand or receptor. These analogs may be peptides or peptide-like compounds. Natural or synthetic small molecules that bind to BR43x2 polypeptides and prevent signaling are also considered antagonists. Thus, BR43x2 antagonists may be useful as therapeutic agents to treat certain diseases where blocking of signaling from BR43x2 receptors or ligands would be therapeutically beneficial. Antagonists may be used as research reagents to study the nature of ligand-receptor interactions. There was expression of BR43x2 on transformed B cell lines including EBV-induced and spontaneous burkitt's lymphoma and several B cell myelomas. It would be useful to inhibit the function of BR43x2 in the treatment of B cell lymphoma or multiple myeloma. BR43x2 antagonists, such as BR43x2 soluble receptors or antibodies, can be used therapeutically to modulate the progression of tumors.

Agonist and antagonist activity can be determined by activity assay assays that determine the potency of receptor/ligand engagement. Stably transfected B cell lines, e.g., Baf 3(a murine pre-B cell line, Palacios and Steinmetz, supra; and Mathey-Prevot, et al, supra), were prepared that expressed BR43X2 and co-expressed high levels of the NfkB, NFAT-1 and AP-1 reporter constructs. TACI and BCMA expressing cell lines were also prepared in a similar manner in Jurkat and other B lymphoma cell lines. Ztnf4 was found to transmit signals through the reporter genes in these structures. Soluble BR43 × 2 and antibodies can be used to measure binding.

In vivo methods for analyzing the proteins of the invention involve viral delivery systems. Examples of viruses used for this purpose include adenovirus, herpes virus, vaccinia virus and adeno-associated virus (AAV). Adenovirus is a double-stranded DNA virus and is currently the most extensively studied gene transfer vector for heterologous nucleic acid delivery (for review see Becker et al, methods in cell biology (meth. cell Biol.) 43: 161-89, 1994; and Douglas and Curiel, Science and Medicine (Science & Medicine) 4: 44-53, 1997). The adenovirus system provides several advantages: adenoviruses are capable of (i) accommodating relatively large DNA inserts; (ii) growing to a high titer; (iii) a broad spectrum of mammalian cell types; and (iv) use with a large number of available vectors containing different promoters. Moreover, since adenovirus is stable in the bloodstream, it can be administered by intravenous injection.

By deleting parts of the adenoviral genome, larger heterologous DNA inserts (up to 7kb) can be accommodated. These inserts can be 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 a host cell (human 293 cell line is an example). When administered intravenously to intact animals, adenoviruses are primarily targeted to the liver. If the adenovirus delivery system carries a deletion in the E1 gene, the virus will not replicate in the host cell. However, the host tissue (e.g., liver) will express and process (and secrete, if present, the signal sequence) the heterologous protein. Proteins secreted in the highly vascularized liver will enter the blood circulation and their effect on infected animals can be determined.

Adenovirus systems can also be used for in vitro production of proteins. By culturing adenovirus-infected non-293 cells under conditions where the cells do not undergo rapid division, the cells are able to produce proteins for an extended period of time. For example, BHK cells are grown to confluence in a cell factory and then exposed to an adenoviral vector encoding the secreted protein of interest. The cells were then cultured under serum-free conditions that allowed the infected cells to survive for several weeks without significant cell division. Alternatively, 293S cells infected with adenoviral vectors can be cultured in suspension culture at relatively high cell densities to produce substantial amounts of protein (see Garnier et al, cell technology 15: 145-55, 1994). Using either procedure, the expressed secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Non-secreted proteins can also be efficiently obtained in the production operations of infected 293S cells.

The effectiveness of the soluble BR43x2, TACI or BCMA polypeptides of the invention in certain disease states can be tested in vivo using established animal models. In particular, soluble BR43x2, TACI or BCMA polypeptides and polypeptide fragments can be tested in vivo in a number of animal models of autoimmune disease, such as the MRL-lpr/lpr or NZB x NZW F1 congenic mouse strains used as SLE (systemic lupus erythematosus) models. These animal models are known in the art, see, for example, autoimmune disease models: guide (A)Autoimmune Disease ModelsA Guideboost), Cohen and Miller, Academic Press. The progeny of the cross between new zealand black mouse (NZB) and new zealand white mouse (NZW) develop a spontaneous form of SLE that closely resembles human SLE. This progeny mouse, called NZBW, begins to produce IgM autoantibodies against T cells at 1 month of age, and by 5-7 months of age Ig anti-DNA autoantibodies are the major immunoglobulins. Hyperactive polyclonal B cells lead to overproduction of autoantibodies. The deposition of these autoantibodies, particularly to single-stranded DNA, is associated with the occurrence of glomerulonephritis which clinically manifests itself as proteinuria, azotemia and renal failure death. Renal failure is the leading cause of death in naturally SLE-infected mice, and in the NZBW strain, the process is chronic and unnoticeable. The disease develops faster and more severely in females than in males, with a mean survival of only 245 days compared to 406 days for males. Although many female mice show symptoms (proteinuria) by the age of 7-9 months, some may show symptoms at a younger or older age. The lethal immunonephritis observed in NZBW mice closely resembles that observed in human SLE, making this spontaneous murine model amenable to testing for possible SLE treatmentAgents are of great interest (Putterman and Naparstek, Murine Models of Spontaneous systemic lupus Erythematosus (Murine Models of spinal systems Erythematosus), autoimmune disease Models: guide, Chapter 14, pp 217-34, 1994; Mohan et al, J.Immunol.). 154: 1470-80, 1995; and Daikh et al, J.Immunol.). 159: 3104-08, 1997). These mice were administered soluble TACI-IG, BR43X 2-Ig, BCMA-Ig or other soluble fusion proteins to evaluate the effectiveness of TACI, BR43X2 or BCMA in ameliorating symptoms and altering the course of the disease, as described in the examples section below.

Mouse models of Experimental Allergic Encephalomyelitis (EAE) have been used as tools to study the mechanisms of immune-mediated diseases and possible therapeutic intervention approaches. This model is similar to human multiple sclerosis and produces demyelination due to T cell activation against neural proteins such as Myelin Basic Protein (MBP) or proteolipid protein (PLP). Vaccination with antigen resulted in the induction of CD4+, MHC class II restricted T cells (Th 1). Modifications to the method of generating EAE can produce acute, chronic-relapsing or passive-transmitting variant forms of this model (Weinberg et al, J Immunol 162: 1818-26, 1999; Mijaba et al, cellular immunology 186: 94-102, 1999; and Glabinski, methods enzymology 288: 182-90, 1997). These mice were administered soluble TACI-IG, BR43X 2-Ig, BCMA-Ig or other soluble fusion proteins to evaluate the effectiveness of TACI, BR43X2 or BCMA in ameliorating symptoms and altering the course of the disease, as described in the examples section below.

In the collagen-induced arthritis (CIA) model, mice develop chronic inflammatory arthritis that closely resembles human Rheumatoid Arthritis (RA). Since CIA has similar immunological and pathological features to RA, this makes this model an ideal model for screening potential human anti-inflammatory compounds. Another advantage of using this CIA model is that the pathogenesis is known. T and B cell epitopes on collagen type II have been identified and various immunological (delayed hypersensitivity and anti-collagen antibodies) and inflammatory (cytokine, chemokine, and matrix degrading enzyme) parameters associated with immune-mediated arthritis have been determined and can be used to evaluate the effectiveness of test compounds in these models (Wooley, curr. Opin. Rheum. 3: 407-20, 1999; Williams et al, immunology (Immunol.) 89: 9784-788, 1992; Myers et al, Life sciences (Life Sci.) 61: 1861-78, 1997; and Wang et al, 92: 8955-959, 1995). These mice were administered soluble TACI-IG, BR43X 2-Ig, BCMA-Ig or other soluble fusion proteins to evaluate the effectiveness of TACI, BR43X2 or BCMA in ameliorating symptoms and altering the course of the disease, as described in the examples section below.

After injection of ovalbumin into mice and re-stimulation nasally with antigens that elicit an asthmatic response in the bronchi similar to asthma, a model of bronchial infection such as asthma can be generated. These mice were administered soluble TACI-IG, BR43X 2-Ig, BCMA-Ig or other soluble fusion proteins to evaluate the effectiveness of TACI, BR43X2 or BCMA in ameliorating symptoms and altering the course of the disease, as described in the examples section below.

Another application in the in vivo model involves antigen stimulation (antigenchallenge) of the animals followed by administration of soluble BR43x 2(TACI) or its ligand ztnf4 and measurement of T and B cell responses.

T cell-dependent and T cell-independent immune responses may be measured by Perez-Melgosa et al, J immunology 163: 1123-7, 1999.

The effect on B cell response can be measured by eliciting an immune response in animals that have been administered BR43x2, TACI or BCMA polypeptides or soluble Ig-fusions following a planned antigen challenge (e.g., ovalbumin or collagen).

Pharmacokinetic studies can be used in conjunction with radiolabeled soluble BR43x2, TACI, or BCMA polypeptides or fusions to determine the distribution and half-life of these polypeptides in vivo. In addition, animal models can be used to determine the effect of soluble BR43x2, TACI or BCMA on tumors and tumor progression in vivo.

The invention also provides the use of a BR43X2, TACI or BCMA polypeptide as a surrogate marker (subvogate marker) in autoimmune diseases, renal diseases, B and T cell diseases. Blood from these patients can be drawn and BR43x2, TACI or BCMA soluble receptors and their ligands can then be detected in the blood.

The invention also provides antibodies. Products such as expression vectors containing the polypeptide of interest or polypeptides isolated from natural sources may be used as antigens to obtain peptides directed against BR43x2 or having the sequence of SEQ ID NO: 8 amino acid sequence of a peptide. And BR43x2 or a polypeptide having the sequence of SEQ ID NO: antibodies that "specifically bind" to peptides of 10 amino acid sequence are particularly useful. If the antibody binds to the BR43x2 polypeptide or SEQ ID NO: 8 has a binding affinity (Ka) of 10⁶M^-1Or higher, preferably 10⁷M^-1Or higher, more preferably 10⁸M^-1Or higher, most preferably 10⁹M^-1Or higher, the antibody is considered to be specifically binding. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art by, for example, Scatchard analysis (Scatchard, ann. ny acad. sci.51: 660, 1949). Suitable antibodies include those that bind to the extracellular domain of BR43X2, particularly BR43X2 (amino acid residues 1-120 of SEQ ID NO: 2), and those that bind to a polypeptide having the amino acid sequence of SEQ ID NO: 10.

anti-BR 43X2 antibodies can be prepared using peptides and polypeptides bearing an antigenic BR43X2 epitope. The peptides and polypeptides with antigenic epitopes of the invention comprise the amino acid sequence of SEQ ID NO: 2, preferably 15 to about 30 amino acids. However, peptides or polypeptides of any length containing a larger portion of the amino acid sequence of the invention, such as 30-50 amino acids, or up to and including the complete amino acid sequence of the polypeptide of the invention, are also useful for inducing antibodies that bind to BR43x 2. Ideally, the amino acid sequence of the epitope-bearing peptide is selected so as to provide substantial solubility in aqueous solution (i.e., the sequence includes relatively hydrophilic residues, while preferably avoiding hydrophobic residues). Hydrophilic peptides can be predicted from hydrophobicity profiles by those skilled in the art, see, for example, Hopp and Woods (Proc. Nat. Acad. Sci. USA) 78: 3824-8, 1981) and Kyte and Doolittle (journal of molecular biology 157: 105-142, 1982). Furthermore, amino acid sequences containing proline residues may also be desirable for antibody production.

Polyclonal antibodies against the recombinant BR43X2 protein or BR43X2 isolated from natural sources can be prepared using methods well known to those skilled in the art. See, for example, Green et al, "Production of Polyclonal Antisera" (Manual of Polyclonal Antisera), A guide to Immunochemical experiments (Manson eds.), pages 1-5 (HumanaPress 1992); and Williams et al, "Expression of foreign proteins and purification of specific polyclonal antibodies in escherichia coli using plasmid vectors (Expression of foreign proteins in e. coli using plasmid vectors and purification of specific polyclonal antibodies)", DNA clone 2: expression system (DNA Cloning 2: Expression Systems, 2 nd edition, Glover et al, eds., page 15 (Oxford university Press 1995). The immunogenicity of BR43x2 polypeptides may be enhanced by the use of adjuvants such as alum (aluminium hydroxide) or freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of BR43x2 or a portion thereof with an immunoglobulin polypeptide or with a maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide moiety is a "hapten-like" the moiety may advantageously be conjugated or linked to a macromolecular carrier such as keyhole limpet hemocyanin (KIH), Bovine Serum Albumin (BSA), or tetanus toxoid for immunization.

Although polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, hamsters, guinea pigs, goats, or sheep, the anti-BR 43X2 antibodies of the present invention may also be derived from non-human primate antibodies. General techniques for generating diagnostically and therapeutically useful antibodies in baboons can be found, for example, in golden berg et al, international patent application WO91/11465, and Losman et al, the journal of international cancer (int.j. cancer) 46: 310, 1990. Antibodies can also be produced in transgenic animals such as transgenic sheep, cows, goats, or pigs, and can be expressed in yeast and fungi in modified form as well as in mammalian and insect cells.

Alternatively, monoclonal antibodies against BR43X2 can be prepared. Rodent monoclonal antibodies directed against specific antigens can be obtained by methods known to those skilled in the art (see, e.g., Kohler et al, Nature 256: 495, 1975, Coligan et al (eds.), Current Protocols in Immunology, Vol.1, Vol.2.5.1-2.6.7 (John Wiley & Sons 1991); Picksley et al, "preparation of monoclonal antibodies against proteins expressed in E.coli (Production of monoclonal antibodies against proteins expressed in E.coli)," DNA clone 2: expression System, Vol.2, Glover et al (eds.), pp.93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by the following steps: injecting a composition containing a BR43x2 gene product into a mouse, verifying the presence of antibody production by removing a serum sample, removing a spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones producing antibodies to the antigen, culturing the clones producing antibodies to the antigen, and isolating the antibodies from the hybridoma culture.

Furthermore, the anti-BR 43X2 antibody of the present invention may also be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice engineered to produce specific human antibodies in response to antigen challenge. In this technique, elements of the human heavy and parental loci are introduced into strains of mice derived from embryonic stem cell lines containing directionally disrupted endogenous heavy and parental loci. The transgenic mouse is capable of synthesizing human antibodies specific for human antigens, and the mouse can be used to prepare hybridomas secreting human antibodies. Methods for obtaining human antibodies from transgenic mice are described, for example, in Green et al, nat. genet, 7: 13, 1994; lonberg et al, Nature 368: 856, 1994, and Taylor et al, int.Immun.6: 579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. These separation techniques include protein A Sepharose (Sepharose) affinity chromatography, size exclusion chromatography and ion exchange chromatography (see, for example, Coligan, pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of immunoglobulin G (IgG)", Methods of molecular biology (Methods in molecular biology, volume 10, pages 79-104 (The Humana Press, 1992)).

For particular applications, it may be desirable to prepare fragments of anti-BR 43 × 2 antibodies. These antibody fragments can be obtained, for example, by proteolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustrative example, an antibody may be cleaved by pepsin to provide a peptide represented as F (ab')₂To the 5S fragment of (a), thereby producing an antibody fragment. This fragment can be further cleaved using a thiol reducing reagent to yield a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction may be performed using a blocking group for a thiol group generated by cleavage of a disulfide bond. As an alternative, enzymatic digestion with pepsin directly produces two monovalent Fab fragments and an Fc fragment. Such methods are described, for example, in golden berg, U.S. Pat. nos. 4,331,647; nisonoff et al, Arch biochem. biophysis.89: 230, 1960; porter, biochem.j.73: 119, 1959; edelman et al, methods enzymology, Vol.1, p.422 (Academic Press 1967); and Coligan, supra.

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 recognized by the intact antibody.

For example, the Fv fragment contains V_HAnd V_LAnd (4) connecting the chains. As with Inbar et al, journal of the national academy of sciences of the united states 69: 2659, 1972, the linkage may be non-covalent. Alternatively, these variable chains may be linked by intermolecular disulfide bonds, or cross-links may be formed by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.12: 437, 1992).

The Fv fragment may comprise V joined by a peptide linker_HAnd V_LAnd (3) a chain. These single-chain antigen-binding proteins (scFv) are constructed by construction to contain encoded V linked by an oligonucleotide_HAnd V_LThe structural gene of the DNA sequence of the region. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E.coli. The recombinant host cell synthesizes a single-chain polypeptide having the two V regions connected by a linker peptide. Methods for preparing scFvs are described, for example, in Whitlow et al, methods: manual of enzymatic Methods (Methods: A company to Methods in enzymology) 2: 97, 1991, see also Bird et al, science 242: 423, 1988; ladner et al, U.S. Pat. Nos. 4,946,778; pack et al, Bio/Technology 11: 1271, 1993; and Sandhu, supra.

As an illustrative example, scfvs may be obtained by in vitro exposure of lymphocytes to BR43x2 polypeptides, and selection of antibody display libraries (e.g., by using immobilized or labeled BR43x2 proteins or peptides) in phage or similar vectors. Genes encoding polypeptides with potential BR43 × 2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or displayed on bacteria such as e.g. e. There are various methods for obtaining a nucleotide sequence encoding the polypeptide, for example by random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides that interact with known targets, which may be proteins or polypeptides such as ligands or receptors, biological or synthetic macromolecules, or organic or inorganic substances. Techniques for generating and screening such random peptide Display libraries are known in the art (Ladner et al, U.S. Pat. No. 5,223,409; Ladner et al, U.S. Pat. No. 4,946,778; Ladner et al, U.S. Pat. No. 5,403,484; Ladner et al, U.S. Pat. No. 5,571,698; and Kay et al, Phage Display of Peptides and proteins (pharmaceutical Display and proteins) (Academic Press, 1996)), and both random peptide Display libraries and kits for screening such libraries are commercially available, for example, from Clontech (Palo Alto, CA), Invitrogen (San Diego, CA), New England Biolabs (Beverly, MA), and Pharmacia LKB Biotechnology (Piscataway, NJ). Random peptide display libraries can be screened using the BR43x2 sequences disclosed herein to identify proteins that bind to BR43x 2.

Another form of antibody fragment is a peptide encoding a single Complementarity Determining Region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of an antibody of interest. These genes can be prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells by the polymerase chain reaction (see, for example, Larrick et al, methods: handbook of methods in enzymology 2: 106, 1991); Courtenay-Luck, "Genetic Manipulation of monoclonal Antibodies" (Genetic Manipulation of monoclonal Antibodies), "monoclonal Antibodies: preparation, modification and clinical applications (Monoclonal Antibodies: Production, Engineering and clinical application), Ritter et al (ed.), page 166 (Cambridge University Press 1995); and Ward et al, "Genetic Manipulation and expression of Antibodies (Genetic Manipulation and expression of Antibodies)", monoclonal Antibodies: principles and Applications (monoclonal antibodies: Principles and Applications), Birch et al (eds.), p 137 (Wiley-Liss 1995)).

Alternatively, the anti-BR 43X2 antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of a mouse immunoglobulin into human variable regions. The framework regions of the mouse counterpart were then replaced with residues typical for human antibodies. The use of antibody components derived from humanized monoclonal antibodies avoids potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable regions are described, for example, in Orlandi et al, Proc. Natl. Acad. Sci. USA 86: 3833, 1989. Techniques for making humanized monoclonal antibodies are described, for example, in Jones et al, nature 321: 522, 1986; carter et al, journal of the national academy of sciences USA 89: 4285, 1992; sandhu, crit.rev.biotech.12: 437, 1992; singer et al, journal of immunity (j.immun.) 150: 2844, 1993; sudhir (eds.), handbook of Antibody Engineering (Antibody Engineering Protocols) (Humana Press 1995); kelley, "engineered therapeutic Antibodies" (engineered therapeutic Antibodies) ", protein engineering: principles and Practice (ProteinEngineering: Principles and Practice), "Cleland et al (eds.), pages 399-434 (John Wiley & Sons, 1996); and Queen et al, U.S. Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotypic antibodies can be prepared by immunizing an animal with an antibody or antibody fragment against BR43X2 using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera", methods of molecular biology: a guide to immunochemistry experiments (Methods in Molecular Biology: Immunochemical Protocols), Manson (eds.), pages 1-12 (Humana Press 1992). See also Coligan, supra, pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypic antibodies can be prepared using an antibody or antibody fragment against BR43X2 as the immunogen and using the techniques described above. As a further alternative, humanized anti-idiotype antibodies or non-human primate anti-idiotype antibodies can be prepared using the techniques described above. Methods for making anti-idiotypic antibodies are described, for example, in Irie, U.S. patent 5,208,146; greene et al, U.S. patent 5,637,677; and Varthakavi and Minocha, journal of general virology (j.gen.virol.) 77: 1875, 1996.

The antibodies or polypeptides herein may also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and such conjugates may be used for in vivo diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the invention may be used to identify or treat tissues or organs that express the corresponding anti-complementary molecule (e.g., receptor or antigen, respectively). More specifically, a BR43x2 polypeptide or an anti-BR 43x2 antibody, or biologically active fragments or portions thereof, may be conjugated to a detectable or cytotoxic molecule and delivered to a mammal having a cell, tissue or organ that expresses the anti-complementary molecule.

Suitable detectable molecules may be linked directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like. Suitable cytotoxic molecules may be linked directly or indirectly to the polypeptide or antibody and include (e.g., directly linked to the polypeptide or antibody, or indirectly linked via a chelating moiety) bacterial or plant toxins such as diphtheria toxin, pseudomonas exotoxin, ricin, abrin, etc., as well as therapeutic radionuclides such as iodine-131, rhenium-188 or yttrium-90. The polypeptide or antibody may also be conjugated to a cytotoxic drug such as doxorubicin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule may be bound to one member of a complement/anti-complement pair, while the other member is bound to a polypeptide or antibody moiety. For these purposes, biotin/streptavidin is a typical complement/anti-complement pair.

Soluble BR43x2 polypeptides or antibodies to BR43x2 may be conjugated directly or indirectly to drugs, toxins, radionuclides, etc., and such conjugates may be used for in vivo diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the invention may be used to identify or treat tissues or organs that express the corresponding anti-complementary molecule (e.g., receptor or antigen, respectively). More specifically, a BR43x2 polypeptide or an anti-BR 43x2 antibody, or biologically active fragments or portions thereof, may be conjugated to a detectable or cytotoxic molecule and delivered to a mammal having a cell, tissue or organ that expresses the anti-complementary molecule.

Suitable detectable molecules may be linked directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like. Suitable cytotoxic molecules may be linked directly or indirectly to the polypeptide or antibody and include (e.g., directly linked to the polypeptide or antibody, or indirectly linked via a chelating moiety) bacterial or plant toxins such as diphtheria toxin, pseudomonas exotoxin, ricin, abrin, etc., as well as therapeutic radionuclides such as iodine-131, rhenium-188 or yttrium-90. The polypeptide or antibody may also be conjugated to a cytotoxic drug such as doxorubicin, an indirect linkage to a detectable molecule or cytotoxic molecule, which may be conjugated to one member of a complement/anti-complement pair, while the other member is conjugated to the polypeptide or antibody moiety. For these purposes, biotin/streptavidin is a typical complement/anti-complement pair.

These polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins can be used for targeted cell or tissue suppression or ablation (e.g., to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e., activation domain or ligand binding domain, plus a targeting domain), a fusion protein that includes only the targeting domain may be suitable for directing a detectable molecule, cytotoxic molecule, or complementing molecule to the cell or tissue type of interest. When the fusion protein having only one such domain includes a complementary molecule, the anti-complementary molecule can bind to the detectable molecule or the cytotoxic molecule. The domain-complementing molecule fusion protein thus represents a general targeting vector for cell/tissue specific delivery of general anti-complementing molecule-detectable/cytotoxic molecule conjugates. The biologically active polypeptide or antibody conjugate described herein may be delivered intravenously, intra-arterially, or intraductally, or may be introduced locally at the desired site of action.

Can be prepared against His or FLAG^TMAn antibody against a labeled soluble BR43X2 polypeptide. Antibodies against MBP fusion proteins produced by E.coli can also be prepared. Alternatively, these polypeptides may include fusion proteins with human Ig. In particular, may contain a tag for His or FLAG^TMAntiserum against labelled soluble polypeptide antibodies BR43X2 for use in therapyBR43x2 tissue distribution analysis by immunohistochemistry on human or primate tissues. These soluble BR43x2 polypeptides may also be used to immunize mice in order to generate monoclonal antibodies against soluble human BR43x2 polypeptides. Monoclonal antibodies directed against the soluble human BR43x2 polypeptide may also be used to mimic ligand/receptor coupling, thereby resulting in activation or inactivation of the ligand/receptor pair. For example, it has been demonstrated that cross-linked anti-soluble CD40 monoclonal antibodies provide a stimulatory signal to B cells that are suboptimal activated by anti-IgM or LPS, resulting in proliferation and production of immunoglobulins. These same monoclonal antibodies can act as antagonists to block activation of the receptor when used in solution. Monoclonal antibodies to BR43X2 can be used to determine the distribution, regulation and biological interaction of this BR43X 2/BR 43X 2-ligand pair on specific cell lineages identified by tissue distribution studies.

The invention also provides isolated and purified BR43x2, TACI, and BCMA polynucleotide probes or primers. These polynucleotide probes may be RNA or DNA. The DNA may be cDNA or genomic DNA. A polynucleotide probe is a single-or double-stranded DNA or RNA, typically a synthetic oligonucleotide, but can also be generated from a cloned cDNA or genomic sequence, and will typically contain at least 16 nucleotides, more typically 17 nucleotides to 25 or more nucleotides, sometimes 40-60 nucleotides, and in some cases a substantial portion, region or even the entire BR43x2 gene or cDNA. Probes and primers are typically synthetic oligonucleotides, but may also be generated from cloned cDNA or genomic sequences or their complements. Although slightly shorter probes (14-17 nucleotides) can be used, the analytical probes are typically at least 20 nucleotides in length. The PCR primers are at least 5 nucleotides long, preferably 15 or more nt, more preferably 20-30 nt. When a small region of a gene is the target of the assay, short polynucleotides may be used. For global analysis of a gene, a polynucleotide probe may contain a complete exon or more. Probes can be labeled to provide a detectable signal using techniques well known in the art, such as enzymes, biotin, radionuclides, fluorophores, chemiluminescent, paramagnetic particles, and the like, commercially available from many sources, such as Molecular Probes (Eugene, OR) and Amersham (Arlington Heights, IL). Preferred regions for constructing probes include ligand binding regions, cysteine-rich pseudo-repeats, signal sequences, and the like. Techniques for preparing polynucleotide probes and hybridization techniques are known in the art, see, e.g., Ausubel et al, Current protocols Molecular Biology, John Wiley and Sons, NY, 1991.

BR43x2, TACI and BCMA polypeptides and antibodies can be used in diagnostic systems to detect the presence of BR43x2, TACI and BCMA and ligand polypeptides of BR43x2, TACI and BCMA, such as ztnf 4. Information derived from these detection methods will provide an understanding of the significance of BR43x2 polypeptides in a variety of diseases and will be useful as diagnostic tools in diseases where changes in BR43x2 levels are significant. Alterations in levels of BR43x2, TACI, and BCMA receptor polypeptides may be indicative of pathological conditions including cancer, autoimmune diseases, and infectious diseases.

In a basic assay, single stranded probe molecules are incubated with RNA isolated from a biological sample under conditions of temperature and ionic strength that promote base pairing between the probe and the target BR43X2, TACI or BCMA RNA species. After separation of unbound probe from the hybridized molecules, the amount of hybrid molecules is detected.

Mature RNA detection hybridization Methods include Northern Analysis and dot/slot blot hybridization (see, e.g., Ausubel, supra; and Wu et al (eds.), "Analysis of Gene Expression at the RNA Level (Analysis of Gene Expression at the RNA Level)," Methods of genetic Biotechnology (Methods in Gene Biotechnology), pp.225-239 (CRC Press, 1997)). Can be used with radioactive isotopes, for example³²P or³⁵S detectably labeling the nucleic acid probe. As an alternative, BR43X 2RNA can be detected by Nonradioactive hybridization methods (see, for example, Isaac (eds.), Experimental procedures for analyzing nucleic acids with Nonradioactive Probes (Protocols for nucleic Acid Analysis by Nonradioactive Probes), HumanaPress Inc., 1993). Typically, non-radioactive detection is achieved by enzymatic conversion of a chromogenic or chemical compound luminescent substrate. Illustrative non-radioactive moieties include biotin, fluorescein, and digoxigenin.

The BR43X2, TACI and BCMA oligonucleotide probes are also useful for in vivo diagnosis. As an illustration, administration to a subject may be¹⁸F-labeled oligonucleotides and visualized by positron emission tomography (Tavitaan et al, Nature Medicine 4: 467, 1998).

Many diagnostic procedures utilize Polymerase Chain Reaction (PCR) to increase the sensitivity of the detection method. Standard techniques for performing PCR are well known (see generally Mathew (eds.), Human Molecular Genetics laboratory Manual (Protocols in Human Molecular Genetics) (HumanaPress, 1991), White (eds.), PCR procedures: Current Methods and Applications (PCR Protocols: Current Methods and Applications) (Humana Press, 1993), Cotter (eds.), Molecular diagnostics of Cancer (Molecular diagnostics of Cancer) (Humana Press, 1996), Hanausek and Walszek (eds.), Tumor markers handbook (Tumor Mark Protocols) (Humana Press, 1998), Lo (eds.), Clinical Applications of PCR (Clinical Applications of PCR) (Humana presses, 1998), and Meltr (eds.), PCR in biological analysis (PCR) (Humana presses, 1998)). PCR primers can be designed to amplify sequences encoding cysteine-rich pseudo-repeats of specific BR43x2 domains or motifs, such as BR43x2, TACI or BCMA.

One PCR variant used in diagnostic assays is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is isolated from a biological sample, reverse-transcribed into cDNA, and the cDNA is incubated with a primer of BR43X2 (see, for example, Wu et al (ed); "Rapid isolation of Specific cDNAs or Genes by PCR", methods of Gene Biotechnology, CRC Press, pp.15-28, 1997). PCR was then performed and the products analyzed using standard techniques.

By way of illustration, RNA is isolated from a biological sample using, for example, the guanidinium thiocyanate cell lysis procedure described above. Alternatively, solid phase techniques can be used to isolate mRNA from cell lysates. Reverse transcription reactions can be initiated using random oligonucleotides, short dT homopolymers, or antisense oligomers of BR43X2, TACI or BCMA, and the isolated RNA. Oligo dT primers have the advantage of amplifying various mRNA nucleotide sequences that can provide control target sequences. The BR43x2, TACI or BCMA sequences were amplified by polymerase chain reaction using two flanking oligonucleotide primers, typically at least 5 bases long.

The PCR amplification product can be detected by various methods. For example, PCR products can be fractionated by gel electrophoresis and visualized by ethidium bromide staining. Alternatively, the fractionated PCR products can be transferred to a membrane, hybridized with a detectably labeled BR43 × 2 probe, and examined by autoradiography. Other alternatives include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescent detection, and the use of C-TRAK colorimetric assays.

Another method is real-time quantitative PCR (Perkin-Elmer Cetus, Norwalk, Ct.). A fluorescent probe consisting of an oligonucleotide and a linked reporter and quencher dye is specifically annealed between the forward and reverse primers. Using the 5' endonuclease activity of Taq DNA polymerase, the quencher dye and reporter dye are separated, and a sequence-specific signal is generated and increased as amplification increases. The fluorescence intensity can be continuously monitored and quantified during the PCR reaction.

Another method for detecting the expression of BR43X2, TACI or BCMA is the Cycling Probe Technology (CPT), in which a single-stranded DNA target is combined with an excess of DNA-RNA-DNA chimeric probes to form a complex, the RNA portion is cleaved with RNase H, and the presence of the cleaved chimeric probes is detected (see, e.g., Beggs et al, J.Clin.Microbiol.) 34: 2985, 1996; and Bekkaoii et al, Biotechnology (Biotechnology) 20: 240, 1996). Other methods for detecting BR43X2, TACI or BCMA sequences can utilize nucleic acid sequence-based amplification (NASBA), template-assisted amplification by cross-hybridization (CATCH) and Ligase Chain Reaction (LCR) among others (see, e.g., Marshall et al, U.S. Pat. No. 5,686,272 (1997); Dyer et al, J.Virol. methods) 60: 161, 1996; Ehricht et al, European journal of biochemistry (Eur. J.biochem.) 243: 358, 1997; and Chadwick et al, J.Virol methods 70: 59, 1998). Other standard methods are known to those skilled in the art.

Probes and primers for BR43x2, TACI, and BCMA can also be used to detect and localize gene expression of BR43x2, TACI, or BCMA in tissue samples. Methods for such In Situ Hybridization are well known to those skilled In the art (see, for example, Choo (eds.), In Situ Hybridization Protocols, Humana Press, 1994; Wu et al (eds), "Analysis of Abundance of cellular DNA or mRNA by Radioactive In Situ Hybridization (RISH)", Methods for genetic biotechnology (Methods In Biotechnology), CRC Press, pp 259-278, 1997; Wu et al (eds), "Localization of DNA Absolution of mRNA by fluorescent In Situ Hybridization" (location of Abundance of mRNA), Methods for genetic biotechnology (RISH) ", Methods for genetic biotechnology, Press, pp 289-279, 1997).

A variety of other diagnostic methods are well known to those skilled in the art (see, e.g., Mathew (eds.), Human Molecular genetics experimental procedures (Protocols in Human Molecular genetics), Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics (Molecular Diagnostics), Humana Press, Inc., 1996; and Elles, Molecular Diagnostics of Genetic Diseases (Molecular Diagnostics of Genetic Diseases), Humana Press, Inc., 1996).

In addition, these polynucleotide probes can also be used to hybridize to corresponding sequences on each chromosome. Chromosomal identification and/or mapping of the BR43x2 gene may provide useful information about the relationship of gene function to disease. Many mapping techniques are available to those skilled in the art, such as somatic cell hybrids and Fluorescence In Situ Hybridization (FISH) mapping. One preferred method is radiation hybrid mapping. Radiation hybrid mapping is a somatic genetics technique developed for constructing high-resolution continuous maps of mammalian chromosomes (Cox et al, science 250: 245-50, 1990). Partial or complete genetic sequence information allows the design of PCR primers suitable for use with the chromosomal radiation hybrid mapping line (panels). Commercially available series of mapping of radiation hybrids covering the entire human genome are available, such as the Stanford G3RH series and the GeneBridge4RH series (Research Genetics, Huntsville, AL). These sets enable rapid PCR-based chromosomal location and ordering of genes, sequence marker sites (STSs) and other non-polymorphic and polymorphic markers in a region of interest. This involves establishing a directly proportional physical distance between the newly discovered gene of interest and the previously mapped markers. Accurate information of gene location can be applied to a number of aspects including: 1) determining whether a sequence is part of an existing contig and obtaining other surrounding genetic sequences in various forms such as YAC-, BAC-or cDNA clones, 2) providing potential candidate genes for showing heritable diseases linked to the same chromosomal region, and 3) for cross-referencing model organisms such as mice, may be advantageous to help determine the functions that a particular gene may have.

Chromosomal mapping can also be achieved using STSs. STS is a unique DNA sequence in the human genome that can be used as a reference point for a particular chromosome or chromosomal region. STS can be determined by oligonucleotide primer pairs that can be used in polymerase chain reaction to specifically detect the site in the presence of all other genomic sequences. Since STS is based only on DNA sequence, the DNA sequence of the DNA sequence can be determined from databases such as the sequence marker site database (dbSTS), GenBank (National Center for Biological Information, National institutes Health, Bethesda, Md.,http://www.ncbi.nlm.nih.gov) They can be fully described and they can be searched with the gene sequence of interest to obtain the short bases contained in themMapping data in the genome tag STS sequence.

The invention also provides reagents for other diagnostic applications. For example, the BR43 × 2 gene, a probe containing BR43 × 2DNA or RNA, or a subsequence thereof may be used to determine whether the BR43 × 2 gene is present on a particular chromosome or whether a mutation has occurred. Detectable chromosomal aberrations at the BR43x2 locus include, but are not limited to, aneuploidy, changes in gene copy number, insertions, deletions, changes in restriction sites, and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences including upstream promoter and regulatory regions, and can manifest themselves as physical changes within the coding sequence or changes in the level of gene expression.

Generally, these diagnostic methods comprise the steps of: (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above under conditions in which the polynucleotide will hybridize to a complementary polynucleotide sequence to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples used in the present invention include genomic DNA, cDNA and RNA. The polynucleotide probe or primer may be RNA or DNA, and will contain SEQ ID NO: 3, SEQ ID NO: 1, or an RNA equivalent thereof. Suitable analytical Methods in this regard include molecular genetic techniques known to those skilled in the art, such as Restriction Fragment Length Polymorphism (RFLP) analysis, Short Tandem Repeat (STR) analysis using PCR techniques or ligation chain reaction (Barany, PCR Methods and applications 1: 5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al, supra; Ausubel et al, supra; Marian, Chest 108: 255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al, supra, Chapter 4) involve hybridization of an RNA probe to an RNA sample from a patient, followed by exposure of the reaction product (RNA-RNA hybrid molecule) to RNase. The hybridizing region of the RNA is protected from digestion. In a PCR assay, a genetic sample from a patient is incubated with a pair of polynucleotide primers, and the region between the two primers is then amplified and recovered. Changes in size and quantity of the recovered product indicate the presence of a mutation in the patient. Another PCR-based technique that may be employed is single-strand conformation polymorphism (SSCP) analysis (Hayashi, PCR methods and uses, 1: 34-8, 1991).

Antisense methodologies can be employed to inhibit gene transcription of BR43x2, TACI, or BCMA in order to inhibit B cell development and interaction with other cells. Polynucleotides complementary to fragments of BR43X2, TACI or BCMA encoding polynucleotides (e.g., the polynucleotides set forth in SEQ ID NO: 3) are designed to bind to and inhibit translation of mRNA encoding BR43X2, TACI or BCMA. These antisense polynucleotides may be used to inhibit the expression of genes encoding BR43x2, TACI, or BCMA in cell culture or in a subject.

Mice engineered to express BR43X2, TACI or BCMA, referred to as "transgenic mice," and mice exhibiting complete loss of BR43X2, TACI or BCMA function, referred to as "knockout mice," can also be prepared (Snouwalert et al, science 257: 1083, 1992; Lowell et al, Nature 366: 740-42, 1993; Capecchi, science 244: 1288-92, 1989; Palmiter et al, Annu Rev Genet.20: 465-99, 1986). For example, transgenic mice that overexpress BR43x2, TACI, or BCMA either extensively or only under tissue-specific or tissue-limiting promoters can be used to determine whether overexpression will result in a phenotype. For example, overexpression of a wild-type BR43x2, TACI or BCMA polypeptide, polypeptide fragment, or mutant thereof may alter normal cellular processes, resulting in a phenotype that can identify tissues functionally associated with expression of BR43x2, TACI or BCMA, and may indicate a therapeutic target for BR43x2, TACI or BCMA, or agonists or antagonists thereof. For example, a preferred transgenic mouse that is engineered is a transgenic mouse that overexpresses soluble BR43x2, TACI, or BCMA. Moreover, this overexpression may result in phenotypes that display human diseases. Similarly, mice that knock out BR43x2, TACI, or BCMA can be used to determine sites where BR43x2 is absolutely required in vivo. The phenotype of the knockout mice predicts the possible in vivo effects of BR43x2, TACI or BCMA antagonists (e.g. as described herein). The cDNA of human BR43x2, TACI or BCMA can be used to isolate mRNA, cDNA and genomic DNA of mouse BR43x2, TACI or BCMA, which are subsequently used to make knockout mice. These mice can be used to study BR43 × 2, TACI or BCMA genes and their encoded proteins in vivo systems and can be used as in vivo models of the corresponding human diseases. Moreover, transgenic expression of BR43x2, TACI or BCMA antisense polynucleotides or ribozymes directed to BR43x2, TACI or BCMA as described herein can be similarly used in the transgenic mice described above.

A pharmaceutically effective amount of a BR43x2, TACI, or BCMA polypeptide of the present invention may be formulated for parenteral, oral, nasal, rectal, topical, or transdermal administration, along with a pharmaceutically acceptable carrier, according to conventional methods. The formulations may also include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in the form of, for example, liquids, powders, emulsions, suppositories, liposomes, transdermal patches (transdermal patches), tablets, and the like. Slow or extended release systems, including any of a variety of biopolymers (biological-based systems), systems employing liposomes, and polymer delivery systems, may also be used with the compositions described herein to provide a continuous or long-term source of BR43x2 polypeptide or antagonist. These sustained release systems can be used, for example, in oral, topical and parenteral formulations. The term "pharmaceutically acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host or patient. Those skilled in the art can use the appropriate methods and techniques described in, for example, Remington: the compounds of The invention are formulated according to accepted experience in pharmaceutical Science and practice (Remington: The Science and practice of Parmecy) (Gennaro eds., Mack Publishing Co., Easton PA, 19 th edition, 1995).

As used herein, a "pharmaceutically effective amount" of a BR43x2, TACI, or BCMA polypeptide, agonist, or antagonist is an amount sufficient to induce a desired biological result. The result may be a reduction in the signs, symptoms, or causes of the disease, or any other desired change in the biological system. For example, an effective amount of a BR43x2, TACI, or BCMA polypeptide will provide subjective relief of symptoms or an objectively identifiable improvement as indicated by a clinician or other qualified observer. For example, such an effective amount of BR43x2, TACI, or BCMA polypeptide or soluble fusion will provide a reduction in B cell response, inhibition or reduction of autoantibody production, inhibition or alleviation of symptoms associated with SLE, MG, or RA during an immune response. An effective amount of BR43x2, TACI, or BCMA will reduce the percentage of B cells in peripheral blood. The effective amount of the BR43x2, TACI, or BCMA polypeptide can vary widely depending on the disease or condition to be treated. The amount of polypeptide to be administered and the concentration in the formulation will depend on the carrier selected, the route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and stability of the compound in the formulation. Thus, the clinician will employ the appropriate formulation at the appropriate concentration in the formulation and the amount of formulation administered, depending on the clinical experience with the patient being treated or with similar patients. These amounts will depend in part on the particular disease being treated, the age, weight, and general health of the patient, as well as other factors apparent to those skilled in the art. Typically a dose will be in the range of 0.1-100mg/kg of subject. The dosage for a particular compound can be determined from in vitro or ex vivo studies as well as experimental animal studies. Finding effective concentrations of compounds in vitro or ex vivo provides guidance for animal studies in which dosages are calculated to provide similar concentrations at the site of action.

The invention is further illustrated by the following non-limiting examples.

Examples

Example 1

Identification of BR43X2

The TACI was cloned from an RPMI array library (array library) using the secretion trapping method. The RPMI1788 (activated B cell line) library was arrayed using 20 96-well plates. Each well contained approximately 100 E.coli colonies, each containing one cDNA clone. DNA was miniprepped using TomTech Quadra 9600 in 96-well plates. The isolated DNA was then pooled into 120 pools (pool), each representing 1600 clones. Cos-7 cells were transfected with these pools and seeded in 12-well plates. Mu.l of pool DNA and 5. mu.l LipofectAMINE were mixed in 92. mu.l serum-free DMEM medium (55 mg sodium pyruvate, 146mg L-glutamine, 5mg transferrin, 2.5mg insulin, 1. mu.g selenium and 5mg fetuin in 500ml DMEM), incubated at room temperature for 30 minutes, after which 400. mu.l serum-free DMEM medium was added. The DNA-LipofectAMINE mixture was added to 220,000 Cos-7 cells/well seeded in 12-well tissue culture plates and incubated at 37 ℃ for 5 hours. After incubation, 500 μ l of 20% FBS DMEM medium (100 ml FBS, 55mg sodium pyruvate, 146mg L-glutamine in 500ml DMEM) was added to each well and the cells were incubated overnight.

This secretion trap screen was performed using biotinylated FLAG marker ztnf 4. The cells were washed with PBS and fixed with 1.8% formaldehyde in PBS for 15 minutes. Then using TNT (H)₂0.1M Tris-HCl in O, 0.15M NaCl, and 0.05% Tween-20). Cells were permeabilized with 0.1% Triton-X in PBS for 15 minutes, followed by one wash in T NT. With TNB (0.1M Tris-HCl, 0.15M NaCl, and 0.5% blocking reagent) and NENTSA-Direct kit (NEN, Boston, MA), cells were blocked for 1 hour according to the manufacturer's instructions. Cells were washed with TNT and blocked with avidin for 15 minutes, then with biotin (Vector Labs product # SP-2001) and washed with TNT in between. Cells were incubated with 1. mu.g/ml ztnf 4/Flag/biotin in TNB for 1 hour, followed by one wash with TNT. Cells were then incubated with a 1:300 streptavidin-HRP (NEN) dilution in TNB for 1 hour and washed with TNT. Hybridization was detected with fluorescein tyramine reagent diluted 1:50 in dilution buffer (NEN) and incubated for 4.4 minutes, followed by a TNT wash. By usingCells were preserved in TNT at 1:5 dilution with Vectashield Mounting Media (Vector Labs, Burlingame, Calif.).

These cells were observed by fluorescence microscopy using a FITC filter. There were 12 pools positive for ztnf4 binding. The D8 pool (representing 1600 clones) was broken down and a single ztnf4 binding positive clone (D8-1) was isolated. Sequencing analysis revealed that the D8-1 clone contained a polypeptide sequence encoding the TACI isoform in which the first cysteine-rich pseudo-repeat of TACI, Phe21-Arg67, was replaced with only one amino acid residue, tryptophan. This isoform is designated BR43 × 2, the polynucleotide sequence of which is shown in SEQ ID NO: 1 in (c).

Example 2

Detection of BR43X 1 in lymphocytes and monocytes

Reverse transcriptase PCR was used to detect expression of BR43x 1 in T and B cells and monocytes. Screening for CD19 using oligonucleotide primers ZC19980(SEQ ID NO: 15) and ZC19981(SEQ ID NO: 16)⁺、CD3⁺And cDNA of monocytes to identify BR 43. The reverse transcriptase reaction is carried out under the conditions: 3 minutes at 94 ℃; followed by 30 cycles of 94 ℃ for 30 seconds, 68 ℃ for 2 minutes and 72 ℃ for 1 minute; after 72 ℃ extension for 7 minutes. A band with the expected size of 720bp was detected only in B cells, but not in activated T cells, consistent with the report of an assay for TACI using antibodies (von Bulow and Bram, supra).

Example 3

B cell proliferation assay with BR43 ligand ztnf4

Will contain 1X 10⁸The vials of harvested frozen Peripheral Blood Mononuclear Cells (PBMCs) were rapidly thawed in a 37 ℃ water bath and resuspended in 25ml of B cell culture medium (Iscove's modified Dulbecco's medium, 10% heat inactivated fetal bovine serum, 5% L-glutamine, 5% Pen/Strep) in a 50ml tube. Using a trypanBlue (GIBCO BRL, Gaithersburg, MD) tests the viability of the cells. Mu.l of Ficoll/Hypaque Plu was plated under cell suspension (pharmaciLKB Biotechnology, Piscataway, NJ), centrifuged at 1800rpm for 30 minutes and stopped with brake off. The interface layer was then removed and transferred to a new 50ml tube, made up to a final volume of 40ml with PBS and centrifuged at 1200rpm for 10 minutes with the brakes turned on. Viability of the isolated B cells was tested using trypan blue. The B cells were resuspended in B cell medium to a final concentration of 1X 10⁶Cells/ml and seeded at 180. mu.l/well in 96-well U-bottom plates (Falcon, VWR, Seattle, WA).

To these cells one of the following stimuli was added to reach a final volume of 200 ml/well:

soluble FLAG markers ztnf4-4sCF or ztnf4-4sNF in a 10-fold diluted form of 1mg to 1ng/ml alone or in addition diluted in NaH₂CO₃(pH9.5) 10. mu.g/ml anti-IgM (goat anti-human IgM) (Southern Biotechnology Associates, Birmingham, AL); or with 10. mu.g/ml anti-IgM and 10ng/ml recombinant human IL4 (diluted in PBS and 0.1% BSA). In addition, other cytokines such as IL-3 and IL-6, as well as soluble CD40(sCD40) antibody (Pharmingen, San Diego, Calif.) were also tested. As a control, cells were incubated with 0.1% Bovine Serum Albumin (BSA) and PBS, 10. mu.g/ml anti-IgM or 10. mu.g/ml anti-IgM and 10ng/ml IL4 (or other cytokines). These cells were then incubated for 72 cells at 37 ℃ in an incubator maintained at humidity. 16 hours before harvest, add 1. mu. Ci to all wells³H thymidine. These cells were collected in a 96-well filter plate (UniFilter GF/C, Packard, Meriden, CT) on which the cells were harvested using a cell harvester (Packard) and collected according to the manufacturer's instructions. The plates were dried at 55 ℃ for 20-30 minutes and the wells were bottom sealed with an opaque plate seal. 0.25ml of scintillation fluid (Microscint-O, Packard) was added to each well and the plate read using a high count microplate scintillation Counter (TopCount microplate scintillation Counter) (Packard).

To measure the induction of mitogenic IgG production in response to various B cells following stimulation of purified B cells, cells were prepared as described and incubated for 9 days. Cell supernatants were collected to determine IgG production.

To measure activation in response to various B cell mitogen cell surface markers following stimulation of purified B cells, cells were prepared as described above and incubated for only 48 hours. Cell surface markers were measured by FACS analysis.

Table 5 summarizes the proliferation of human purified B cells stimulated with various B cells mitogens:

TABLE 5

Irritant substance Coefficient of proliferation

ztnf4 1.5

ztnf4+IL4 9.9

ztnf4+ anti-IgM + IL 415.8

Synergistic effects of ztnf4 with IL4, IL3 (10. mu.g/ml) and IL6 (10. mu.g/ml) on B cell proliferation were observed. A two-fold increase in B cell signaling was observed when sCD40 was used.

Table 6 summarizes the induction of mitogenic IgG production (ng/ml) in response to various B cells following stimulation of purified B cells.

TABLE 6

Irritant substance Control ztnf4

anti-IgM 37.5

Anti IgM + IL-41332

Anti IgM + IL-4+ IL-51045

An increase in cell surface activation labeling was observed after stimulation of purified B cells with ztnf4 alone, or with anti-IgM or anti-IgM + IL-4. There was no effect on the proliferation of PBMNCs in the presence of the best or suboptimal T cell mitogens. Moreover, no effect on TNF α production was observed in purified monocytes in response to LPS stimulation.

Figure 3 shows the co-stimulation of human B lymphocytes by soluble ztnf4 to allow their proliferation and secretion of immunoglobulins. FIG. 3A shows the proliferation of purified human peripheral blood B cells after 5 days in culture in response to stimulation with soluble ztnf4(25ng/ml) in the presence of IL-4 alone, and in the presence of IL-4 and anti-IgM, anti-CD 40, or anti-CD 19. FIG. 3B shows IgM and IgG levels measured in supernatants obtained from human B cells stimulated with soluble ztnf4 in the presence of IL-4 or IL-4+ IL-5 after 9 days of culture.

These results suggest that soluble ztnf4 is a B cell activating molecule that acts synergistically with other B cell stimulators, but weak alone. Soluble ztnf4 promotes B cell proliferation and Ig production. Upregulation of adhesion molecules, co-stimulatory molecules and activation receptors suggests a role in promoting APC function in B cells.

FIG. 4 shows the stimulation of human peripheral blood B cells in vitro within 5 days of soluble ztnf4(25ng/ml) or control protein (ubiquitin) in the presence of 10ng/ml IL-4. Purified TACI-Ig, BCMA-Ig or control Fc was tested for inhibition of soluble ztnf 4-specific proliferation.

Example 4

Selection of TACI and BCMA transformed BHK cells Using ztnf4 in combination

BHK cells expressing high levels of TACI protein were selected by dilution cloning of the transfectant pool. In binding buffer (PBS, 2% BSA, 0.02% NaN)₃) In (1) willg/ml biotinylated ztnf4 and transfectant cells (2X 10)⁵) Incubate together on ice for 30 minutes. Cells were washed 2 times with binding buffer and then incubated with SA-PE (Caltag) (1:1000 dilution in binding buffer) on ice for 30 minutes. The cells were then washed 2 times in binding buffer and resuspended in binding buffer before being read by FACS (FACS Vantage, Becton Dickinson). Clones with the highest binding to TNF4 were selected.

BHK cells expressing high levels of BCMA protein were selected by surface labeling of BCMA expressing transfectant pools with biotinylated ztnf 4. Bright cells in FL2 were then aseptically sorted by streptavidin-phycoerythrin (SA-PE Caltag Burlingame, CA) on a FACS Vantage (becton dickinson). These single colonies were then screened for ztnf4 binding.

Example 5

Tissue distribution

Human multi-tissue Northern blots (MTN I, MTN II and MTN III; Clontech) were probed to determine the tissue distribution of human BR43X2 and TACI expression. A PCR-derived probe of about 500bp (SEQ ID NO: 21) was amplified using BR43X 2(SEQ ID NO: 1) as a template and the oligonucleotides ZC20061(SEQ ID NO: 22) and ZC20062(SEQ ID NO: 23) as primers. This sequence is identical to the homologous region of TACI. The amplification was performed as follows: 1.0 minute at 94 ℃ for 1 cycle; 30 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 30 seconds; after 10 minutes at 72 ℃ for 1 cycle. The PCR product was visualized by agarose gel electrophoresis and the 500bp PCR product was purified using a gel extraction kit (Qiagen, Chatsworth, Calif.) according to the manufacturer's instructions. The probes were radiolabeled using the MULTIPRIMEDNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The probe was purified using a NUCTRAP push column (push column) (Stratagene). Prehybridization was performed using EXPRESSHYB (Clontech) solution and used as hybridization solution for Northern blotting. Hybridization uses 10⁶cpm/ml labeled probe was performed overnight at 65 ℃. Then in 2 x SSC and 0.1% SDS middle chamberThese blots were washed warm, followed by two washes in 0.1 XSSC and 0.1% SDS at 50 ℃. Transcripts of approximately 1.5kb were detected in the spleen, lymph nodes and small intestine.

Human multi-tissue Northern blots (MTN I, MTN II and MTN III; Clontech) were probed to determine the tissue distribution of human BCMA expression. An approximately 257bp probe derived from PCR (SEQ ID NO: 24) was amplified using cDNA from Daudi cells as a template and the oligonucleotides ZC21065(SEQ ID NO: 25) and ZC21067(SEQ ID NO: 26) as primers. The amplification was performed as follows: 1.0 minute at 94 ℃ for 1 cycle; 30 seconds at 94 ℃,30 seconds at 60 ℃ and 30 seconds at 72 ℃, for 35 cycles; after 10 minutes at 72 ℃ for 1 cycle. The PCR product was visualized by agarose gel electrophoresis and the 257bp PCR product was purified using a gel extraction kit (Qiagen, Chatsworth, Calif.) according to the manufacturer's instructions. The probes were radiolabeled using the MULTIPRIMEDNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The probe was purified using a NUCTRAP push column (Stratagene). Prehybridization was performed using EXPRESSHYB (Clontech) solution and used as hybridization solution for Northern blotting. Hybridization uses 10⁶cpm/ml labeled probe was performed overnight at 65 ℃. These blots were then washed in 2 XSSC and 0.1% SDS at room temperature followed by two washes in 0.1 XSSC and 0.1% SDS at 50 ℃. An approximate 1.2kb transcript was detected in stomach, small intestine, lymph node, trachea, spleen and testis.

RNA Master dotbats (Clontech) containing RNA from various tissues, which was tagged with 8 housekeeping genes, were also probed by hybridization as described above using either the TACI probe (SEQ ID NO: 21) or the BCMA probe (SEQ ID NO: 24). Expression of BR43X 2/TACI was observed in the spleen, lymph nodes, small intestine, stomach, salivary glands, appendix, lung, bone marrow and fetal spleen. BCMA expression was detected in the small intestine, spleen, stomach, colon, lymph nodes and appendix.

Human tumor series blots V (Invitrogen, San Diego, Calif.) and human lymphoma blots (Invitrogen) were probed with either a Br43X 2/TACI probe (SEQ ID NO: 21) or a BCMA probe (SEQ ID NO: 24) as described above. A1.5 kb transcript corresponding to TACI was found in non-Hodgkin's lymphoma and parotid tumors. A1.2 kb transcript corresponding to BCMA was found in glandular, non-Hodgkin's, and parotid tumors.

Total RNA WAs prepared from CD4+, CD8+, CD19+ and mixed lymphocyte reactive cells (CellPro, Bothell, WA) using guanidinium isothiocyanate (Chirgwin et al, Biochemistry 18: 52-94, 1979) followed by a CsCl centrifugation step. Poly (A) + RNA was isolated by chromatography on oligo d (T) cellulose (Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Northern blot analysis was then performed as follows.

Approximately 2mg of each poly A + RNA in 2.2M Formaldehyde/phosphate buffer (50mM Na)₂HPO₄，50mM NaH₂PO4, 50mM NaOAc, 1mM EDTA and 2.2M formaldehyde) and separated by electrophoresis on 1.5% agarose mini-gels (Stratagene Cloning Systems, La Jolla, Calif.) in formaldehyde/phosphate buffer. The RNA was blotted onto nytran filters (Schleicher) overnight&Schuell, Keene, NH) and inThe filters were UV cross-linked (1,200m joints) in a UV cross-linker (Stratagene cloning Systems) and then baked at 80 ℃ for one hour.

These blots were probed with either a TACI probe (SEQ ID NO: 21) or a BCMA probe (SEQ ID NO: 24). In CD19 only⁺A1.5 kb band representing TACI was detected in the cells. In CD8⁺、CD19⁺And a 1.2kb transcript representing BCMA was weakly detected in MLR cells.

Blots were prepared with poly (A) RNAs from K-562 cells (erythroid, ATCC CCL243), HUT78 cells (T cells, ATCC TIB-161), Jurkat cells (T cells), DAUDI (Burkitt's human lymphoma, Clontech, Palo Alto, Calif.), RAJI (Burkitt's human lymphoma, Clontech), and HL60 (monocytes), and Northern blot analysis was performed on these blots as described above. These blots were probed with either a TACI probe (SEQ ID NO: 21) or a BCMA probe (SEQ ID NO: 24). A1.5 kb transcript corresponding to TACI was detected in Raji cells. A1.2 kb transcript corresponding to BCMA was detected in Daudi, Raji and Hut78 cells.

Tissues expressing human or murine TACI and human BCMA were identified using PCR-based screening. Screening of human and murine Rapid-Scan according to manufacturer's instructions^TMGene expression series (OriGene technologies, Rockville, Md.). Oligonucleotide primers ZC24200(SEQ ID NO: 27) and ZC24201(SEQ ID NO: 28) were designed to span one exon junction and generate a 272bp fragment corresponding to murine TACI. Expression is detected in the spleen, thymus, lung, breast, heart, muscle, skin, adrenal gland, stomach, small intestine, brain, ovary, prostate and embryo. Other bands of-500 and 800bp were detected in many tissues.

Oligonucleotide primers ZC24198(SEQ ID NO: 29) and ZC24199(SEQ ID NO: 30) were designed to span one exon junction and generate a 204bp fragment corresponding to human TACI. Expression was detected in spleen, brain, heart, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, adrenal gland, pancreas, prostate, peripheral blood lymphocytes and bone marrow.

Oligonucleotide primers ZC24271(SEQ ID NO: 31) and ZC24272(SEQ ID NO: 32) were designed to span one exon junction and generate a 329bp fragment corresponding to human BCMA. Expression was detected in brain, spleen, colon, lung, small intestine, stomach, ovary, testis, salivary gland, adrenal gland, pancreas, prostate, peripheral blood lymphocytes, bone marrow and fetal liver.

Oligonucleotide primers ZC24495(SEQ ID NO: 33) and ZC24496(SEQ ID NO: 34) were designed to span one exon junction, generating a 436bp fragment corresponding to murine BCMA. Expression was detected in the liver.

Example 6

Preparation of TACI-Ig and BCMA-Ig fusion vectors

Construction of Ig Gamma 1Fc4 fragment

To prepare TACI-Ig fusion proteins, the Fc region (hinge region and CH2 and CH3 regions) of human IgG1 were modified to remove the receptor (FcgRI) and complement (C1q) binding functions of Fc. This modified version of human IgG1Fc is referred to as Fc 4.

The Fc region was isolated from a human fetal liver library (Clontech) by PCR using oligomer primers ZC10, 134(SEQ ID NO: 43) and ZC10, 135(SEQ ID NO: 44). PCR was used to introduce mutations in this Fc region to reduce FcgRI binding. The FcgRI binding site (Leu-Leu-Gly-Gly) was mutated to Ala-Glu-Gly-Ala (amino acid residues 38-41 of SEQ ID NO: 45) to reduce FcR1 binding according to Baum et al (EMBO J.13: 3992-4001, 1994) (Duncan et al, Nature 332: 563-4, 1988). The mutations were introduced using oligonucleotide primers ZC15, 345(SEQ ID NO: 46) and ZC15, 347(SEQ ID NO: 47). In a final volume of 50. mu.l, 570ng of IgFc template, 5. mu.l of 10 XPfu reaction buffer (Stratagene), 8. mu.l of 1.25mM dNTPs, 31. mu.l of dH were added₂O, 2. mu.l of 20mM ZC15, 345(SEQ ID NO: 46) and ZC15, 347(SEQ ID NO: 47). An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 1 minute. Pfu polymerase (2.5 units, Stratagene) was added followed by 25 cycles of: 30 seconds at 94 ℃,30 seconds at 55 ℃ and 1 minute at 72 ℃ followed by 7 minutes of extension at 72 ℃. The reaction product was electrophoresed, and a band corresponding to an expected size of about 676bp was detected. The band was cut from the gel and washed with QIAGEN QIAquick^TMThe gel extraction kit (Qiagen) was recovered according to the manufacturer's instructions.

Mutations Ala to Ser (amino acid residue 134 of SEQ ID NO: 45) and Pro to Ser (amino acid residue 135 of SEQ ID NO: 45) were also introduced by PCR to reduce complement Clq binding and/or complement fixation (Duncan and Winter, Nature 332: 788, 1988), as well as the stop codon TAA. Two first rounds of reactions were performed using the mutated IgFc sequence of the Fc γ RI binding site as template. Mu.l of IgFc template with a final volume containing 1. mu.l of Fc. gamma.RI binding site mutation, 5. mu.l of 10X Rfu reaction buffer (Stratagene), 8. mu.l of 1.25mM dNTPs、31μl dH₂O, 2. mu.l of 20mM ZC15, 517(SEQ ID NO: 48) (a 5 'primer starting at nucleotide 26 of SEQ ID NO: 45) and 2. mu.l of 20mM ZC15, 530(SEQ ID NO: 49) (a 3' primer starting from the complementary strand of nucleotide 405 of SEQ ID NO: 45). The second reaction contained 20mM stock solutions of oligonucleotide primers ZC15, 518(SEQ ID NO: 50) (5 'primer starting at nucleotide 388 of SEQ ID NO: 45) and ZC15, 347(SEQ ID NO: 47) (3' primer) each 2. mu.l to introduce the Ala to Ser mutation, the Xba I restriction site and the stop codon. An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 1 minute. Pfu polymerase (2.5 units, Stratagene) was added followed by 25 cycles of: 30 seconds at 94 ℃,30 seconds at 55 ℃,2 minutes at 72 ℃ and then 7 minutes at 72 ℃. The reaction product was electrophoresed and bands corresponding to expected sizes of-370 bp and-395 bp, respectively, were detected. These bands were cut from the gel and the gel was washed with QIAGEN QIAquick^TMThe gel extraction kit (Qiagen) was recovered according to the manufacturer's instructions. A second round of reaction was performed to ligate the above fragments and add a 5' BamHI restriction site. 30 μ l dH was added to 50ul of final volume₂O, 8. mu.l of 1.25mM dNTPs, 5. mu.l of 10 XPfu polymerase reaction buffer (Stratagene) and 1. mu.l each of the two first PCR products. An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 1 minute. Pfu polymerase (2.5 units, Stratagene) was added followed by 5 cycles of: 30 seconds at 94 ℃,30 seconds at 55 ℃ and 2 minutes at 72 ℃. The temperature was again raised to 94 ℃ and 2. mu.l each of 20mM ZC15, 516(SEQ ID NO: 51) (5' primer starting at nucleotide 1 of SEQ ID NO: 45) and ZC15, 347(SEQ ID NO: 47) stocks were added, followed by 25 cycles of: 30 seconds at 94 ℃,30 seconds at 55 ℃,2 minutes at 72 ℃ and finally 7 minutes at 72 ℃. A portion of the reaction product was observed by gel electrophoresis. A789 bp band corresponding to the expected size was detected.

Construction of TACI-Fc4 and BCMA-Fc4 expression vectors

Construction of an expression plasmid containing the TACI-Fc4 and BCMA-Fc4 fusion proteins by homologous recombination in Yeast. Isolation of a nucleic acid comprising SEQ ID NO: 5 from nucleotide 15 to nucleotide 475. The two primers used to generate the TACI fragment were: (1) contains 40bp of 5' vector flanking sequence and 17bp of primer (SEQ ID NO: 52) corresponding to the amino terminal of the TACI fragment; (2) the 40bp 3' end corresponding to the flanking Fc4 sequence and 17bp (SEQ ID NO: 53) corresponding to the carboxy terminus of the TACI fragment. Mu.l of the final volume was loaded with 10ng TACI template, 10. mu.l of 10 XTaq polymerase reaction buffer (Perkin Elmer), 8. mu.l of 2.5 nNTPs, 78. mu.l dH₂O, 20mM oligonucleotide primer SEQ ID NO: 52 and SEQ ID NO: 53 stocks 2. mu.l each, and Taq polymerase (2.5 units, Life Technology). An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 2 minutes, after which 25 cycles were performed, each cycle being: 30 seconds at 94 ℃,30 seconds at 65 ℃,1 minute at 72 ℃, followed by 5 minutes of extension at 72 ℃.

Isolation of a nucleic acid comprising SEQ ID NO: 7 from nucleotide 219 to nucleotide 362. The two primers used for this BCMA fragment preparation were: oligonucleotide primers (SEQ ID NO: 54) of 17bp containing a 40bp 5' vector flanking sequence and corresponding to the amino terminus of the BCMA fragment; and oligonucleotide primers (SEQ ID NO: 55) containing 40bp 3' ends corresponding to the flanking Fc4 sequences and 17bp corresponding to the carboxy terminus of the BCMA fragment. Mu.l of the final volume was added 10ng of BCMA template, 10. mu.l of 10 XTaq polymerase reaction buffer (Perkin Elmer), 8. mu.l of 2.5mM dNTPs, 78. mu.l dH₂O, 20mM oligonucleotide primer seq id NO: 54 and SEQ ID NO: 55 stock solutions were 2. mu.l each. An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 2 minutes, after which 25 cycles were performed, each cycle being: 30 seconds at 94 ℃,30 seconds at 65 ℃,1 minute at 72 ℃ followed by 5 minutes of extension at 72 ℃.

Fragments containing the cDNA encoding the Fc4 fragment for each TACI and BCMA fusion construct were constructed in a similar manner. For TACI, the two primers used for Fc4 fragment generation were (upstream and downstream): oligonucleotide primers (SEQ ID NO: 56) containing 40bp 5' TACI flanking sequence and corresponding to 17bp of the amino terminal of the Fc4 fragment; and an oligonucleotide primer (SEQ ID NO: 57) containing the 40bp 3' end corresponding to the flanking vector sequence and the 17bp end corresponding to the carboxy terminus of the Fc4 fragment. For BCMA, the upstream primer for the generation of the Fc4 fragment was an oligonucleotide primer (SEQ ID NO: 58) containing the 40bp 5' BCMA flanking sequence and the 17bp corresponding to the amino terminus of the Fc4 fragment. The downstream primer for Fc4 of the BCMA structure was the same as described for TACI-Fc 4(SEQ ID NO: 57).

To 100. mu.l of the final volume were added 10ng of the above Fc4 template, 10. mu.l of 10 XTAQQ polymerase reaction buffer (Perkin Elmer), 8. mu.l of 2.5nM dNTPs, and 78. mu.l of dH₂O, SEQ ID NO for TACI 20mM oligonucleotide: 56 and SEQ ID NO: 57 stock of 2. mu.l each and 20mM for BCMA oligonucleotides SEQ ID NO: 58 and SEQ ID NO: 57 stocks 2. mu.l each, and Taq polymerase (2.5 units, Life Technology). An equal volume of mineral oil was added and the reaction was heated to 94 ℃ for 2 minutes, after which 25 cycles were performed, each cycle being: 30 seconds at 94 ℃,30 seconds at 65 ℃,1 minute at 72 ℃ followed by 5 minutes of extension at 72 ℃.

For analysis, 10. mu.l of each of the above 100. mu.l PCR reactions were electrophoresed in 1 XTBE buffer on 0.8% LMP agarose gel (Seaplaque GTG). The remaining 90. mu.l of each PCR reaction was precipitated by adding 5. mu.l of 1M NaCl and 250. mu.l of absolute ethanol. Plasmid pZMP6 was cut with SmaI to linearize it at the polylinker. Plasmid pZMP6 is derived from plasmid pCZR199 (American type culture Collection, Manassas, VA, ATCC #98668), a mammalian expression vector containing the CMV immediate early promoter, a consensus intron from the variable region of the mouse immunoglobulin heavy chain locus, restriction sites for insertion of coding sequences, a stop codon and a human growth hormone terminator. The plasmid also has an E.coli origin of replication, a mammalian selectable marker expression unit with the SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator. The vector pZMP6 was constructed from pCZR199 by replacing the metallothionein promoter with the CMV immediate early promoter and the Kozac sequence at the 5' end of the open reading frame.

Mu.l of competent yeast cells (Saccharomyces cerevisiae) were mixed with 10. mu.l of a solution containing approximately 1. mu.g each of the TACI or BCMA extracellular domains and the Fc4PCR fragment suitable for recombination with them, and 100ng of SmaI digested pZMP6 vector, and then transferred to a 0.2cm electroporation cuvette. The yeast/DNA mixture was electrically pulsed at 0.75kV (5kV/cm), ∞ ohms, 25. mu.F. 600. mu.l of 1.2M sorbitol was added to each cup and two 300. mu.l aliquots of yeast were plated on URA-D plates and incubated at 30 ℃.

Approximately 48 hours later, from a single plate Ura + yeast transformants were resuspended in 1ml H₂O, briefly centrifuged to pellet the yeast cells. The cell pellet was resuspended in 1ml lysis buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris pH8.0, 1mM EDTA). Mu.l of this lysis mixture was added to an Eppendorf tube containing 300. mu.l of acid-washed glass beads and 200. mu.l of phenol-chloroform, vortexed for 1 minute with two or three intermediate intervals, and then centrifuged at maximum speed for 5 minutes in an Eppendorf centrifuge. Mu.l of the aqueous phase was transferred to a new tube, and the DNA was precipitated with 600. mu.l of ethanol (EtOH) and centrifuged at 4 ℃ for 10 minutes. The DNA pellet was resuspended in 100. mu. l H₂And (4) in O.

Electrocompetent E.coli cells (DH10B, GibcoBRL) were transformed with 0.5-2ml of yeast DNA preparation and 40. mu.l of DH10B cells. The cells were electrically pulsed at 2.0kV, 25mF and 400 ohms. After electroporation, 1ml SOC (2% bactopeptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10mM NaCl, 2, 5mM KCl, 10mM MgCl₂，10mM MgSO₄20mM glucose) was plated in 250. mu.l aliquots onto 4 LB AMP plates (LB medium (Lennox), 1.8% Bacto agar (Difco), 100mg/L ampicillin).

The presence of the insert was verified by restriction digestion and the various DNA sequences were confirmed to be correctly ligated to each other to identify a single clone containing the correct expression structure of TACI-Fc4 or BCMA-Fc 4. The inserts of positive clones were subjected to sequence analysis. Plasmid DNA was isolated on a larger scale using the Qiagen Maxi kit (Qiagen) according to the manufacturer's instructions.

Example 7

Mammalian expression of TACI-Fc4 and BCMA-Fc4

BHK570 cells (ATCC CRL-10314) were plated in 10cm tissue culture dishes and plated in DMEM/FBS medium (DMEM, Gibco/BRL high glucose (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone, Logan, UT), 1mM L-glutamine (JRH Biosciences, Lenexa, KS), 1mM sodium pyruvate (Gibco BRL)), 37 ℃, 5% CO₂Grow overnight to approximately 50-70% confluence. Then in serum-free (SF) medium preparation (DMEM, 10mg/ml transferrin, 5mg/ml insulin, 2mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate), using plasmids TACI-Fc4/pZMP6 or BCMA-Fc4/pZMP6 and Lipofectamine^TM(Gibco BRL) transfected cells. TACI-Fc4/pZMP6 or BCMA-Fc4/pZMP6 to 640. mu.l total final volume were diluted with SF medium in 15ml tubes. Collecting 35 μ l Lipofectamine^TM(Gibco BRL) was mixed with 605. mu.l of SF medium. Adding the Lipofectamine to the DNA mixture^TMThe mixture was incubated at room temperature for about 30 minutes. To this DNA: lipofectamine^TMThe mixture was added to 5ml of SF medium. The cells were washed once with 5ml SF medium, aspirated, and the DNA: lipofectamine^TMAnd (3) mixing. Cells were incubated at 37 ℃ for 5 hours, and then 6.4ml of DMEM/10% FBS, 1% PSN medium was added to each plate. The plates were incubated overnight at 37 ℃ and then the next day the DNA was replaced with fresh 5% FBS/EMEM medium: lipofectamine^TMAnd (3) mixing. On day 5 post-transfection, the cell fractions were placed in T-162 shake flasks and cultured in selection medium (DMEM/5% FBS, 1% L-GLU, 1% NaPyr). Approximately 10 days after transfection, methotrexate resistant colonies from two 150mm dishes per transfection were trypsinized, the cells were pooled and seeded in a T-162 shake flask, and then transferred to large scale culture.

Example 9

Transgenic expression of ztnf4

Transgenic animals expressing the ztnf4 gene were prepared using adult fertile males (B6C3f1), prepubertal fertile females (B6C3f1), vasectomized males (B6D2f1) and adult fertile females (B6D2f1), all from Taconn farm, Germantown, NY. Pregnant mare serum gonadotropin (Sigma, st. louis, MO) and human chorionic gonadotropin (hCG (Sigma)) were used to superovulate fertile females well before puberty. Adult fertile males are then mated with the superovulated females and mating is verified by the presence or absence of vaginal plugs.

Fertilized eggs were collected under a surgical scope (Leica MZ12 Stereo Microscope, Leica, Wetzlar, germany). Then after having been at 5% CO₂、5％ O₂And 90% N₂The eggs were washed with hyaluronidase incubated at 37 ℃ and Whitten's W640 medium (Table 8; all reagents were available from Sigmachemical Co.). Eggs were stored at 37 ℃/5% CO prior to microinjection₂An incubator.

TABLE 8

WHITTEN 640 culture medium

mgs/200ml mgs/500ml

NaCl 1280 3200

KCl 72 180

KH₂PO₄ 32 80

MgSO₄-7H₂O 60 150

Glucose 200500

Calcium lactate 106265

Penicillin G1537.5

Streptomycin sulfate 1025

NaHCO₃ 380 950

Sodium pyruvate 512.5

H₂O 200ml 500ml

500mM EDTA 100μl 250μl

5% phenol Red 200. mu.l 500. mu.l

BSA 600 1500

Oligonucleotide primers SEQ ID NO: 36 and SEQ ID NO: 37, the 858bp open reading frame encoding the full-length human TACI ligand Blys (SEQ ID NO: 35) was amplified by PCR to introduce the optimal start codon and flanking 5 'PmeI and 3' AscI sites. Subcloning the PmeI/AscI fragment into pKF024, pKF024 being a B and/or T cell restricted transgenic vector containing Ig Em enhancer (690 bp NotI/XbaI from pEmSRd; Bodrug et al, EMBO J.13: 2124-30, 1994), Ig V_hThe promoter (536 bpHincII/XhoI fragment from pJH1X (-); Hu et al, J.Exp.Med.) -177: 1681-90, 1993), the 16S intron of SV40 (171 bp XhoI/HindIII fragment from pEmSR), a PmeI/AscI polylinker, and the polyadenylation signal of the human growth hormone gene (627bpSmaI/EcoRI fragment; Seeburg, DNA 1: 239-49, 1982). The transgenic insert was isolated from the plasmid backbone by NotI digestion and agarose gel purification, and fertilized eggs from the above described B6C3F1Tac mouse mating were microinjected and transplanted into pseudopregnant females essentially as described previously (Malik et al, molecular cell. biol. 15: 2349-58, 1995).

The recipient animals are returned in pairs to their cages and allowed to become pregnant for 19-21 days. After birth, sex was identified and weaned 19-21 days after birth, and the tail was cut with clean scissors to obtain 0.5cm biopsy (for genotype analysis)

Genomic DNA was prepared from the excised tail pieces using a commercially available kit (DNeasy 96 Tissue kit; Qiagen, Valencia, Calif.) according to the manufacturer's instructions. Genomic DNA was analyzed by PCR using primers designed for the 3' UTR portion of human growth hormone (hGH) of the transgenic vector. Primers ZC17251(SEQ ID NO: 38) and ZC17252(SEQ ID NO: 39) amplify a 368 base pair hGH fragment. Regions unique to the human sequence (identified from the alignment of human and mouse growth hormone 3' UTR DNA sequences) were used to ensure that the PCR reaction did not amplify mouse sequences. In addition, primers ZC17156(SEQ ID NO: 40) and ZC17157(SEQ ID NO: 41) which hybridize to the vector sequence and amplify the cDNA insert may also be used with the hGH primer. In these experiments, DNA from the transgenic positive animals produced two bands, one corresponding to the 368 base pair band of the hGH 3' UTR fragment and one to the variable size band of the cDNA insert.

Once the animals were confirmed to be Transgenic (TG), TG animals were backcrossed to inbred lines by placing one TG female and one wild type male, or one TG male and one or two wild type females together. After birth and weaning, pups were sexed and their tails clipped for genotyping.

To examine transgene expression in live animals, experiments were performed on surviving biopsies. The mRNA expression level of each transgene was analyzed using RNA solution hybridization experiments or real-time PCR performed on ABI Prism 7700(PE Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

Preparation of cells and flow cytometry

Starting mice of different ages were analyzed. For flow cytometry (FACS) analysis of lymphoid tissues, Bone Marrow (BM) cells were isolated by carefully disrupting the femur and tibia in Phosphate Buffered Saline (PBS) using a mortar and pestle. The cells were resuspended, bone debris removed by passive sedimentation, and the cells were then pelleted at 1000 Xg. Splenocytes, thymocytes, or lymph node cells were obtained by compressing the whole tissue between two slides and then resuspended and then pelleted as for BM. Prior to staining, cells were resuspended in FACS washing buffer (FACSWB) (Hank's balanced salt solution, 1% BSA, 10mM Hepes, pH7.4) at a concentration of 20X 10⁶Individual cells/ml. For dyeing, 1X 10⁶Individual cells were transferred to 5ml tubes and washed with 1ml facs WB, then pelleted at 1000 × g. Cells were then incubated on ice for 20 minutes in a total volume of 100ml FACS WB in the presence of saturating amounts of appropriate FITC, PE and/or Tricholor (TC) bound mAbs. Cells were washed with 1.5ml WB, pelleted, then resuspended in 400ml WB and analyzed on a FACSCalibur flow cytometer using CellQuest software (becton dickinson, Mountain View, CA). The detectors for the Front (FSC) and side (SSC) light scatter are set on a linear range, while all three fluorescence channels (FL-1, FL-2 and FL-3) employ logarithmic detectors.

Each experiment corrected for spectral overlap between FL channels using a single color-stained cell population. All uncancelled (ungated) cells were collected on plates and the data were analyzed using CellQuest software. RBCs and dead cells are excluded by electronic threshold (electronically gating) data based on FSC versus SSC profiles.

Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 8 monoclonal antibody (mAb) (clone 53-6.7) and phycoerythrin (phytoerthyrin) (PE) -conjugated anti-CD 4 (clone RM4-5), anti-CD 5 (clone 53-7.3), anti-CD 19 (clone 1D3), and anti-syndecan (clone 281-2) mAbs were purchased from PharMingen (San Di ego, CA). TriColor (TC) conjugated anti-CD 45R/B220mAb (clone RA3-6B2) was purchased from Caltag.

Transgenic mice overexpressing ztnf4 in the lymphoid compartment showed increased peripheral B cell numbers, increased plasma cells, and increased serum immunoglobulin levels. These transgenic animals had increased numbers of B200+ cells in the spleen, lymph nodes and thymus. Increased numbers of splenic B cells include regular B-2 cells and normally rare B-1 cell populations. In general, B-1 cells are primarily confined to the peritoneal and other body cavities, produce low affinity, self-reactive antibodies, and are often associated with the development of autoimmune diseases such as SLE, systemic lupus erythematosus.

Older transgenic animals produce autoantibodies that are characteristic of systemic lupus erythematosus: proteinuria and hardened glomeruli.

FIG. 5A shows a single cell suspension of spleen (top panel), mesenteric lymph node (middle panel) and bone marrow (bottom panel) stained with anti-B220-TC and analyzed by flow cytometry, prepared as described below. The number of B220+ cells in each tissue was calculated by multiplying the percentage of B220+ cells by the total number of hepatocytes (except trypan blue stained cells) counted by a hemocytometer. Each bar represents data obtained from a single ztnf4 transgenic (Tg, shaded bar) or non-Tg littermates (blank bar) control mice.

Fig. 5B shows cells isolated from lymph nodes (upper row), spleen (middle row) and thymus (bottom row) of ztnf4TG (left panel) or non-TG littermates (right panel), stained with mabs to the indicated molecules (DC5, CD4 and CD8) and analyzed by flow cytometry. The data shown are limited to exclude dead cells and RBCs.

Figure 5C shows total IgG, IgM, and IgE levels in serum of ztnf4 transgenic mice over the 6-23 week age range.

Fig. 5D shows glomerular amyloid deposition and glomerular mesangial thickening identified in H & E stained kidney sections of ztnf4 transgenic mice compared to normal glomeruli of control littermates.

FIG. 5E shows an increase in effector T cells in ztnf4 transgenic mice, similar to that reported by Mackay et al (J.Exp.Med.) 190: 1697-1710, 1999).

Soluble TACI (BR43 x 2) or BCMA-Ig fusions were injected into transgenic mice overexpressing ztnf 4. Lymphoid tissues were analyzed by flow cytometry (FACS) to identify any changes in the number of B220+ B cells in the spleen, lymph nodes and thymus.

Example 10

Direct binding ELISA

A direct binding ELISA was developed to characterize the ability of soluble TACI-Ig or soluble BCMA-Ig to bind ztnf4 and inhibit the biological activity of ztnf4 in vitro.

Using ELISA A buffer (0.1M Na2 HCO)₃pH9.6，0.02％ NaN₃) Mu.g/ml goat anti-human Ig (Jackson Labs, Bar Harbor, MA) in (1) coated 96-well plates and incubated overnight at 4 ℃. TACI, BCMA and an unrelated TNF receptor such as ztnfr10(SEQ ID NO: 42) as a control were diluted 5-fold from 10. mu.g/ml, titrated to 320ng/ml plus one zero, and then co-incubated with either 2.5, 0.5 or 0.1. mu.g/ml biotinylated ztnf4 or ovalbumin as a negative control, and incubated at room temperature for 1 hour.

Then subjecting the co-incubated material toThe mixture of the body-biotinylated ligands was added to goat anti-human Ig coated 96-well plates. Then, 200mg of NaN was applied using ELISA C (500. mu.l of Tween20(Sigma Chemical Co., St. Louis, Mo.)₃PBS, final volume 1 liter) and blocked with Superblock (Pierce, Rockford, IL). The plates were then incubated at 37 ℃ for 2 hours.

These plates were washed once more with ELISA C, after which 100. mu.l/well of the solution present in ELISA B (5 or 10. mu.g BSA (Sigma) constituted 1% or 2% BSA, respectively, 250. mu.l Tween20 (Sigma), 100mg NaN were added₃Phosphate buffered saline (PBS, Sigma) pH7.2, final volume 500 ml; alternatively, the buffer may consist of 1% or 2% BSA in ELISA C buffer. ) 1:10,000 neutr-avidin-HRP in (1). The plates were then developed with OPD at room temperature for 10 minutes and read at 492.

Example 11

Assay for biological Activity

A biological activity assay was developed to measure the inhibition of soluble TACI-FC on human B cell stimulation by soluble ztnf 4. B cells were isolated from peripheral blood mononuclear cells (PBMNC) using CD19 magnetic beads and a VarioMacs magnetic separation system (Miltenyi Biotec Auburn, CA) according to the manufacturer's instructions. Purified B cells were mixed with soluble ztnf4(25ng/ml) and recombinant human IL-4(10ng/ml Pharmingen) and (in triplicate) at 1X 10 per well⁵Individual cells were seeded in round-bottomed 96-well plates.

Soluble TACI-FC was diluted from 5. mu.g/ml to 6ng/ml and incubated with the B cells for 5 days with 1. mu. Ci per well on day 4³H thymidine (Amersham) treatment was performed overnight. As a control, soluble TACI-FC was incubated with B cells and IL-4 in the absence of ztnf 4.

Cells in the plates were harvested using a Packard plate harvester and then counted using a Packard reader. In vitro, TACI-Ig soluble receptors inhibit the ability of soluble ztnf4 to stimulate B cell proliferation in a dose-dependent manner. A10-fold molar excess of TACI-Ig completely inhibited the proliferation of human B cells in response to soluble ztnf4 in the presence of IL-4.

Example 12

ORIGIN assay

The level of ztnf4 in patients with disease conditions (e.g., SLE, rheumatoid arthritis, etc.) relative to normal individuals is determined using an electrochemiluminescence assay. Standard curves were prepared from 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml and Ong/ml soluble human ztnf4 prepared in ORIGIN buffer (Igen, Gaithersburg, Md.). Serum samples were diluted in ORIGIN buffer. Standards and samples were incubated with biotinylated rabbit anti-human ztnf4-NF BV antibody diluted to 1. mu.g/ml in Origin assay buffer (IGEN) and ruthenated rabbit anti-human ztnf4-NF BV polyclonal antibody diluted to 1. mu.g/ml in Origin assay buffer (IGEN) for 2 hours at room temperature. After incubation, the samples were vortexed and 50. mu.l/tube of 0.4mg/ml streptavidin Dynabeads (Dynal, Oslo, Norway) was added to each standard and sample, followed by incubation at room temperature for 30 minutes. The samples were then vortexed and read on an Origin analyzer (Igen) according to the manufacturer's instructions. The Origin assay is based on electrochemiluminescence, producing a result output in the ECL-what this is, how it works and what this tells you what.

In the cases of NZBEW 1/J and MRL/Mpj-Fas from advanced stages of development into glomerulonephritis and autoimmune diseases^1prElevated levels of ztnf4 were detected in serum samples from mice.

Example 13

Soluble TACI-Ig in spontaneous SLE model

NZBW mice develop symptoms of spontaneous SLE at approximately 7-9 months of age. When the production of B cell autoantibodies was considered to be high in the NZBW mice on average, TACI-Fc was administered to the NZBW mice over a 5-week period and its inhibitory effect on B cells was monitored.

100 females (NZB × NZW) F of 8 weeks of age₁The mice (Jackson laboratory) were divided into 6 groups of 15 mice each. Before treatment, the proteins in the mouse urine were monitored monthly and blood was drawn for the construction of CBC and serum pools. Sera were screened for the presence of autoantibodies. Since proteinuria is a marker signal for glomerulonephritis, protein levels in urine were monitored at time intervals in dipsticks during the study. Animals were weighed before treatment. Dosing was initiated when mice were approximately 5 months of age. Mice were injected intraperitoneally 3 times weekly for 5 weeks with vehicle alone (PBS) or human IgG-FC (control protein) or TACI-FC4 (test protein).

Group (5 mice per group)	Treatment of	Dosage form
			1	Untreated control
2	Simple carrier
			3	Human IgG-FC	20μg
4	Human IgG-FC	100μg
			5	Human TACI-FC4	20μg
6	Human TACI-FC4	100μg

Blood was collected twice during dosing and at least two collections were performed after dosing. The urinary volume bar value and body weight for proteinuria were measured every two weeks after the start of the administration. Blood, urine volume bar values and body weight values were collected at euthanasia. The spleen, thymus, liver, gallbladder, left kidney, and brain were weighed. Both spleen and thymus were divided into two portions for FACS analysis and histological analysis. Submandibular salivary glands, mesenteric lymph node chains, liver lobes and gallbladder, cecum and large intestine, stomach, small intestine, pancreas, right kidney, adrenal gland, tongue and trachea and esophagus, heart, and lung were also collected for histological analysis.

FIG. 6 shows elevated levels of ztnf4 in serum of NZBWF1 and MRL/lpr/lpr mice that are associated with the development of SLE. The upper panel of FIG. 6A shows that serum levels of ztnf4 are age-related in 68 NZBKF 1 mice in the range of 10-40 weeks of age and in NZB/B mice at 10 and 30 weeks of age. The middle panel shows the correlation of proteinuria in NZBKF 1 mice with three ranges, trace-20 mg/dl (T-30), 100-300ng/dl, and 200mg/dl, compared to control NZB/B mice. The lower panel shows the level of ztnf4 in nzbff 1 mice as a function of various titers of anti-ds DNA antibody compared to control NZB/B mice.

FIG. 6B shows that a similar relationship was obtained in 23 MRL/lpr/lpr mice over the age range of 18-24 weeks and 10 control MRL/MpJ mice at 11 weeks of age.

Fig. 7 shows the results of urinalysis. If the dipstick reads ≧ 100mg/dl, the mouse is considered to have proteinuria. (A) PBS, (B) human IgG FC, 100mg, (C) human IgGFC, 20mg, (D) human TACI-IgG, 100mg, and (E) human TACI-IgG, 20 mg. Mice treated with soluble TACI-IgG fusions exhibited a reduction in proteinuria.

Peripheral blood analysis of treated animals revealed that white blood cell and lymphocyte counts were reduced in TACI-FC treated mice (20 and 100mg) compared to FC (20 and 100mg) and PBS treated mice two weeks after treatment began. FAC analysis (lymphocytes as a threshold) of peripheral blood drawn 6 weeks after the start of treatment (two weeks after the last treatment administered) showed a drastic reduction in the percentage of B cells present in the sample. B cell levels were still decreasing, but not dramatically, 5 weeks after the last treatment. Table 9 provides the mean (and standard deviation) of mice in each treatment group (table 9). A decrease in the percentage of B cells in peripheral blood was also observed two weeks into the treatment.

TABLE 9

Example 14

Soluble TACI-Ig in Normal mice

TACI-FC was administered to Blab/C mice to monitor their effect on normal mice. 60 8-week-old female Balb/C mice (HSD) were divided into 12 groups of 5 mice each. Before treatment, mice were weighed and bled for CBC and serum bank construction. Groups 1-9 received 12 days of daily intraperitoneal Injections (IP) of vehicle alone (PBS) or human IgG-FC (control protein) or TACI-FC4 (test protein) and were sacrificed on day 14. Groups 10-11 received 3 IP injections weekly for two weeks and were sacrificed on day 14.

Group (5 pieces each)	Treatment of	Dosage form
			1	Human TACI-FC4	200mg
2	Human TACI-FC4	100mg
			3	Human TACI-FC4	20μg
4	Human TACI-FC4	5μg
			5	Human FC4	200μg
6	Human FC4	100mg
			7	Human FC4	20mg
8	Human FC4	5mg
			9	Simple carrier	In the same amount as used
10	Human TACI-FC4	100mg
			11	Human FC4	100mg
12	Untreated control

Blood was collected on day 7 and day 12. At euthanasia, blood was collected and weighed. The spleen, thymus, and brain were weighed. Both spleen and thymus were divided into two portions for FACS analysis and histological analysis. Skin, spleen, mesenteric LN chains, submandibular salivary glands, ovaries, uterus, cervix, bladder, mesenteric lymph node chains, liver lobes and gall bladder, cecum and large intestine, stomach, small intestine, pancreas, right kidney, adrenal gland, tongue and trachea and esophagus, heart, thymus, thigh muscle, left and right thigh, brain will also be collected for histological analysis.

As described in example 13, a significant reduction in the percentage of B cells in peripheral blood cells was observed at day 7 (by CBC) and 12 (by FACS) from all TACI-FC4 treated samples, compared to those samples treated with FC4 or PBS alone and analyzed by CBC or FACS. Furthermore, B cells were reduced by nearly 50% in the spleen from TACI-FC4 treated animals compared to FC4 treated mice on day 14.

Example 15

anti-dsDNA ELISA

Autoimmunity was characterized by high levels of anti-double stranded DNA antibodies. To measure the anti-dsDNA antibody levels in transgenic mice overexpressing ztnf4 and NZBW mice, an ELISA assay was developed. Coating with poly-L-lysine (Sigma) (20. mu.l/ml in 0.1M Tris buffer pH 7.3) at 75. mu.l/well96-well microtiter plates (Nunc) were incubated overnight at room temperature. Then at dH₂The plates were washed in O and coated with poly dAdT (Sigma) (20. mu.l/ml in 0.1M Tris buffer pH 7.3) at 75. mu.l/well and incubated for 60 min at room temperature. Then by dH₂The plates were O-washed and blocked with 2% BSA in Tris buffer (Sigma) for 30 min at room temperature, followed by dH₂Final wash in O.

Serum samples were taken from the ztnf4 transgenic mice described in example 10 and from the NZBW mice described in example 11. The serum samples were diluted 1:50 in 1% BSA/2% BGG (Calbiochem) in Tris buffer. These diluted samples were then titrated into the coated plate at 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400(50 μ l/well) and incubated for 90 minutes at room temperature.

Then at dH₂Plates were washed in O and goat anti-mouse IgG-Fc-HRP (Cappel) diluted 1:1000 in 1% BSA/2% BGG was added at 50. mu.l/well. The plate was incubated at room temperature for 60 minutes. At dH₂Plates were washed 5 times in O and developed with OPD (1 plate/10 ml Novo D, plated at 100. mu.l/well). Using 100. mu.l/well 1N H₂SO₄The color development was terminated and the OD value was read at 492 nm.

FIG. 8 shows the anti-dsDNA levels in two ztnf4 transgenic mice (23 weeks old), two non-transgenic littermates, compared to the anti-dsDNA levels detected in the serum of NZBWF1(32 weeks old) and MRL/lpr/lpr (19 weeks old) mice.

Example 16

Soluble TACI-Ig in spontaneous ELE model

25 female PLxSJL F1 mice (12 weeks old, Jackson laboratory) were injected subcutaneously with 125 μ g of antigen (myelin proteolipid protein, PLP, residues 139-151) formulated in Freund's complete adjuvant. These mice were divided into 5 groups of 5 mice each. Pertussis toxin (400ng) was injected intraperitoneally on days 0 and 2. Some groups were given 1, 10 or 100-fold doses of TACI, BCMA or BR43x2, one group received vehicle alone, and one group received no treatment. Prophylactic treatment will begin on day 0, and intervention treatment will begin on day 7 or at the onset of clinical symptoms. Symptoms of the disease, weight loss, and paralysis occur on about days 10-14 and persist for about 1 week. Animals were evaluated daily by weighing and assigning a clinical score that corresponds to the extent of symptoms. Clinical symptoms of EAE occur within days 10-14 of vaccination and persist for approximately 1 week. At the end of the study, all animals were euthanized by administration of excess gas and necropsy was performed. Brains and spines were collected for histological analysis, or frozen for mRNA analysis. Body weight and clinical score data were plotted for individual and groups.

Clinical scoring

0 normal

0.5 Weak, the tail tension may be reduced but not absent

1 Tail Soft (when picking up mouse at tail base, can not lift tail)

2 Soft tail, weak leg (unable to lift tail, able to use back leg to erect but shaky leg)

3 local paralysis (squat with legs, walking with back legs in a paddling manner)

4 paralysis (inability to move the hind legs, dragging the legs when trying to walk)

Paralysis of the extremities (paralysis of the front legs or walking in a loop, with possible head inclination)

6 moribund (complete paralysis, inability to obtain food or water, sacrifice animal)

Example 17

CIA model of TACI-FC and rheumatoid arthritis

Male DBA/1J mice (Jackson laboratories) at 8 weeks of age were grouped into groups of 5 mice each, and these mice were given two subcutaneous injections of 50-100. mu.l of 1mg/ml collagen (of chicken or bovine origin) 3 weeks apart. One control will not receive collagen injections. The first injection was formulated in freund's complete adjuvant, while the second injection was formulated in freund's incomplete adjuvant. TACI-FC is administered prophylactically at or before the second injection, or after the animal has developed a clinical score of 2 or more for at least 24 hours. After the second collagen injection, the animals began to exhibit symptoms of arthritis, usually within 2-3 weeks. The extent of disease was assessed from each paw by measuring the thickness of the paw with calipers and giving each paw a clinical score (0-3). Clinical scoring: 0 normal, 1 red swelling of one or more toes, 2 mild paw inflammation, 3 moderate paw inflammation, 4 severe paw inflammation. Animals were euthanized after a period of disease establishment. The paw was collected for histological or mRNA analysis and serum was collected for immunoglobulin and cytokine analysis.

Example 18

Neutralization of TACI antibodies

Polyclonal anti-peptide antibodies were prepared by immunizing 2 female New Zealand white rabbits with the peptides huztnf4-1 SAGIAKLEEGPELQLAIPRE (SEQ ID NO: 59) or huztnf4-2SFKRGSALEEKENKELVKET (SEQ ID NO: 60). These peptides were synthesized using an Applied Biosystems model 431A peptide synthesizer (Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. These peptides were then conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) by activation with maleimide. Each of the rabbits was initially injected intraperitoneally (ip) with 200. mu.g of peptide formulated in Freund's complete adjuvant, followed by a three-week booster intraperitoneal injection of 100. mu.g of peptide formulated in Freund's incomplete adjuvant. 7-10 days after administration of the second booster injection (3 injections in total), blood was drawn from the animals and serum was collected. The animals were then boosted and blood was drawn from the animals every three weeks.

Ztnf4 peptide-specific rabbit sera were qualitatively analyzed by ELISA titration detection using 1. mu.g/ml of the peptide used to make the antibodies (SEQ ID NOs: 59 and 60) as antibody target. The 2 rabbit sera against huztnf4-1 peptide (SEQ ID NO: 59) had titers of binding to their specific peptides at a dilution of 1:1E5(1: 100000). The 2 rabbit sera against huztnf4-2 peptide (SEQ ID NO: 60) had titers of binding to their specific peptides at a dilution of 1:5E6 and to recombinant full-length protein (N-terminal FLAG marker ztnf4 prepared in baculovirus (huztnf4s-NF-Bv) and C-terminal FLAG marker ztnf4 prepared in BHK cells) at a dilution of 1:5E 6.

The ztnf4 peptide-specific polyclonal antibody was affinity purified from rabbit sera using a CNBR-SEPHAROSE48 protein column (Pharmacia LKB) prepared with CNBR-SEPHAROSE10 mg-specific peptide (SEQ ID NO: 59 or 60) per gram, followed by 20-fold dialysis in PBS overnight. Antibodies specific for ztnf4 were qualitatively assayed by ELISA titration using 1 μ g/ml of the appropriate peptide antigen or recombinant full-length protein (huztnf4-NF-Bv) as the target for the antibody. The rabbit anti-huztnf 4-1 affinity purified antibody has a Lower Limit of Detection (LLD) of 5ng/ml for its specific antigen (huztnf4-1 peptide, SEQ ID NO: 59). The rabbit anti-huztnf 4-2 affinity purified antibody has a Lower Limit of Detection (LLD) of 0.5ng/ml for its specific antigen (huztnf4-2 peptide, SEQ ID NO: 60). The rabbit anti-huztnf 4-2 affinity purified antibody has a Lower Limit of Detection (LLD) of 5ng/ml for recombinant protein huztnf4 s-NF-Bv.

Mouse monoclonal antibodies were also prepared and selected for inhibition of the inhibitory effect of the biotin label soluble ztnf 4. None of the TACI monoclonal antibodies (248.14, 248.23 or 246.3) blocked the binding of ztnf4 to BCMA. When the conditioned medium was diluted to 1:243, monoclonal 248.23 reduced binding to about 50% for 10ng/ml ztnf 4-biotin, and to about 2-fold in undiluted medium. Monoclonal 246.3 reduced binding to about 50% of 10ng/ml ztnf 4-biotin in conditioned medium dilutions 1:243-1:181, while reducing binding 5-fold in undiluted medium.

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.

The application also discloses the following:

1. a method of inhibiting ztnf4 activity in a mammal, comprising administering to said mammal an amount of a compound selected from the group consisting of:

a) a polypeptide comprising the extracellular domain of BR43x 2;

b) a polypeptide comprising an extracellular domain of TACI;

c) a polypeptide comprising the extracellular domain of BCMA;

d) comprises the amino acid sequence shown in SEQ ID NO: 10;

e) and SEQ ID NO: 2 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

f) and SEQ ID NO: 4 or an antibody or antibody fragment to which the polypeptide of (4) specifically binds;

g) and SEQ ID NO: 6 or an antibody or antibody fragment to which the polypeptide of (a);

h) and SEQ ID NO: 8 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

i) and SEQ ID NO: 10 or an antibody or antibody fragment to which the polypeptide of claim 10 specifically binds;

k) SEQ ID NO: 4;

l) SEQ ID NO: 6 at amino acid residues 1-166; and

m) SEQ ID NO: 8 from 1 to 150.

2. The method according to item 1, wherein said compound is a fusion protein consisting of a first part and a second part joined together by a peptide bond, said first part comprising a polypeptide selected from the group consisting of:

a) comprises the amino acid sequence shown in SEQ ID NO: 8;

b) comprises the amino acid sequence shown in SEQ ID NO: 2 at amino acid residues 25-58;

c) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 34-66;

d) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 71-104;

e) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 25-104;

f) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8 to 37;

g) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 41-88;

h) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8-88; and is

The second portion comprises another polypeptide.

3. The method according to item 2, wherein the first part further comprises a polypeptide selected from the group consisting of:

a) SEQ ID NO: 2 amino acid residues 59 to 120;

b) SEQ ID NO: 6 amino acid residues 105-166; and

c) SEQ ID NO: 8, 89-150 amino acid residues.

4. The method of clause 2, wherein the first portion is selected from the group consisting of:

a) a polypeptide comprising BR43x2 extracellular domain;

b) a polypeptide comprising a TACI extracellular domain; and

c) a polypeptide comprising a BCMA extracellular domain.

5. The method of clause 2, wherein the first portion is selected from the group consisting of:

a) SEQ ID NO: 4;

b) SEQ ID NO: 6 at amino acid residues 1-154; and

c) SEQ ID NO: 8 from position 1 to 48.

6. The method according to item 2, wherein the second portion is a heavy chain constant region of an immunoglobulin.

7. The method according to item 1, wherein the antibody or antibody fragment is selected from the group consisting of:

a) a polyclonal antibody;

b) a murine monoclonal antibody;

c) a humanized antibody derived from b); and

d) a human monoclonal antibody.

8. The method according to item 7, wherein the antibody fragment is selected from the group consisting of: f (ab '), F (ab), Fab', Fab, Fv, scFv and minimal recognition units.

9. The method according to item 1, wherein the mammal is a primate.

10. The method according to clause 1, wherein the ztnf4 activity is associated with B lymphocytes.

11. The method according to clause 1, wherein the ztnf4 activity is associated with activated B lymphocytes.

12. The method according to clause 1, wherein the ztnf4 activity is associated with resting B lymphocytes.

13. The method according to clause 1, wherein the ztnf4 activity is associated with the production of antibodies.

14. The method of clause 13, wherein the production of the antibody is associated with an autoimmune disease.

15. The method of item 14, wherein the autoimmune disease is systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, or rheumatoid arthritis.

16. The method of clause 1, wherein the ztnf4 activity is associated with asthma, bronchitis, or emphysema.

17. The method according to clause 1, wherein the ztnf4 activity is associated with end-stage renal failure.

18. The method according to clause 1, wherein the ztnf4 activity is associated with renal disease.

19. The method according to item 18, wherein the kidney disease is glomerulonephritis, vasculitis, nephritis or pyelonephritis.

20. A method according to item 1, wherein said is associated with renal tumor, multiple myeloma, lymphoma, light chain neuropathy, or amyloidosis.

21. The method according to clause 1, wherein the ztnf4 activity is associated with effector T cells.

22. The method of clause 21, wherein the ztnf4 activity is associated with modulating an immune response.

23. The method of clause 21, wherein the activity is associated with immunosuppression.

24. The method of clause 21, wherein the immunosuppression is associated with transplant rejection, graft-versus-host disease, or inflammation.

25. The method of clause 24, wherein the activity is associated with an autoimmune disease.

26. The method of clause 25, wherein the autoimmune disease is insulin-dependent diabetes mellitus or Crohn's disease.

27. The method according to clause 26, wherein the ztnf4 activity is associated with inflammation.

28. The method of item 27, wherein the inflammation is associated with joint pain, swelling, anemia, or septic shock.

29. A method for inhibiting BR34 x2, TACI, or BCMA receptor-ligand engagement comprising administering an amount of a compound selected from the group consisting of:

a) a polypeptide comprising BR43x2 extracellular domain;

b) a polypeptide comprising a TACI extracellular domain;

c) a polypeptide comprising a BCMA extracellular domain;

d) comprises the amino acid sequence shown in SEQ ID NO: 10;

e) and SEQ ID NO: 2 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

f) and SEQ ID NO: 4 or an antibody or antibody fragment to which the polypeptide of (4) specifically binds;

g) and SEQ ID NO: 6 or an antibody or antibody fragment to which the polypeptide of (a);

h) and SEQ ID NO: 8 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

i) and SEQ ID NO: 10 or an antibody or antibody fragment to which the polypeptide of claim 10 specifically binds;

j) and SEQ ID NO: 18 or an antibody or antibody fragment to which the polypeptide of 18 specifically binds;

k) and SEQ ID NO: 20 or an antibody or antibody fragment to which the polypeptide of 20 specifically binds;

k) SEQ ID NO: 4;

l) SEQ ID NO: 6 at amino acid residues 1-166; and

m) SEQ ID NO: 8 from 1 to 150.

30. The method of item 29, wherein said compound is a fusion protein consisting of a first portion and a second portion joined together by a peptide bond, said first portion comprising a polypeptide selected from the group consisting of:

a) comprises the amino acid sequence shown in SEQ ID NO: 8;

b) comprises the amino acid sequence shown in SEQ ID NO: 2 at amino acid residues 25-58;

c) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 34-66;

d) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 71-104;

e) comprises the amino acid sequence shown in SEQ ID NO: 6 at amino acid residues 25-104;

f) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8 to 37;

g) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 41-88;

h) comprises the amino acid sequence shown in SEQ ID NO: 8, amino acid residues 8-88; and is

The second portion comprises another polypeptide.

31. A method according to clause 30, wherein the first portion further comprises a polypeptide selected from the group consisting of:

a) SEQ ID NO: 2, amino acid residues 59 to 120;

b) SEQ ID NO: 6 at amino acid residues 105-166; and

c) SEQ ID NO: 8 at amino acid residues 89-150.

32. The method of item 30, wherein the first portion is selected from the group consisting of:

a) a polypeptide comprising BR43x2 extracellular domain;

b) a polypeptide comprising a TACI extracellular domain; and

c) a polypeptide comprising a BCMA extracellular domain.

33. The method of item 30, wherein the first portion is selected from the group consisting of:

a) SEQ ID NO: 4;

b) SEQ ID NO: 6 at amino acid residues 1-154; and

c) SEQ ID NO: 8 from position 1 to 48.

34. The method of clause 30, wherein the second portion is a heavy chain constant region of an immunoglobulin.

35. The method according to item 29, wherein the antibody or antibody fragment is selected from the group consisting of:

a) a polyclonal antibody;

b) a murine monoclonal antibody;

c) a humanized antibody derived from b); and

d) a human monoclonal antibody.

36. The method of clause 35, wherein the antibody fragment is selected from the group consisting of: f (ab '), F (ab), Fab', Fab, Fv, scFv and minimal recognition units.

37. The method of clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with B lymphocytes.

38. The method of clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with activated B lymphocytes.

39. The method of clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with resting B lymphocytes.

40. The method of clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with antibody production.

41. The method of clause 29, wherein the production of the antibody is associated with an autoimmune disease.

42. A method according to item 41, wherein the autoimmune disease is systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, or rheumatoid arthritis.

43. A method according to item 29, wherein said BR43x2, TACI, or BCMA receptor-ligand engagement is associated with asthma, bronchitis, or emphysema.

44. The method according to clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with end-stage renal failure.

45. The method according to clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with renal disease.

46. The method according to item 45, wherein the kidney disease is glomerulonephritis, vasculitis, nephritis or pyelonephritis.

47. A method according to clause 29, wherein said receptor-ligand engagement is associated with nephroma, multiple myeloma, lymphoma, light chain neuropathy, or amyloidosis.

48. The method of clause 29, wherein the BR43x2, TACI, or BCMA receptor-ligand engagement is associated with effector T cells.

49. The method according to item 48, wherein said BR43x2, TACI or BCMA receptor-ligand engagement is associated with modulation of an immune response.

50. The method of clause 49, wherein the receptor-ligand engagement is associated with immunosuppression.

51. A method according to clause 50, wherein said immunosuppression is associated with transplant rejection, graft-versus-host disease, or inflammation.

52. A method according to clause 50, wherein said receptor-ligand engagement is associated with an autoimmune disease.

53. The method of clause 52, wherein the autoimmune disease is insulin-dependent diabetes mellitus or Crohn's disease.

54. The method according to item 50, wherein said BR43x2, TACI or BCMA receptor-ligand engagement is associated with inflammation.

55. The method of item 54, wherein the inflammation is associated with joint pain, swelling, anemia, or septic shock.

56. Encoding the amino acid sequence of SEQ ID NO: 2.

57, SEQ ID NO: 1.

58. An expression vector comprising the following operably linked elements:

a transcription promoter;

a polynucleotide molecule according to item 56; and

a transcription terminator.

59. An expression vector according to clause 58, further comprising a secretory receptor-ligand engagement sequence operably linked to the polynucleotide molecule.

60. A cultured cell into which has been introduced an expression vector according to item 58, wherein the cultured cell expresses the polypeptide encoded by the polynucleotide fragment.

61. A method of producing a polypeptide comprising:

culturing a cell into which an expression vector according to item 58 has been introduced;

whereby said cell expresses said polypeptide encoded by said polynucleotide molecule; and

recovering the expressed polypeptide.

62. Has the sequence shown in SEQ ID NO: 2.

63. The polypeptide of item 62 in combination with a pharmaceutically acceptable carrier.

Sequence listing

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<213> Artificial sequence

<220>

<223> FLAG tag

<400>13

<210>14

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> Glu-Glu labeling

<400>14

<210>15

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC19980

<400>15

<210>16

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC19981

<400>16

<210>17

<211>1149

<212>DNA

<213> human

<220>

<221>CDS

<222>(236)...(1027)

<400>17

<210>18

<211>264

<212>PRT

<213> human

<400>18

<210>19

<211>1430

<212>DNA

<213> Tragulus taiwanensis

<220>

<221>CDS

<222>(102)...(848)

<400>19

<210>20

<211>249

<212>PRT

<213> Tragulus taiwanensis

<400>20

<210>21

<211>473

<212>DNA

<213> Artificial sequence

<220>

<223> Northern blot Probe

<400>21

<210>22

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223>ZC20061

<400>22

<210>23

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC20062

<400>23

<210>24

<211>256

<212>DNA

<213> Artificial sequence

<220>

<223> Northern blot Probe

<400>24

<210>25

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC21065

<400>25

<210>26

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC21067

<400>26

<210>27

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24200

<400>27

<210>28

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24201

<400>28

<210>29

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24198

<400>29

<210>30

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24199

<400>30

<210>31

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24271

<400>31

<210>32

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24272

<400>32

<210>33

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24495

<400>33

<210>34

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC24496

<400>34

<210>35

<211>1090

<212>DNA

<213> human

<400>35

<210>36

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400>36

<210>37

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400>37

<210>38

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC17251

<400>38

<210>39

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC17252

<400>39

<210>40

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC17156

<400>40

<210>41

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC17157

<400>41

<210>42

<211>813

<212>DNA

<213> human

<400>42

<210>43

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC10134

<400>43

<210>44

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC10135

<400>44

<210>45

<211>768

<212>DNA

<213> human

<220>

<221>CDS

<222>(7)...(759)

<223> Ig Fc sequences

<400>45

<210>46

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15345

<400>46

<210>47

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15347

<400>47

<210>48

<211>55

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15517

<400>48

<210>49

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15530

<400>49

<210>50

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15518

<400>50

<210>51

<211>57

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide ZC15516

<400>51

<210>52

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>52

<210>53

<211>48

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>53

<210>54

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>54

<210>55

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>55

<210>56

<211>60

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>56

<210>57

<211>56

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>57

<210>58

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>58

<210>59

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> antibody peptide

<400>59

<210>60

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> antibody peptide

<400>60

Claims (10)

1. A method of inhibiting ztnf4 activity in a mammal, comprising administering to said mammal an amount of a compound selected from the group consisting of:

a) a polypeptide comprising the extracellular domain of BR43x 2;

b) a polypeptide comprising an extracellular domain of TACI;

c) a polypeptide comprising the extracellular domain of BCMA;

d) comprises the amino acid sequence shown in SEQ ID NO: 10;

e) and SEQ ID NO: 2 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

f) and SEQ ID NO: 4 or an antibody or antibody fragment to which the polypeptide of (4) specifically binds;

g) and SEQ ID NO: 6 or an antibody or antibody fragment to which the polypeptide of (a);

h) and SEQ ID NO: 8 or an antibody or antibody fragment that specifically binds to the polypeptide of (a);

i) and SEQ ID NO: 10 or an antibody or antibody fragment to which the polypeptide of claim 10 specifically binds;

k) SEQ ID NO: 4;

l) SEQ ID NO: 6 at amino acid residues 1-166; and

m) SEQ ID NO: 8 from 1 to 150.

2. The method according to claim 1, wherein said compound is a fusion protein consisting of a first portion and a second portion joined together by a peptide bond, said first portion comprising a polypeptide selected from the group consisting of:

j) comprises the amino acid sequence shown in SEQ ID NO: 8;

k) comprises the amino acid sequence shown in SEQ ID NO: 2 at amino acid residues 25-58;

l) comprises SEQ ID NO: 6 at amino acid residues 34-66;

m) comprises SEQ ID NO: 6 at amino acid residues 71-104;

n) comprises SEQ ID NO: 6 at amino acid residues 25-104;

o) a polypeptide comprising SEQ ID NO: 8, amino acid residues 8 to 37;

p) comprises SEQ ID NO: 8, amino acid residues 41-88;

q) comprises SEQ ID NO: 8, amino acid residues 8-88; and is

The second portion comprises another polypeptide.

3. The method according to claim 2, wherein said first portion further comprises a polypeptide selected from the group consisting of:

r) SEQ ID NO: 2 amino acid residues 59 to 120;

s) SEQ ID NO: 6 amino acid residues 105-166; and

t) SEQ ID NO: 8, 89-150 amino acid residues.

4. The method according to claim 2, wherein the first moiety is selected from the group consisting of:

u) a polypeptide comprising the extracellular domain of BR43x 2;

v) a polypeptide comprising a TACI extracellular domain; and

w) a polypeptide comprising a BCMA extracellular domain.

5. The method according to claim 2, wherein the first moiety is selected from the group consisting of:

x) SEQ ID NO: 4;

y) SEQ ID NO: 6 at amino acid residues 1-154; and

z) SEQ ID NO: 8 from position 1 to 48.

6. The method according to claim 2, wherein the second portion is a heavy chain constant region of an immunoglobulin.

7. The method according to claim 1, wherein the antibody or antibody fragment is selected from the group consisting of:

aa) polyclonal antibodies;

bb) murine monoclonal antibody;

cc) a humanized antibody derived from b); and

dd) human monoclonal antibodies.

8. The method according to claim 7, wherein the antibody fragment is selected from the group consisting of: f (ab '), F (ab), Fab', Fab, Fv, scFv and minimal recognition units.

9. The method according to claim 1, wherein the mammal is a primate.

10. The method according to claim 1, wherein said ztnf4 activity is associated with B lymphocytes.