Abstract:
The present invention concerns therapeutic agents that modulate the activity of TALL-1. In accordance with the present invention, modulators of TALL-1 may comprise an amino acid sequence Dz 2 Lz 4  wherein z 2  is an amino acid residue and z 4  is threonyl or isoleucyl. Exemplary molecules comprise a sequence of the formulae 
                         (SEQ. ID. NO: 100)         a 1 a 2 a 3 CDa 6 La 8 a 9 a 10 Ca 12 a 13 a 14 ,                   (SEQ. ID. NO: 104)         b 1 b 2 b 3 Cb 6 Db 8 Lb 10 b 11 b 12 b 13 b 14 Cb 16 b 17 b 18                     (SEQ. ID. NO: 105)         c 1 c 2 c 3 Cc 5 Dc 7 Lc 9 c 10 c 11 c 12 c 13 c 14 Cc 16 c 17 c 18                     (SEQ. ID. NO: 106)         d 1 d 2 d 3 Cd 5 d 6 d 7 WDd 10 Ld 13 d 14 d 15 Ld 16 d 17 d 18                     (SEQ. ID. NO: 107)         e 1 e 2 e 3 Ce 5 e 6 e 7 De 9 Le 11 Ke 13 ce 15 e 16 e 17 e 18                     (SEQ. ID NO: 109)         f 1 f 2 f 3 Kf 5 Df 7 Lf 9 f 10 Qf 12 f 13 f 14                                 
wherein the substituents are as defined in the specification. The invention further comprises compositions of matter of the formula
 
       (X 1 ) a —V 1 —(X 2 ) b  
 
     wherein V1 is a vehicle that is covalently attached to one or more of the above TALL-1 modulating compositions of matter. The vehicle and the TALL-1 modulating composition of matter may be linked through the N- or C-terminus of the TALL-1 modulating portion. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain.

Description:
RELATED APPLICATIONS 
       [0001]    The present application is a continuation of U.S. patent application Ser. No. 13/938,141, filed Jul. 9, 2013, which is a divisional of U.S. patent application Ser. No. 12/788,137, filed May 26, 2010, issued as U.S. Pat. No. 8,507,426, issued Aug. 13, 2013, which is a continuation of U.S. patent application Ser. No. 11/272,521, filed Nov. 10, 2005, issued as U.S. Pat. No. 7,737,111, issued Jun. 15, 2010, which is a divisional of U.S. patent application Ser. No. 10/145,206, filed May 13, 2002, issued as U.S. Pat. No. 7,259,137, issued Aug. 21, 2007, which claims priority to U.S. Provisional Application No. 60/290,196, filed May 11, 2001, the disclosures of which are incorporated by reference herein in their entirety, including drawings. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    After years of study in necrosis of tumors, tumor necrosis factors (TNFs) α and β were finally cloned in 1984. The ensuing years witnessed the emergence of a superfamily of TNF cytokines, including fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L), TNF-related apoptosis-inducing ligand (TRAIL, also designated AGP-1), osteoprotegerin binding protein (OPG-BP or OPG ligand), 4-1BB ligand, LIGHT, APRIL, and TALL-1. Smith et al. (1994), Cell 76: 959-962; Lacey et al. (1998), Cell 93: 165-176; Chichepotiche et al. (1997), J. Biol. Chem. 272: 32401-32410; Mauri et al. (1998), Immunity 8: 21-30; Hahne et al. (1998), J. Exp. Med. 188: 1185-90; Shu et al. (1999), J. Leukocyte Biology 65: 680-3. This family is unified by its structure, particularly at the C-terminus. In addition, most members known to date are expressed in immune compartments, although some members are also expressed in other tissues or organs, as well. Smith et al. (1994), Cell 76: 959-62. All ligand members, with the exception of LT-α, are type II transmembrane proteins, characterized by a conserved 150 amino acid region within C-terminal extracellular domain. Though restricted to only 20-25% identity, the conserved 150 amino acid domain folds into a characteristic β-pleated sheet sandwich and trimerizes. This conserved region can be proteolytically released, thus generating a soluble functional form. Banner et al. (1993), Cell 73: 431-445. 
         [0003]    Many members within this ligand family are expressed in lymphoid enriched tissues and play important roles in the immune system development and modulation. Smith et al. (1994). For example, TNFα is mainly synthesized by macrophages and is an important mediator for inflammatory responses and immune defenses. Tracey &amp; Cerami (1994), Ann. Rev. Med. 45: 491-503. Fas-L, predominantly expressed in activated T cell, modulates TCR-mediated apoptosis of thymocytes. Nagata, S. &amp; Suda, T. (1995) Immunology Today 16: 39-43; Castrim et al. (1996), Immunity 5: 617-27. CD40L, also expressed by activated T cells, provides an essential signal for B cell survival, proliferation and immunoglobulin isotype switching. Noelle (1996), Immunity 4: 415-9. 
         [0004]    The cognate receptors for most of the TNF ligand family members have been identified. These receptors share characteristic multiple cysteine-rich repeats within their extracellular domains, and do not possess catalytic motifs within cytoplasmic regions. Smith et al. (1994). The receptors signal through direct interactions with death domain proteins (e.g. TRADD, FADD, and RIP) or with the TRAF proteins (e.g. TRAF2, TRAF3, TRAF5, and TRAF6), triggering divergent and overlapping signaling pathways, e.g. apoptosis, NF- K B activation, or JNK activation. Wallach et al. (1999), Annual Review of Immunology 17: 331-67. These signaling events lead to cell death, proliferation, activation or differentiation. The expression profile of each receptor member varies. For example, TNFR1 is expressed on a broad spectrum of tissues and cells, whereas the cell surface receptor of OPGL is mainly restricted to the osteoclasts. Hsu et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3540-5. 
         [0005]    A number of research groups have recently identified TNF family ligands with the same or substantially similar sequence. The ligand has been variously named neutrokine a (WO 98/18921, published May 7, 1998), 63954 (WO 98/27114, published Jun. 25, 1998), TL5 (EP 869 180, published Oct. 7, 1998), NTN-2 (WO 98/55620 and WO 98/55621, published Dec. 10, 1998), TNRL1-alpha (WO 9911791, published Mar. 11, 1999), kay ligand (WO99/12964, published Mar. 18, 1999), and AGP-3 (U.S. Prov. App. Nos. 60/119,906, filed Feb. 12, 1999 and 60/166,271, filed Nov. 18, 1999, respectively); and TALL-1 (WO 00/68378, published Nov. 16, 2000). Each of these references is hereby incorporated by reference. Hereinafter, the ligands reported therein are collectively referred to as TALL-1. 
         [0006]    TALL-1 is a member of the TNF ligand superfamily that is functionally involved in B cell survival and proliferation. Transgenic mice overexpressing TALL-1 had severe B cell hyperplasia and lupus-like autoimmune disease. Khare et al. (2000) PNAS 97(7):3370-3375). Both TACI and BCMA serve as cell surface receptors for TALL-1. Gross et al. (2000), Nature 404: 995-999; Ware (2000), J. Exp. Med. 192(11): F35-F37; Ware (2000), Nature 404: 949-950; Xia et al. (2000), J. Exp. Med. 192(1):137-143; Yu et al. (2000), Nature Immunology 1(3):252-256; Marsters et al. (2000), Current Biology 10:785-788; Hatzoglou et al. (2000) J. of Immunology 165:1322-1330; Shu et al. (2000) PNAS 97(16):9156-9161; Thompson et al. (2000) J. Exp. Med. 192(1):129-135; Mukhopadhyay et al. (1999) J. Biol. Chem. 274(23): 15978-81; Shu et al. (1999) J. Leukocyte Biol. 65:680-683; Gruss et al. (1995) Blood 85(12): 3378-3404; Smith et al. (1994), Cell 76: 959-962; U.S. Pat. No. 5,969,102, issued Oct. 19, 1999; WO 00/67034, published Nov. 9, 2000; WO 00/40716, published Jul. 13, 2000; WO 99/35170, published Jul. 15, 1999. Both receptors are expressed on B cells and signal through interaction with TRAF proteins. In addition, both TACI and BCMA also bind to another TNF ligand family member, APRIL. Yu et al. (2000), Nature Immunology 1(3):252-256. APRIL has also been demonstrated to induce B cell proliferation. 
         [0007]    To date, no recombinant or modified proteins employing peptide modulators of TALL-1 have been disclosed. Recombinant and modified proteins are an emerging class of therapeutic agents. Useful modifications of protein therapeutic agents include combination with the “Fc” domain of an antibody and linkage to polymers such as polyethylene glycol (PEG) and dextran. Such modifications are discussed in detail in a patent application entitled, “Modified Peptides as Therapeutic Agents,” publicshed WO 00/24782, which is hereby incorporated by reference in its entirety. 
         [0008]    A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”). 
         [0009]    Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference in its entirety). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an immobilized target protein. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24. 
         [0010]    Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity. 
         [0011]    Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in  E. coli . Another  E. coli -based method allows display on the cell&#39;s outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “ E. coli  display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ peptides linked to RNA; for example, PROfusion technology, Phylos, Inc. See, for example, Roberts &amp; Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells &amp; Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62. Conceptually, one may discover peptide mimetics of any protein using phage display, RNA-peptide screening, and the other methods mentioned above. 
       SUMMARY OF THE INVENTION 
       [0012]    The present invention concerns therapeutic agents that modulate the activity of TALL-1. In accordance with the present invention, modulators of TALL-1 may comprise an amino acid sequence Dz 2 Lz 4  (SEQ ID NO: 108) wherein z 2  is an amino acid residue and z 4  is threonyl or isoleucyl. Such modulators of TALL-1 comprise molecules of the following formulae: 
         [0000]                            I(a)            (SEQ. ID. NO: 100)           a 1 a 2 a 3 CDa 6 La 8 a 9 a 10 Ca 12 a 13 a 14              
wherein:
       a 1 , a 2 , a 3  are each independently absent or amino acid residues;   a 6  is an amino acid residue;   a 9  is a basic or hydrophobic residue;   a 8  is threonyl or isoleucyl;   a 12  is a neutral hydrophobic residue; and   a 13  and a 14  are each independently absent or amino acid residues.       
 
         [0000]                            I(b)            (SEQ. ID. NO: 104)           b 1 b 2 b 3 Cb 5 b 6 Db 8 Lb 10 b 11 b 12 b 13 b 14 Cb 16 b 17 b 18              
wherein:
       b 1  and b 2  are each independently absent or amino acid residues;   b 3  is an acidic or amide residue;   b 5  is an amino acid residue;   b 6  is an aromatic residue;   b 8  is an amino acid residue;   b 10  is T or I;   b 11  is a basic residue;   b 12  and b 13  are each independently amino acid residues;   b 14  is a neutral hydrophobic residue; and   b 16 , b 17 , and b 18  are each independently absent or amino acid residues.       
 
         [0000]                            I(c)           (SEQ. ID. NO: 105)           c 1 c 2 c 3 Cc 5 Dc 7 Lc 9 c 10 c 11 c 12 c 13 c 14 Cc 16 c 17 c 18              
wherein:
       c 1 , c 2 , and c 3  are each independently absent or amino acid residues;   c 5  is an amino acid residue;   c 7  is an amino acid residue;   c 9  is T or I;   c 10  is a basic residue;   c 11  and c 12  are each independently amino acid residues;   c 13  is a neutral hydrophobic residue;   c 14  is an amino acid residue;   c 16  is an amino acid residue;   c 17  is a neutral hydrophobic residue; and   c 18  is an amino acid residue or is absent.       
 
         [0000]                            I(d)           (SEQ. ID. NO: 106)           d 1 d 2 d 3 Cd 5 d 6 d 7 WDd 10 Ld 12 d 13 d 14 Cd 15 d 16 d 17              
wherein:
       d 1 , d 2 , and d 3  are each independently absent or amino acid residues;   d 5 , d 6 , and d 7  are each independently amino acid residues;   d 10  is an amino acid residue;   d 13  is T or I;   d 14  is an amino acid residue; and   d 16 , d 17 , and d 18  are each independently absent or amino acid residues.       
 
         [0000]                            I(e)           (SEQ. ID. NO: 107)           e 1 e 2 e 3 Ce 5 e 6 e 7 De 9 Le 11 Ke 13 Ce 15 e 16 e 17 e 18              
wherein:
       e 1 , e 2 , and e 3  are each independently absent or amino acid residues;   e 5 , e 6 , e 7 , e 9 , and e 13  are each independently amino acid residues;   e 11  is T or I; and   e 15 , e 16 , and e 17  are each independently absent or amino acid residues.       
 
         [0000]                            I(f)           (SEQ. ID NO: 109)           f 1 f 2 f 3 Kf 5 Df 7 Lf 9 f 10 Qf 12 f 13 f 14              
wherein:
       f 1 , f 2 , and f 3  are absent or are amino acid residues (with one of f 1 , f 2 , and f 3  preferred to be C when one of f 12 , f 13 , and f 14  is C);   f 5  is W, Y, or F (W preferred);   f 7  is an amino acid residue (L preferred);   f 9  is T or I (T preferred);   f 10  is K, R, or H (K preferred);   f 12  is C, a neutral hydrophobic residue, or a basic residue (W, C, or R preferred);   f 13  is C, a neutral hydrophobic residue or is absent (V preferred); and   f 14  is any amino acid residue or is absent;   provided that only one of f 1 , f 2 , and f 3  may be C, and only one of f 12 , f 13 , and f 14  may be C.       
 
         [0059]    Compounds of formulae I(a) through I(f) above incorporate Dz 2 Lz 4 , as well as SEQ ID NO: 63 hereinafter. The sequence of I(f) was derived as a consensus sequence as described in Example 1 hereinbelow. Of compounds within formula I(f), those within the formula 
         [0000]                            I(f&#39;)           (SEQ ID NO: 125)           f 1 f 2 f 3 KWDf 7 L 9 KQf 12 f 13 f 14              
are preferred. Compounds falling within formula I(f) include SEQ ID NOS: 32, 58, 60, 62, 63, 66, 67, 69, 70, 114, 115, 122, 123, 124, 147-150, 152-177, 179, 180, 187.
 
         [0060]    Also in accordance with the present invention are compounds having the consensus motif: 
         [0000]                            (SEQ ID NO: 110)           PFPWE            
which also bind TALL-1.
 
         [0061]    Further in accordance with the present invention are compounds of the formulae: 
         [0000]                            I(g)           (SEQ. ID. NO. 101)           g 1 g 2 g 3 Cg 5 PFg 8 Wg 10 Cg 11 g 12 g 13              
wherein:
       g 1 , g 2  and g 3  are each independently absent or amino acid residues;   g 5  is a neutral hydrophobic residue;   g 8  is a neutral hydrophobic residue;   g 10  is an acidic residue;       
 
         [0000]                            I(h)           (SEQ. ID. NO: 102)           h 1 h 2 h 3 CWh 6 h 7 WGh 10 Ch 12 h 13 h 14              
wherein:
       h 1 , h 2 , and h 3  are each independently absent or amino acid residues;   h 6  is a hydrophobic residue;   h 7  is a hydrophobic residue;   h 10  is an acidic or polar hydrophobic residue; and   h 12 , h 13 , and h 14  are each independently absent or amino acid residues.       
 
         [0000]                            I(i)           (SEQ. ID. NO: 103)           i 1 i 2 i 3 Ci 5 i 6 i 7 i 8 i 9 i 10 Ci 12 i 13 i 14              
wherein:
       i 1  is absent or is an amino acid residue;   i 2  is a neutral hydrophobic residue;   i 3  is an amino acid residue;   i 5 , i 6 , i 7 , and i 8  are each independently amino acid residues;   i 9  is an acidic residue;   i 10  is an amino acid residue;   i 12  and i 13  are each independently amino acid residues; and   i 14  is a neutral hydrophobic residue.       
 
         [0079]    The compounds defined by formulae I(g) through I(i) also bind TALL-1. 
         [0080]    Further in accordance with present invention, modulators of TALL-1 comprise:
       a) a TALL-1 modulating domain (e.g., an amino acid sequence of Formulae I(a) through I(i)), preferably the amino acid sequence Dz 2 Lz 4 , or sequences derived therefrom by phage display, RNA-peptide screening, or the other techniques mentioned above; and   b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred;
 
wherein the vehicle is covalently attached to the TALL-1 modulating domain. The vehicle and the TALL-1 modulating domain may be linked through the N- or C-terminus of the TALL-1 modulating domain, as described further below. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain. Such Fc-linked peptides are referred to herein as “peptibodies.” Preferred TALL-1 modulating domains comprise the amino acid sequences described hereinafter in Tables 1 and 2. Other TALL-1 modulating domains can be generated by phage display, RNA-peptide screening and the other techniques mentioned herein.
       
 
         [0083]    Further in accordance with the present invention is a process for making TALL-1 modulators, which comprises:
       a. selecting at least one peptide that binds to TALL-1; and   b. covalently linking said peptide to a vehicle.
 
The preferred vehicle is an Fc domain. Step (a) is preferably carried out by selection from the peptide sequences in Table 2 hereinafter or from phage display, RNA-peptide screening, or the other techniques mentioned herein.
       
 
         [0086]    The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable. 
         [0087]    The primary use contemplated for the compounds of this invention is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity comparable to—or even greater than—the natural ligand mimicked by the peptide. 
         [0088]    The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention. 
         [0089]    Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0090]      FIG. 1A  shows a single disulfide-bonded dimer with the Fc domain linked at the amino terminus of the peptides or peptide-linker combinations of X 2  as defined below.  FIG. 1B  shows a doubly disulfide bonded dimer with the Fc domain linked at the amino terminus of the peptides or peptide-linker combinations of X 2  as defined below.  FIG. 1C  shows a noncovalent dimer.  FIG. 1D  shows a single disulfide-bonded dimer with the Fc domain linked at the carboxyl terminus of the peptides or peptide-linker combinations of X 1  as defined below.  FIG. 1E  shows a double disulfide bonded dimer with the Fc domain linked at the carboxyl terminus of the peptides or peptide-linker combinations of X 1  as defined below.  FIG. 1F  shows a noncovalent dimer. IgG1 antibodies typically have two disulfide bonds at the hinge region of the antibody. The Fc domain in  FIGS. 1A and 1D  may be formed by truncation between the two disulfide bond sites or by substitution of a cysteinyl residue with an unreactive residue (e.g., alanyl). The Fc domain in  FIGS. 1B and 1E  may be formed by truncation of the parent antibody to retain both cysteinyl residues in the Fc domain chains or by expression from a construct including a sequence encoding such an Fc domain. The Fc domain in  FIGS. 1C and 1F  may be formed by elimination of the cysteinyl residues by either truncation or substitution. One may desire to eliminate the cysteinyl residues to avoid impurities formed by reaction of the cysteinyl residue with cysteinyl residues of other proteins present in the host cell. The noncovalent bonding of the Fc domains is sufficient to hold together the dimer. Other dimers may be formed by using Fc domains derived from different types of antibodies (e.g., IgG2, IgM). 
           [0091]      FIG. 2A  shows the structure of a single chain molecule featuring tandem repeats of the pharmacologically peptides P 1  and P 2  as defined below, and may also represent the DNA construct for the molecule.  FIG. 2B  shows a dimer in which the linker-peptide portion is present on only one chain of the dimer.  FIG. 2C  shows a dimer having the peptide portion on both chains. The dimer of  FIG. 2C  will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in  FIG. 3A . In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro. 
           [0092]      FIGS. 3A  and B show exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in this invention.  FIG. 3A  shows nucleotides 1-360 and encoded amino acids 1-120.  FIG. 3B  shows nucleotides 361-684 and encoded amino acids 121-228. 
           [0093]      FIGS. 4A-F  show the nucleotide and amino acid sequences (SEQ ID NOS: 3-26) of NdeI to SalI fragments encoding peptide and linker.  FIG. 4A  shows the nucleotide and amino acid sequences of AGP3-8-1-a (SEQ ID NOs:3 and 4) and AGP3-8-2-a (SEQ ID NOs:5 and 6).  FIG. 4B  shows the nucleotide and amino acid sequences of AGP3-8-4-a (SEQ ID NOs:7 and 8) and AGP3-12-4-a (SEQ ID NOs:9 and 10).  FIG. 4C  shows the nucleotide and amino acid sequences of AGP3-12-3-a (SEQ ID NOs:11 and 12) and AGP3-12-5-a (SEQ ID NOs:13 and 14).  FIG. 4D  shows the nucleotide and amino acid sequences of AGP3-12-8-a (SEQ ID NOs:15 and 16) and AGP3-12-9-a (SEQ ID NOs:17 and 18).  FIG. 4E  shows the nucleotide and amino acid sequences of AGP3-12-10-a (SEQ ID NOs:19 and 20) and AGP3-12-11-a (SEQ ID NOs:21 and 22).  FIG. 4F  shows the nucleotide and amino acid sequences of AGP3-12-14-a (SEQ ID NOs:23 and 24) and AGP3 consensus (SEQ ID NOs:25 and 26). 
           [0094]      FIGS. 5A-M  show the nucleotide sequence (SEQ ID NO: 28) of pAMG21-RANK-Fc vector, which was used to construct Fc-linked molecules of the present invention.  FIG. 5A  shows nucleotides 1-480, which include enzyme restriction sites Pfl 1108I, GblII, and ScaI, promoter region PcopB, and the coding region and corresponding encoded amino acid sequence for copB protein.  FIG. 5B  shows nucleotides 481-560, which include enzyme restriction sites BmnI, DrdII, and DraIII, promoter region PrepA, a binding site for copB, and the coding regions and corresponding encoded amino acid sequences for copT and repA1.  FIG. 5C  shows nucleotides 961-1500, which include enzyme restriction sites BstBI, AceIII, and AflII.  FIG. 5D  shows nucleotides 1501-2220, which include enzyme restriction site PflMI and the coding region and corresponding encoded amino acid sequence for repA4.  FIG. 5E  shows nucleotides 2221-2760, which include enzyme restriction sites BglI, SfiI, BstEII, and BspLullI.  FIG. 5F  shows nucleotides 2761-3480, which include enzyme restriction sites NspV and BplI and the coding region and corresponding encoded amino acid sequence for APHII.  FIG. 5G  shows nucleotides 3481-4080, which include enzyme restriction sites EagI and BcgI, promoter region APHII, APHII mRNA, and the coding region and corresponding encoded amino acid sequence for APHII.  FIG. 5H  shows nucleotides 4081-4620, which include enzyme restriction sites NsiI, BsaI, and Psp1406I and T1 and T2 hairpins.  FIG. 5I  shows nucleotides 4621-5220, which include enzyme restriction sites AatII and BsmI and the coding region and corresponding encoded amino acid sequence for luxR.  FIG. 5J  shows nucleotides 5221-5760, which include enzyme restriction sites NruI, ClaI, BbaI, and NdeI, the coding region and corresponding amino acid sequence of luxR and RANK, promoter regions luxPL and luxPR, and a CRP binding site.  FIG. 5K  shows nucleotides 5761-6240, which include enzyme restriction sites ApaLI, Acc65I, KpnI, SalI, and AccI, and the coding region and corresponding amino acid sequence for Fc.  FIG. 5L  shows nucleotides 6241-6780, which include enzyme restriction sites BspEI, AhdI, BspHI, EconI, BsrGI, BmaI, SmaI, and SexAI.  FIG. 5M  shows nucleotides 6781-7285, which include enzyme restriction sites BamHI and BlpI and T7 and toop hairpins. 
           [0095]      FIGS. 6A  and B show the DNA sequence (SEQ ID NO: 97) inserted into pCFM1656 between the unique  Aat II (position #4364 in pCFM1656) and  Sac II (position #4585 in pCFM1656) restriction sites to form expression plasmid pAMG21 (ATCC accession no. 98113).  FIG. 6A  shows the first part of the DNA sequence.  FIG. 6B  shows the second part of the DNA sequence. 
           [0096]      FIG. 7  shows that the TALL-1 peptibody (SEQ ID NO: 70) inhibits TALL-1-mediated B cell proliferation. Purified B cells (10 5 ) from B6 mice were cultured in triplicates in 96-well plated with the indicated amounts of TALL-1 consensus peptibody in the presence of 10 ng/ml TALL-1 plus 2 μg/ml anti-IgM antibody. Proliferation was measured by radioactive [ 3 H]thymidine uptake in the last 18 h of pulse. Data shown represent mean±SD triplicate wells. 
           [0097]      FIG. 8  shows that a TALL-1 N-terminal tandem dimer peptibodies (SEQ ID NO: 123, 124 in Table 5B hereinafter) are preferable for inhibition of TALL-1-mediated B cell proliferation. Purified B cells (10 5 ) from B6 mice were cultured in triplicates in 96-well plated with the indicated amounts of TALL-1 12-3 peptibody and TALL-1 consensus peptibody (SEQ ID NOS: 115 and 122 of Table 5B) or the related dimer peptibodies (SEQ ID NOS: 123, 124) in the presence of 10 μg/ml TALL-1 plus 2 μg/ml anti-IgM antibody. Proliferation was measured by radioactive [ 3 H]thymidine uptake in the last 18 h of pulse. Data shown represent mean±SD triplicate wells. 
           [0098]      FIG. 9  shows that AGP3 peptibody binds to AGP3 with high affinity. Dissociation equilibrium constant (K D ) was obtained from nonlinear regression of the competition curves using a dual-curve one-site homogeneous binding model (KinEx™ software). K D  is about 4 pM for AGP3 peptibody binding with human AGP3 (SEQ ID NO: 123). 
           [0099]      FIGS. 10A  and B show that AGP3 peptibody blocks both human and murine AGP3 in the Biacore competition assay. Soluble human TACI protein was immobilized to B1 chip. 1 nM of recombinant human AGP3 protein ( FIG. 10A ) or 5 nM of recombinant murine AGP3 protein ( FIG. 10B ) was incubated with indicated amount of AGP3 peptibody before injected over the surface of receptor. Relative human AGP3 and murine AGP3 (binding response was shown (SEQ ID NO: 123).  FIG. 10A  shows results for human AGP3.  FIG. 10B  shows results for murine AGP3. 
           [0100]      FIG. 11A  shows that AGP3 peptibody blocked AGP3 binding to all three receptors TACI, BCMA and BAFFR in Biacore competition assay. Recombinant soluble receptor TACI, BCMA and BAFFR proteins were immobilized to CM5 chip. 1 nM of recombinant human AGP3 (upper panel) were incubated with indicated amount of AGP3 peptibody before injected over each receptor surface. Relative binding of AGP3 was measured. Similarly, 1 nM of recombinant APRIL protein was incubated with indicated amount of AGP3 peptibody before injected over each receptor surface.  FIG. 11B  shows that AGP3 peptibody didn&#39;t inhibit APRIL binding to all three receptors (SEQ ID NO: 123). 
           [0101]      FIGS. 12A  and B show that AGP3 peptibody inhibits mouse serum immunoglobulin level increase induced by human AGP3 challenge. Balb/c mice received 7 daily intraperitoneal injections of 1 mg/Kg human AGP3 protein along with saline, human Fc, or AGP3 peptibody at indicated doses, and were bled on day 8. Serum total IgM and IgA level were measured by ELISA (SEQ ID NO: 123).  FIG. 12A  shows IgM levels.  FIG. 12B  shows IgA levels. 
           [0102]      FIG. 13  shows that AGP3 peptibody treatment reduced arthritis severity in the mouse CIA model. Eight to 12 weeks old DBA/1 male mice were immunized with bovine collagen type II (bCII) emulsified in complete freunds adjuvant intradermally at the base of tail, and were boosted 3 weeks after the initial immunization with bCII emulsified in incomplete freunds adjuvant. Treatment with indicated dosage of AGP3 peptibody was begun from the day of booster immunization for 4 weeks. As described before (Khare et al.,  J. Immunol.  155: 3653-9, 1995), all four paws were individually scored from 0-3 for arthritis severity (SEQ ID NO: 123). 
           [0103]      FIG. 14  shows that AGP3 peptibody treatment inhibited anti-collagen antibody generation in the mouse CIA model. Serum samples were taken one week after final treatment (day 35) as described above. Serum anti-collagen II antibody level was determined by ELISA analysis (SEQ ID NO: 123). 
           [0104]      FIGS. 15A  and B show that AGP3 peptibody treatment delayed proteinuria onset and improved survival in NZB/NZW lupus mice. Five-month-old lupus prone NZBx NZBWF1 mice were treated i.p. 3×/week for 8 weeks with PBS or indicated doses of AGP3 peptibody (SEQ ID NO: 123) or human Fc proteins. Protein in the urine was evaluated monthly throughout the life of the experiment with Albustix reagent strips (Bayer AG).  FIG. 15A  shows delayed proteinuria onset.  FIG. 15B  shows prolonged survival. 
           [0105]      FIG. 16A  shows the nucleic acid and amino acid sequences of a preferred TALL-1-binding peptibody (SEQ ID NOS: 189 and 123).  FIG. 16A  shows nucleotides 1-480 and amino acids 1-160.  FIG. 16B  shows nucleotides 481-882 and amino acids 161-293. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definition of Terms 
       [0106]    The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. 
       General Definitions 
       [0107]    The term “comprising” means that a compound may include additional amino acids on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compound. 
         [0108]    Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. The term “physiologically acceptable salts” refers to any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate. 
         [0109]    Amino Acids 
         [0110]    The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E. 
         [0111]    The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q. 
         [0112]    The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W. 
         [0113]    The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, and R. 
         [0114]    The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q. 
         [0115]    The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle). 
         [0116]    The term “neutral hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral hydrophobic amino acid residues include A, V, L, I, P, W, M, and F. 
         [0117]    The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N. 
         [0118]    The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N. 
         [0119]    Peptides 
         [0120]    The term “peptide” refers to molecules of 1 to 40 amino acids, with molecules of 5 to 20 amino acids preferred. Exemplary peptides may comprise the TALL-1 modulating domain of a naturally occurring molecule or comprise randomized sequences. 
         [0121]    The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods or RNA-peptide screening) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display,  E. coli  display, ribosome display, RNA-peptide screening, chemical screening, and the like. 
         [0122]    The term “TALL-1 modulating domain” refers to any amino acid sequence that binds to the TALL-1 and comprises naturally occurring sequences or randomized sequences. Exemplary TALL-1 modulating domains can be identified or derived by phage display or other methods mentioned herein. 
         [0123]    The term “TALL-1 antagonist” refers to a molecule that binds to the TALL-1 and increases or decreases one or more assay parameters opposite from the effect on those parameters by full length native TALL-1. Such activity can be determined, for example, by such assays as described in the subsection entitled “Biological activity of AGP-3” in the Materials &amp; Methods section of the patent application entitled, “TNF-RELATED PROTEINS”, WO 00/47740, published Aug. 17, 2000. 
         [0124]    Vehicles and Peptibodies 
         [0125]    The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain (which is preferred) as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide (e.g., dextran); any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor; albumin, including human serum albumin (HSA), leucine zipper domain, and other such proteins and protein fragments. Vehicles are further described hereinafter. 
         [0126]    The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc&#39;s are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms. 
         [0127]    The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference in their entirety. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter. 
         [0128]    The term “Fe domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fe&#39;s, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. 
         [0129]    The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc. 
         [0130]    The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in  FIG. 1 . 
         [0131]    The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR 1 , NRC(O)R 1 , —NRC(O)OR 1 , —NRS(O) 2 R 1 , —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R 1  and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R 2  or —NR 3 R 4  wherein R 2 , R 3  and R 4  are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter. 
         [0132]    The terms “peptibody” and “peptibodies” refer to molecules comprising an Fc domain and at least one peptide. Such peptibodies may be multimers or dimers or fragments thereof, and they may be derivatized. In the present invention, the molecules of formulae II through VI hereinafter are peptibodies when VI is an Fc domain. 
         [0133]    Structure of Compounds 
         [0134]    In General. 
         [0135]    The present inventors identified sequences capable of binding to and modulating the biological activity of TALL-1. These sequences can be modified through the techniques mentioned above by which one or more amino acids may be changed while maintaining or even improving the binding affinity of the peptide. 
         [0136]    In the compositions of matter prepared in accordance with this invention, the peptide(s) may be attached to the vehicle through the peptide&#39;s N-terminus or C-terminus. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers. Thus, the vehicle-peptide molecules of this invention may be described by the following formula: 
         [0000]      (X 1 ) a —V 1 —(X 2 ) b   II
 
         [0000]    wherein:
       V 1  is a vehicle (preferably an Fc domain);   X 1  and X 2  are each independently selected from -(L 1 ) c -P 1 , -(L 1 ) c -P 1 -(L 2 ) d -P 2 , -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 , and -(L 2 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 -(L 4 ) f -P 4      P 3  and P 4  are each independently sequences of TALL-1 modulating domains, such as those of Formulae I(a) through I(i);   L 1 , L 2 , L 3 , and L 4  are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.       
 
         [0141]    Thus, compound II comprises preferred compounds of the formulae 
         [0000]      X 1 —V 1   III
 
         [0000]    and multimers thereof wherein V 1  is an Fc domain and is attached at the C-terminus of A 1 ; 
         [0000]      V 1 —X 2   IV
 
         [0000]    and multimers thereof wherein V 1  is an Fc domain and is attached at the N-terminus of A 2 ; 
         [0000]      V 1 -(L 1 ) c P 1   V
 
         [0000]    and multimers thereof wherein V 1  is an Fc domain and is attached at the N-terminus of -(L 1 ) c -P 1 ; and 
         [0000]      V 1 -(L 1 ) c -P 1 -(L 2 ) d -P 2   VI
 
         [0000]    and multimers thereof wherein V 1  is an Fc domain and is attached at the N-terminus of -L 1 -P 1 -L 2 -P 2 . 
         [0142]    Peptides. 
         [0143]    The peptides of this invention are useful as TALL-1 modulating peptides or as TALL-1 modulating domains in the molecules of formulae II through VI. Molecules of this invention comprising these peptide sequences may be prepared by methods known in the art. 
         [0144]    Preferred peptide sequences are those of the foregoing formulae I(a) having the substituents identified below. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Preferred peptide substituents 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Formula I(a) 
                 a 8  is T; 
               
               
                   
                 a 9  is a basic residue (K most preferred); and 
               
               
                   
                 a 12  is a neutral hydrophobic residue (F most preferred). 
               
               
                 Formula I(b) 
                 b 3  is D, Q, or E; 
               
               
                   
                 b 6  is W or Y; 
               
               
                   
                 b 10  is T; 
               
               
                   
                 b 11  is K or R; and 
               
               
                   
                 b 14  is V or L. 
               
               
                 Formula I(c) 
                 c 9  is T; 
               
               
                   
                 c 10  is K or R; 
               
               
                   
                 c 13  is a I, L, or V; and 
               
               
                   
                 c 17  is A or L. 
               
               
                 Formula I(d) 
                 d 13  is T. 
               
               
                 Formula I(e) 
                 e 11  is T. 
               
               
                 Formula I(f) 
                 f 6  is T; 
               
               
                   
                 f 7  is K; and 
               
               
                   
                 f 10  is V. 
               
               
                 Formula I(g) 
                 g 5  is W; 
               
               
                   
                 g 8  is P; 
               
               
                   
                 g 10  is E; and 
               
               
                   
                 g 13  is a basic residue. 
               
               
                 Formula I(h) 
                 h 1  is G; 
               
               
                   
                 h 6  is A; 
               
               
                   
                 h 7  is a neutral hydrophobic residue; and 
               
               
                   
                 h 10  is an acidic residue. 
               
               
                 Formula I(i) 
                 i 2  is W; and 
               
               
                   
                 i 14  is W. 
               
               
                   
               
             
          
         
       
     
         [0145]    Preferred peptide sequences appear in Table 2 below. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2  
               
             
             
               
                   
               
               
                 Preferred TALL-1 modulating domains 
               
             
          
           
               
                   
                   
                 SEQ 
               
               
                   
                 Sequence 
                 ID NO: 
               
               
                   
                   
               
             
          
           
               
                   
                 PGTCFPFPWECTHA 
                 29 
               
               
                   
                   
               
               
                   
                 WGACWPFPWECFKE 
                 30 
               
               
                   
                   
               
               
                   
                 VPFCDLLTKHCFEA 
                 31 
               
               
                   
                   
               
               
                   
                 GSRCKYKWDVLTKQCFHH 
                 32 
               
               
                   
                   
               
               
                   
                 LPGCKWDLLIKQWVCDPL 
                 33 
               
               
                   
                   
               
               
                   
                 SADCYFDILTKSDVCTSS 
                 34 
               
               
                   
                   
               
               
                   
                 SDDCMYDQLTRMFICSNL 
                 35 
               
               
                   
                   
               
               
                   
                 DLNCKYDELTYKEWCQFN 
                 36 
               
               
                   
                   
               
               
                   
                 FHDCKYDLLTRQMVCHGL 
                 37 
               
               
                   
                   
               
               
                   
                 RNHCFWDHLLKQDICPSP 
                 38 
               
               
                   
                   
               
               
                   
                 ANQCWWDSLTKKNVCEFF 
                 39 
               
               
                   
                   
               
               
                   
                 YKGRQMWDILTRSWVVSL 
                 126 
               
               
                   
                   
               
               
                   
                 QDVGLWWDILTRAWMPNI 
                 127 
               
               
                   
                   
               
               
                   
                 QNAQRVWDLLIRTWVYPQ 
                 128 
               
               
                   
                   
               
               
                   
                 GWNEAWWDELTKIWVLEQ 
                 129 
               
               
                   
                   
               
               
                   
                 RITCDTWDSLIKKCVPQS 
                 130 
               
               
                   
                   
               
               
                   
                 GAIMQFWDSLTKTWLRQS 
                 131 
               
               
                   
                   
               
               
                   
                 WLHSGWWDPLTKHWLQKV 
                 132 
               
               
                   
                   
               
               
                   
                 SEWFFWFDPLTRAQLKFR 
                 133 
               
               
                   
                   
               
               
                   
                 GVWFWWFDPLTKQWTQAG 
                 134 
               
               
                   
                   
               
               
                   
                 MQCKGYYDILTKWCVTNG 
                 135 
               
               
                   
                   
               
               
                   
                 LWSKEVWDILTKSWVSQA 
                 136 
               
               
                   
                   
               
               
                   
                 KAAGWWFDWLTKVWVPAP 
                 137 
               
               
                   
                   
               
               
                   
                 AYQTWFWDSLTRLWLSTT 
                 138 
               
               
                   
                   
               
               
                   
                 SGQHFWWDLLTRSWTPST 
                 139 
               
               
                   
                   
               
               
                   
                 LGVGQKWDPLTKQWVSRG 
                 140 
               
               
                   
                   
               
               
                   
                 VGKMCQWDPLIKRTVCVG 
                 141 
               
               
                   
                   
               
               
                   
                 CRQGAKFDLLTKQCLLGR 
                 142 
               
               
                   
                   
               
               
                   
                 GQAIRHWDVLTKQWVDSQ 
                 143 
               
               
                   
                   
               
               
                   
                 RGPCGSWDLLTKHCLDSQ 
                 144 
               
               
                   
                   
               
               
                   
                 WQWKQQWDLLTKQMVWVG 
                 145 
               
               
                   
                   
               
               
                   
                 PITICRKDLLTKQVVCLD 
                 146 
               
               
                   
                   
               
               
                   
                 KTCNGKWDLLTKQCLQQA 
                 147 
               
               
                   
                   
               
               
                   
                 KCLKGKWDLLTKQCVTEV 
                 148 
               
               
                   
                   
               
               
                   
                 RCWNGKWDLLTKQCIHPW 
                 149 
               
               
                   
                   
               
               
                   
                 NRDMRKWDPLIKQWIVRP 
                 150 
               
               
                   
                   
               
               
                   
                 QAAAATWDLLTKQWLVPP 
                 151 
               
               
                   
                   
               
               
                   
                 PEGGPKWDPLTKQFLPPV 
                 152 
               
               
                   
                   
               
               
                   
                 QTPQKKWDLLTKQWFTRN 
                 153 
               
               
                   
                   
               
               
                   
                 IGSPCKWDLLTKQMICQT 
                 154 
               
               
                   
                   
               
               
                   
                 CTAAGKWDLLTKQCIQEK 
                 155 
               
               
                   
                   
               
               
                   
                 VSQCMKWDLLTKQCLQGW 
                 156 
               
               
                   
                   
               
               
                   
                 VWGTWKWDLLTKQYLPPQ 
                 157 
               
               
                   
                   
               
               
                   
                 GWWEMKWDLLTKQWYRPQ 
                 158 
               
               
                   
                   
               
               
                   
                 TAQVSKWDLLTKQWLPLA 
                 159 
               
               
                   
                   
               
               
                   
                 QLWGTKWDLLTKQYIQIM 
                 160 
               
               
                   
                   
               
               
                   
                 WATSQKWDLLTKQWVQNM 
                 161 
               
               
                   
                   
               
               
                   
                 QRQCAKWDLLTKQCVLFY 
                 162 
               
               
                   
                   
               
               
                   
                 KTTDCKWDLLTKQRICQV 
                 163 
               
               
                   
                   
               
               
                   
                 LLCQGKWDLLTKQCLKLR 
                 164 
               
               
                   
                   
               
               
                   
                 LMWFWKWDLLTKQLVPTF 
                 165 
               
               
                   
                   
               
               
                   
                 QTWAWKWDLLTKQWIGPM 
                 166 
               
               
                   
                   
               
               
                   
                 NKELLKWDLLTKQCRGRS 
                 167 
               
               
                   
                   
               
               
                   
                 GQKDLKWDLLTKQYVRQS 
                 168 
               
               
                   
                   
               
               
                   
                 PKPCQKWDLLTKQCLGSV 
                 169 
               
               
                   
                   
               
               
                   
                 GQIGWKWDLLTKQWIQTR 
                 170 
               
               
                   
                   
               
               
                   
                 VWLDWKWDLLTKQWIHPQ 
                 171 
               
               
                   
                   
               
               
                   
                 QEWEYKWDLLTKQWGWLR 
                 172 
               
               
                   
                   
               
               
                   
                 HWDSWKWDLLTKQWVVQA 
                 173 
               
               
                   
                   
               
               
                   
                 TRPLQKWDLLTKQWLRVG 
                 174 
               
               
                   
                   
               
               
                   
                 SDQWQKWDLLTKQWFWDV 
                 175 
               
               
                   
                   
               
               
                   
                 QQTFMKWDLLTKQWIRRH 
                 176 
               
               
                   
                   
               
               
                   
                 QGECRKWDLLTKQCFPGQ 
                 177 
               
               
                   
                   
               
               
                   
                 GQMGWRWDPLIKMCLGPS 
                 178 
               
               
                   
                   
               
               
                   
                 QLDGCKWDLLTKQKVCIP 
                 179 
               
               
                   
                   
               
               
                   
                 HGYWQKWDLLTKQWVSSE 
                 180 
               
               
                   
                   
               
               
                   
                 HQGQCGWDLLTRIYLPCH 
                 181 
               
               
                   
                   
               
               
                   
                 LHKACKWDLLTKQCWPMQ 
                 182 
               
               
                   
                   
               
               
                   
                 GPPGSVWDLLTKIWIQTG 
                 183 
               
               
                   
                   
               
               
                   
                 ITQDWRFDTLTRLWLPLR 
                 184 
               
               
                   
                   
               
               
                   
                 QGGFAAWDVLTKMWITVP 
                 185 
               
               
                   
                   
               
               
                   
                 GHGTPWWDALTRIWILGV 
                 186 
               
               
                   
                   
               
               
                   
                 VWPWQKWDLLTKQFVFQD 
                 187 
               
               
                   
                   
               
               
                   
                 WQWSWKWDLLTRQYISSS 
                 188 
               
               
                   
                   
               
               
                   
                 NQTLWKWDLLTKQFITYM 
                 60 
               
               
                   
                   
               
               
                   
                 PVYQGWWDTLTKLYIWDG 
                 61 
               
               
                   
                   
               
               
                   
                 WLDGGWRDPLIKRSVQLG 
                 62 
               
               
                   
                   
               
               
                   
                 GHQQFKWDLLTKQWVQSN 
                 63 
               
               
                   
                   
               
               
                   
                 QRVGQFWDVLTKMFITGS 
                 64 
               
               
                   
                   
               
               
                   
                 QAQGWSYDALIKIWIRWP 
                 65 
               
               
                   
                   
               
               
                   
                 GWMHWKWDPLTKQALPWM 
                 66 
               
               
                   
                   
               
               
                   
                 GHPTYKWDLLTKQWILQM 
                 67 
               
               
                   
                   
               
               
                   
                 WNNWSLWDPLTKLWLQQN 
                 68 
               
               
                   
                   
               
               
                   
                 WQWGWKWDLLTKQWVQQQ 
                 69 
               
               
                   
                   
               
               
                   
                 GQMGWRWDPLTKMWLGTS 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0146]    It is noted that the known receptors for TALL-1 bear some sequence homology with preferred peptides: 
         [0000]    
       
         
               
               
             
               
             
           
               
                 12-3 
                                        LPGCK WDLL I K QWVCDP L   
               
               
                   
               
               
                 BAFFR 
                    MRRGPRSLRGRDAPVPTPCVPTEC YDLL V R KCVDCR L L 
               
               
                   
               
               
                 TACI 
                 TICNHQSQRTCAAFCRSLSCRKEQGKF YD H L L R DCISCASI 
               
               
                   
               
               
                 BCMA 
                 FVSPSQEIRGRFRRMLQMAGQCSQNEY FD S L L H ACPCQ L RC 
               
             
          
           
               
                 (SEQ ID NOS: 33, 195, 196, and 197, respectively). 
               
             
          
         
       
     
         [0147]    Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well. Any of these peptides may be derivatized as described hereinafter. 
         [0148]    Additional useful peptide sequences may result from conservative and/or non-conservative modifications of the amino acid sequences of the sequences in Table 2. 
         [0149]    Conservative modifications will produce peptides having functional and chemical characteristics similar to those of the peptide from which such modifications are made. In contrast, substantial modifications in the functional and/or chemical characteristics of the peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule. 
         [0150]    For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alanine scanning mutagenesis). 
         [0151]    Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-peptide molecules (see preceding formulae) described herein. Exemplary amino acid substitutions are set forth in Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Amino Acid Substitutions 
               
             
          
           
               
                   
                 Original 
                 Exemplary 
                 Preferred 
               
               
                   
                 Residues 
                 Substitutions 
                 Substitutions 
               
               
                   
                   
               
               
                   
                 Ala (A) 
                 Val, Leu, Ile 
                 Val 
               
               
                   
                 Arg (R) 
                 Lys, Gln, Asn 
                 Lys 
               
               
                   
                 Asn (N) 
                 Gln 
                 Gln 
               
               
                   
                 Asp (D) 
                 Glu 
                 Glu 
               
               
                   
                 Cys (C) 
                 Ser, Ala 
                 Ser 
               
               
                   
                 Gln (Q) 
                 Asn 
                 Asn 
               
               
                   
                 Glu (E) 
                 Asp 
                 Asp 
               
               
                   
                 Gly (G) 
                 Pro, Ala 
                 Ala 
               
               
                   
                 His (H) 
                 Asn, Gln, Lys, Arg 
                 Arg 
               
               
                   
                 Ile (I) 
                 Leu, Val, Met, Ala, Phe, 
                 Leu 
               
               
                   
                   
                 Norleucine 
                   
               
               
                   
                 Leu (L) 
                 Norleucine, Ile, Val, Met, 
                 Ile 
               
               
                   
                   
                 Ala, Phe 
                   
               
               
                   
                 Lys (K) 
                 Arg, 1,4 Diamino-butyric 
                 Arg 
               
               
                   
                   
                 Acid, Gln, Asn 
                   
               
               
                   
                 Met (M) 
                 Leu, Phe, Ile 
                 Leu 
               
               
                   
                 Phe (F) 
                 Leu, Val, Ile, Ala, Tyr 
                 Leu 
               
               
                   
                 Pro (P) 
                 Ala 
                 Gly 
               
               
                   
                 Ser (S) 
                 Thr, Ala, Cys 
                 Thr 
               
               
                   
                 Thr (T) 
                 Ser 
                 Ser 
               
               
                   
                 Trp (W) 
                 Tyr, Phe 
                 Tyr 
               
               
                   
                 Tyr (Y) 
                 Trp, Phe, Thr, Ser 
                 Phe 
               
               
                   
                 Val (V) 
                 Ile, Met, Leu, Phe, Ala, 
                 Leu 
               
               
                   
                   
                 Norleucine 
               
               
                   
                   
               
             
          
         
       
     
         [0152]    In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. 
         [0153]    As noted in the foregoing section “Definition of Terms,” naturally occurring residues may be divided into classes based on common sidechain properties that may be useful for modifications of sequence. For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the peptide that are homologous with non-human orthologs, or into the non-homologous regions of the molecule. In addition, one may also make modifications using P or G for the purpose of influencing chain orientation. 
         [0154]    In making such modifications, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). 
         [0155]    The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157: 105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 
         [0156]    It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. a biological property of the protein. 
         [0157]    The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.” 
         [0158]    A skilled artisan will be able to determine suitable variants of the polypeptide as set forth in the foregoing sequences using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a peptide to similar peptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a peptide that are not conserved relative to such similar peptides would be less likely to adversely affect the biological activity and/or structure of the peptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the peptide structure. 
         [0159]    Additionally, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues that are important for activity or structure in similar peptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the peptides. 
         [0160]    One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a peptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays know to those skilled in the art. Such data could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. 
         [0161]    A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974); Chou et al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide&#39;s or protein&#39;s structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will gain dramatically in accuracy. 
         [0162]    Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl et al., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-8 (1987)), and “evolutionary linkage” (See Home, supra, and Brenner, supra). 
         [0163]    Vehicles. 
         [0164]    This invention requires the presence of at least one vehicle (V 1 ) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used; e.g., Fc&#39;s at each terminus or an Fc at a terminus and a PEG group at the other terminus or a sidechain. Exemplary vehicles include:
       an Fc domain;   other proteins, polypeptides, or peptides capable of binding to a salvage receptor;   human serum albumin (HSA);   a leucine zipper (LZ) domain;   polyethylene glycol (PEG), including 5 kD, 20 kD, and 30 kD PEG, as well as other polymers;   dextran;
 
and other molecules known in the art to provide extended half-life and/or protection from proteolytic degradation or clearance.
       
 
         [0171]    An Fc domain is the preferred vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. Fusion to the N terminus is preferred. 
         [0172]    As noted above, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:
   1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.   2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in  E. coli  such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as  E. coli . The Fc domain of SEQ ID NO: 2 is one such Fc variant.   3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.   4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).   5. Sites involved in interaction with complement, such as the Clq binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.   6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.   7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.   8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.   
 
         [0181]    Preferred Fc variants include the following. In SEQ ID NO: 2 ( FIGS. 3A  and B), the leucine at position 15 may be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues. 
         [0182]    An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display or RNA-peptide screening for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization). 
         [0183]    As noted above, polymer vehicles may also be used for V 1 . Various means for attaching chemical moieties useful as vehicles are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins. 
         [0184]    A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kD to about 50 kD, most preferably from about 5 kD to about 10 kD. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group). 
         [0185]    A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis. The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry. 
         [0186]    Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulin has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention. 
         [0187]    Linkers. 
         [0188]    Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 30 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly) 4 , (Gly) 5 ), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are: 
         [0000]                            (SEQ ID NO: 40)           (Gly) 3 Lys(Gly) 4 ;                       (SEQ ID NO: 41)           (Gly) 3 AsnGlySer(Gly) 2 ;                       (SEQ ID NO: 42)           (Gly) 3 Cys(Gly) 4 ;            and                       (SEQ ID NO: 43)           GlyProAsnGlyGly.            
To explain the above nomenclature, for example, (Gly) 3 Lys(Gly) 4  means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO: 40). Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.
 
         [0189]    Preferred linkers are amino acid linkers comprising greater than 5 amino acids, with suitable linkers having up to about 500 amino acids selected from glycine, alanine, proline, asparagine, glutamine, lysine, threonine, serine or aspartate. Linkers of about 20 to 50 amino acids are most preferred. One group of preferred linkers are those of the formulae 
         [0000]                    (SEQ ID NO: 193)       GSGSATGGSGSTASSGSGSATx 1 x 2         and       (SEQ ID NO: 194)       GSGSATGGSGSTASSGSGSATx 1 x 2 GSGSATGGSGSTASSGSGSATx 3 x 4              
wherein x 1  and x 3  are each independently basic or hydrophobic residues and x 2  and x 4  are each independently hydrophobic residues. Specific preferred linkers are:
 
         [0000]    
       
         
               
             
           
               
                 (SEQ ID NO: 59) 
               
               
                 GSGSATGGSGSTASSGSGSATHM 
               
               
                   
               
               
                 (SEQ ID NO: 190) 
               
               
                 GSGSATGGSGSTASSGSGSATGM 
               
               
                   
               
               
                 (SEQ ID NO: 191) 
               
               
                 GSGSATGGSGSTASSGSGSATGS, 
               
               
                 and 
               
               
                   
               
               
                 (SEQ ID NO: 192) 
               
               
                 GSGSATGGSGSTASSGSGSATHMGSGSATGGSGSTASSGSGSATHM. 
               
             
          
         
       
     
         [0190]    Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH 2 ) s —C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C 1 -C 6 ) lower acyl, halogen (e.g., Cl, Br), CN, NH 2 , phenyl, etc. An exemplary non-peptide linker is a PEG linker, 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above. 
         [0191]    Derivatives. 
         [0192]    The inventors also contemplate derivatizing the peptide and/or vehicle portion of the compounds. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:
   1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.   2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.   
 
         [0000]    
       
                 
         
             
             
         
       
       
          In Formula VIII, each “V 1 ” may represent typically one strand of the Fc domain. 
         3. One or more peptidyl [—C(O)NR-] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH 2 — carbamate [—CH 2 —OC(O)NR—], phosphonate, —CH 2 -sulfonamide [—CH 2 —S(O) 2 NR-1, urea [—NHC(O)NH—], —CH 2 -secondary amine, and alkylated peptide [—C(O)NR 6 — wherein R 6  is lower alkyl]. 
         4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR 1  (other than —NH 2 ), —NRC(O)R 1 , —NRC(O)OR 1 , —NRS(O) 2 R 1 , —NHC(O)NHR 1 , succinimide, or benzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R 1  are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C 1 -C 4  alkyl, C 1 -C 4  alkoxy, chloro, and bromo. 
         5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. Exemplary C-terminal derivative groups include, for example, —C(O)R 2  wherein R 2  is lower alkoxy or —NR 3 R 4  wherein R 3  and R 4  are independently hydrogen or C 1 -C 8  alkyl (preferably C 1 -C 4  alkyl). 
         6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9. 
         7. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below. 
       
     
         [0200]    Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. 
         [0201]    Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. 
         [0202]    Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively. 
         [0203]    Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R—N═C═N—R) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. 
         [0204]    Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. 
         [0205]    Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9. 
         [0206]    Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming cross-links in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization. 
         [0207]    Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art. 
         [0208]    Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &amp; Co., San Francisco), pp. 79-86 (1983). 
         [0209]    Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For  E. coli , which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes. 
         [0210]    Methods of Making 
         [0211]    The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used. 
         [0212]    The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. 
         [0213]    The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art. 
         [0214]    Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as  E. coli  sp.), yeast (such as  Saccharomyces  sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art. 
         [0215]    Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art. 
         [0216]    The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques. 
         [0217]    Uses of the Compounds 
         [0218]    Compounds of this invention may be particularly useful in treatment of B-cell mediated autoimmune diseases. In particular, the compounds of this invention may be useful in treating, preventing, ameliorating, diagnosing or prognosing lupus, including systemic lupus erythematosus (SLE), and lupus-associated diseases and conditions. Other preferred indications include B-cell mediated cancers, including B-cell lymphoma. 
         [0219]    The compounds of this invention can also be used to treat inflammatory conditions of the joints. Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees, millions of people worldwide. Rheumatoid arthritis is a disease of articular joints in which the cartilage and bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived from the synovial membrane. The disease may involve peri-articular structures such as bursae, tendon sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985), Anderson&#39;s Pathology, Kissane (ed.), 11:1828). Osteoarthritis is a common joint disease characterized by degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint. Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of chondrocytes to catabolic and anabolic stimuli. Changes in some matrix molecules of articular cartilage reportedly occur in early osteoarthritis (Thonar et al. (1993), Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657 and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1, TALL-1R and modulators thereof are believed to be useful in the treatment of these and related conditions. 
         [0220]    Compounds of this invention may also be useful in treatment of a number of additional diseases and disorders, including:
       acute pancreatitis;   ALS;   Alzheimer&#39;s disease;   asthma;   atherosclerosis;   autoimmune hemolytic anemia;   cancer, particularly cancers related to B cells;   cachexia/anorexia;   chronic fatigue syndrome;   cirrhosis (e.g., primary biliary cirrhosis);   diabetes (e.g., insulin diabetes);   fever;   glomerulonephritis, including IgA glomerulonephritis and primary glomerulonephritis;   Goodpasture&#39;s syndrome;   Guillain-Barre syndrome;   graft versus host disease;   Hashimoto&#39;s thyroiditis;   hemorrhagic shock;   hyperalgesia;   inflammatory bowel disease;   inflammatory conditions of a joint, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis;   inflammatory conditions resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease processes;   insulin-dependent diabetes mellitus;   ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration);   learning impairment;   lung diseases (e.g., ARDS);   multiple myeloma;   multiple sclerosis;   Myasthenia gravis;   myelogenous (e.g., AML and CIVIL) and other leukemias;   myopathies (e.g., muscle protein metabolism, esp. in sepsis);   neurotoxicity (e.g., as induced by HIV);   osteoporosis;   pain;   Parkinson&#39;s disease;   Pemphigus;   polymyositis/dermatomyositis;   pulmonary inflammation, including autoimmune pulmonary inflammation;   pre-term labor;   psoriasis;   Reiter&#39;s disease;   reperfusion injury;   septic shock;   side effects from radiation therapy;   Sjogren&#39;s syndrome;   sleep disturbance;   temporal mandibular joint disease;   thrombocytopenia, including idiopathic thrombocytopenia and autoimmune neonatal thrombocytopenia;   tumor metastasis;   uveitis; and   vasculitis.       
 
         [0272]    Compounds of this invention may be administered alone or in combination with a therapeutically effective amount of other drugs, including analgesic agents, disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and any immune and/or inflammatory modulators. Thus, compounds of this invention may be administered with:
       Modulators of other members of the TNF/TNF receptor family, including TNF antagonists, such as etanercept (Enbrel™), sTNF-RI, onercept, D2E7, and Remicade™.   Nerve growth factor (NGF) modulators.   IL-1 inhibitors, including IL-1ra molecules such as anakinra and more recently discovered IL-1ra-like molecules such as IL-1Hy1 and IL-1Hy2; IL-1 “trap” molecules as described in U.S. Pat. No. 5,844,099, issued Dec. 1, 1998; IL-1 antibodies; solubilized IL-1 receptor, and the like.   IL-6 inhibitors (e.g., antibodies to IL-6).   IL-8 inhibitors (e.g., antibodies to IL-8).   IL-18 inhibitors (e.g., IL-18 binding protein, solubilized IL-18 receptor, or IL-18 antibodies).   Interleukin-1 converting enzyme (ICE) modulators.   insulin-like growth factors (IGF-1, IGF-2) and modulators thereof.   Transforming growth factor-β (TGF-β), TGF-β family members, and TGF-β modulators.   Fibroblast growth factors FGF-1 to FGF-10, and FGF modulators.   Osteoprotegerin (OPG), OPG analogues, osteoprotective agents, and antibodies to OPG-ligand (OPG-L).   bone anabolic agents, such as parathyroid hormone (PTH), PTH fragments, and molecules incorporating PTH fragments (e.g., PTH (1-34)-Fc).   PAF antagonists.   Keratinocyte growth factor (KGF), KGF-related molecules (e.g., KGF-2), and KGF modulators.   COX-2 inhibitors, such as Celebrex™ and Vioxx™   Prostaglandin analogs (e.g., E series prostaglandins).   Matrix metalloproteinase (MMP) modulators.   Nitric oxide synthase (NOS) modulators, including modulators of inducible NOS.   Modulators of glucocorticoid receptor.   Modulators of glutamate receptor.   Modulators of lipopolysaccharide (LPS) levels.   Anti-cancer agents, including inhibitors of oncogenes (e.g., fos, jun) and interferon.   Noradrenaline and modulators and mimetics thereof.       
 
         [0296]    Pharmaceutical Compositions 
         [0297]    In General. 
         [0298]    The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington&#39;s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference in their entirety. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations. 
         [0299]    Oral Dosage Forms. 
         [0300]    Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington&#39;s Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine. 
         [0301]    Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-83; Newmark, et al. (1982), I. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties. 
         [0302]    For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods”. 
         [0303]    The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression. 
         [0304]    Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. 
         [0305]    One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. 
         [0306]    Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. 
         [0307]    Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic. 
         [0308]    An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. 
         [0309]    Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate. 
         [0310]    To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios. 
         [0311]    Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid. 
         [0312]    Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms; e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. 
         [0313]    Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating. 
         [0314]    Pulmonary Delivery Forms. 
         [0315]    Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl. 5): s. 143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Gin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), I. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor). 
         [0316]    Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. 
         [0317]    All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy. 
         [0318]    The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung. 
         [0319]    Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. 
         [0320]    Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. 
         [0321]    Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol. 
         [0322]    Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2- tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. 
         [0323]    Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. 
         [0324]    Nasal Delivery Forms. 
         [0325]    Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated. 
         [0326]    Dosages. 
         [0327]    The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram. 
       Specific Preferred Embodiments 
       [0328]    The inventors have determined preferred structures for the preferred peptides listed in Table 4 below. The symbol “A” may be any of the linkers described herein or may simply represent a normal peptide bond (i.e., so that no linker is present). Tandem repeats and linkers are shown separated by dashes for clarity. 
         [0000]                                  TABEL 4                    Preferred embodiments                    SEQ ID           Sequence/structure   NO:                       LPGCKWDLLIKQWVCDPL-Λ-V 1     44                       V 1 -Λ-LPGCKWDLLIKQWVCDPL   45                       LPGCKWDLLIKQWVCDPL-Λ-   46           LPGCKWDLLIKQWVCDPL-Λ-V 1                             V 1 -Λ-LPGCKWDLLIKQWVCDPL-Λ-   47           LPGCKWDLLIKQWVCDPL                           SADCYFDILTKSDVCTSS-Λ-V 1     48                       V 1 -Λ-SADCYFDILTKSDVCTSS   49                       SADCYFDILTKSDVTSS-Λ-SADCYF   50           DILTKSDVTSS-Λ-V 1                             V 1 -Λ-SADCYFDILTKSDVTSS-Λ-   51           SADCYFDILTKSDVTSS                           FHDCKWDLLTKQWVCHGL-Λ-V 1     52                       V 1 -Λ-FHDCKWDLLTKQWVCHGL   53                       FHDCKWDLLTKQWVCHGL-Λ-   54           FHDCKWDLLTKQWVCHGL-Λ-V 1                             V 1 -Λ-FHDCKWDLLTKQWVCHGL-Λ-   55           FHDCKWDLLTKQWVCHGL                        
“V 1 ” is an Fc domain as defined previously herein. In addition to those listed in Table 4, the inventors further contemplate heterodimers in which each strand of an Fc dimer is linked to a different peptide sequence; for example, wherein each Fc is linked to a different sequence selected from Table 2.
 
         [0329]    All of the compounds of this invention can be prepared by methods described in PCT appl. no. WO 99/25044. 
         [0330]    The invention will now be further described by the following working examples, which are illustrative rather than limiting. 
       Example 1 
     Peptides 
     Peptide Phage Display 
       [0331]    1. Magnetic Bead Preparation 
         [0332]    A. Fc-TALL-1 Immobilization on Magnetic Beads 
         [0333]    The recombinant Fc-TALL-1 protein was immobilized on the Protein A Dynabeads (Dynal) at a concentration of 8 μg of Fc-TALL-1 per 100 μl of the bead stock from the manufacturer. By drawing the beads to one side of a tube using a magnet and pipetting away the liquid, the beads were washed twice with the phosphate buffer saline (PBS) and resuspended in PBS. The Fc-TALL-1 protein was added to the washed beads at the above concentration and incubated with rotation for 1 hour at room temperature. The Fc-TALL-1 coated beads were then blocked by adding bovine serum albumin (BSA) to 1% final concentration and incubating overnight at 4° C. with rotation. The resulting Fe-TALL-1 coated beads were then washed twice with PB ST (PBS with 0.05% Tween-20) before being subjected to the selection procedures. 
         [0334]    B. Negative Selection Bead Preparation 
         [0335]    Additional beads were also prepared for negative selections. For each panning condition, 250 μl of the bead stock from the manufacturer was subjected to the above procedure (section 1A) except that the incubation step with Fc-TALL-1 was omitted. In the last washing step, the beads were divided into five 50 μl aliquots. 
         [0336]    2. Selection of TALL-1 Binding Phage 
         [0337]    A. Overall Strategy 
         [0338]    Two filamentous phage libraries, TN8-IX (5×10 9  independent transformants) and TN12-I (1.4×10 9  independent transformants) (Dyax Corp.), were used to select for TALL-1 binding phage. Each library was subjected to either pH 2 elution or ‘bead elution’ (section 2E). Therefore, four different panning conditions were carried out for the TALL-1 project (TN8-IX using the pH2 elution method, TN8-IX using the bead elution method, TN12-I the using p112 elution method, and TN 12-I using the bead elution method). Three rounds of selection were performed for each condition. 
         [0339]    B. Negative Selection 
         [0340]    For each panning condition, about 100 random library equivalent (5×10 11  pfu for TN8-IX and 1.4×10 11  pfu for TN12-I) was aliquoted from the library stock and diluted to 300 μl with PB ST. After the last washing liquid was drawn out from the first 50 μl aliquot of the beads prepared for negative selections (section 1B), the 300 μl diluted library stock was added to the beads. The resulting mixture was incubated for 10 minutes at room temperature with rotation. The phage supernatant was drawn out using the magnet and added to the second 50 μl aliquot for another negative selection step. In this way, five negative selection steps were performed. 
         [0341]    C. Selection Using the Fc-TALL-1 Protein Coated Beads 
         [0342]    The phage supernatant after the last negative selection step (section 1B) was added to the Fc-TALL-1 coated beads after the last washing step (section 1A). This mixture was incubated with rotation for two hours at room temperature, allowing specific phage to bind to the target protein. After the supernatant is discarded, the beads were washed seven times with PBST. 
         [0343]    D. pH2 Elution of Bound Phage 
         [0344]    After the last washing step (section 2C), the bound phages were eluted from the magnetic beads by adding 200 μl of CBST (50 mM sodium citrate, 150 mM sodium chloride, 0.05% Tween-20, pH2). After 5 minute incubation at room temperature, the liquid containing the eluted phage were drawn out and transferred to another tube. The elution step was repeated again by adding 200 μl of CBST and incubating for 5 minutes. The liquids from two elution steps were added together, and 100 μl of 2 M Tris solution (pH 8) was added to neutralize the pH. 500 μl of Min A Salts solution (60 mM K 2 HPO 4 , 33 mM KH 2 PO 4 , 7.6 mM (NH 4 )SO 4 , and 1.7 mM sodium citrate) was added to make the final volume to 1 ml. 
         [0345]    E. ‘Bead Elution’ 
         [0346]    After the final washing liquid was drawn out (section 2C), 1 ml of Min A salts solution was added to the beads. This bead mixture was added directly to a concentrated bacteria sample for infection (section 3A and 3B). 
         [0347]    3. Amplification 
         [0348]    A. Preparation of Plating Cells 
         [0349]    Fresh  E. Coli . (XL-1 Blue MRF′) culture was grown to OD 600 =0.5 in LB media containing 12.5 μg/ml tetracycline. For each panning condition, 20 ml of this culture was chilled on ice and centrifuged. The bacteria pellet was resuspended in 1 ml of the Min A Salts solution. 
         [0350]    B. Transduction 
         [0351]    Each mixture from different elution methods (section 2D and 2E) was added to a concentrated bacteria sample (section 3A) and incubated at 37° C. for 15 minutes. 2 ml of NZCYM media (2λNZCYM, 50 μg/ml ampicillin) was added to each mixture and incubated at room temperature for 15 minutes. The resulting 4 ml solution was plated on a large NZCYM agar plate containing 50 μg/ml ampicillin and incubated overnight at 37° C. 
         [0352]    C. Phage Harvesting 
         [0353]    Each of the bacteria/phage mixture that was grown overnight on a large NZCYM agar plate (section 3B) was scraped off in 35 ml of LB media, and the agar plate was further rinsed with additional 35 ml of LB media. The resulting bacteria/phage mixture in LB media was centrifuged to pellet the bacteria away. 50 ml the of the phage supernatant was transferred to a fresh tube, and 12.5 ml of PEG solution (20% PEG8000, 3.5M ammonium acetate) was added and incubated on ice for 2 hours to precipitate phages. Precipitated phages were centrifuged down and resuspended in 6 ml of the phage resuspension buffer (250 mM NaCl, 100 mM Tris pH8, 1 mM EDTA). This phage solution was further purified by centrifuging away the remaining bacteria and precipitating the phage for the second time by adding 1.5 ml of the PEG solution. After a centrifugation step, the phage pellet was resuspended in 400 IA of PBS. This solution was subjected to a final centrifugation to rid of remaining bacteria debris. The resulting phage preparation was titered by a standard plaque formation assay (Molecular Cloning, Maniatis et al 3 rd  Edition). 
         [0354]    4. Two More Rounds of Selection and Amplification. 
         [0355]    In the second round, the amplified phage (10 10  pfu) from the first round (section 3C) was used as the input phage to perform the selection and amplification steps (sections 2 and 3). The amplified phage (10 10  pfu) from the 2 nd  round in turn was used as the input phage to perform 3 rd  round of selection and amplification (sections 2 and 3). After the elution steps (sections 2D and 2E) of the 3 rd  round, a small fraction of the eluted phage was plated out as in the plaque formation assay (section 3C). Individual plaques were picked and placed into 96 well microtiter plates containing 100 μl of TE buffer in each well. These master plates were incubated in a 37° C. incubator for 1 hour to allow phages to elute into the TE buffer. 
         [0356]    5. Clonal Analysis (Phage ELISA and Sequencing) 
         [0357]    The phage clones were analyzed by phage ELISA and sequencing methods. The sequences were ranked based on the combined results from these two assays. 
         [0358]    A. Phage ELISA 
         [0359]    An XL-1 Blue MRF′ culture was grown until OD 600 , reaches 0.5. 30 μl of this culture was aliquoted into each well of a 96 well microtiter plate. 10 μl of eluted phage (section 4) was added to each well and allowed to infect bacteria for 15 min at room temperature. 130 μl of LB media containing 12.5 μl of tetracycline and 50 μg/ml of ampicillin was added to each well. The microtiter plate was then incubated overnight at 37° C. The recombinant TALL-1 protein (1 μg/ml in PBS) was allowed to coat onto the 96-well Maxisorp plates (NUNC) overnight and 4° C. As a control, the recombinant Fc-Trail protein was coated onto a separate Maxisorp plate at the same molar concentration as the TALL-1 protein. 
         [0360]    On the following day, liquids in the protein coated Maxisorp plates were discarded, and each well was blocked with 300 μl of 2% BSA solution at 37° C. for one hour. The BSA solution was discarded, and the wells were washed three times with the PBST solution. After the last washing step, 50 μl of PBST was added to each well of the protein coated Maxisorp plates. Each of the 50 μl overnight cultures in the 96 well microtiter plate was transferred to the corresponding wells of the TALL-1 coated plates as well as the control Fc-Trail coated plates. The 100 μl mixtures in the two kinds of plates were incubated for 1 hour at room temperature. The liquid was discarded from the Maxisorp plates, and the wells were washed five times with PBST. The HRP-conjugated anti-M13 antibody (Pharmacia) was diluted to 1:7,500, and 100 μl of the diluted solution was added to each well of the Maxisorp plates for 1 hour incubation at room temperature. The liquid was again discarded and the wells were washed seven times with PBST. 100 μl of tetramethylbenzidine (TMB) substrate (Sigma) was added to each well for the color reaction to develop, and the reaction was stopped with 50 μl of the 5 N H 2 SO 4  solution. The OD 450  was read on a plate reader (Molecular Devices). 
         [0361]    B. Sequencing of the Phage Clones. 
         [0362]    For each phage clone, the sequencing template was prepared by a PCR method. The following oligonucleotide pair was used to amplify about 500 nucleotide fragment: 
         [0000]                            primer #1           (SEQ ID NO: 56)           (5′-CGGCGCAACTATCGGTATCAAGCTG-3′)           and                        primer #2           (SEQ ID NO: 57)           (5′-CATGTACCGTAACACTGAGTTTCGTC-3′).            
The following mixture was prepared for each clone.
 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Reagents 
                 volume (μL)/tube 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 dH 2 0 
                 26.25 
               
               
                   
                 50% glycerol 
                 10 
               
               
                   
                 10B PCR Buffer (w/o MgCl 2 ) 
                 5 
               
               
                   
                 25 mM MgCl 2   
                 4 
               
               
                   
                 10 mM dNTP mix 
                 1 
               
               
                   
                 100 μM primer 1 
                 0.25 
               
               
                   
                 100 μM primer 2 
                 0.25 
               
               
                   
                 Taq polymerase 
                 0.25 
               
               
                   
                 Phage in TE (section 4) 
                 3 
               
               
                   
                 Final reaction volume 
                 50 
               
               
                   
                   
               
             
          
         
       
     
         [0363]    The thermocycler (GeneAmp PCR System 9700, Applied Biosystems) was used to run the following program: 94° C. for 5 min; [94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 45 sec.]×30 cycles; 72° C. for 7 min; cool to 4° C. The PCR product was checked by running 5 μl of each PCR reaction on a 1% agarose gel. The PCR product in the remaining 45 μl from each reaction was cleaned up using the QlAquick Multiwell PCR Purification kit (Qiagen), following the manufacturer&#39;s protocol. The resulting product was then sequenced using the ABI 377 Sequencer (Perkin-Elmer) following the manufacturer recommended protocol. 
         [0364]    6. Sequence Ranking and Consensus Sequence Determination 
         [0365]    A. Sequence Ranking 
         [0366]    The peptide sequences that were translated from variable nucleotide sequences (section 5B) were correlated to ELISA data. The clones that showed high OD 450  in the TALL-1 coated wells and low OD 450  in the Fc-Trail coated wells were considered more important. The sequences that occur multiple times were also considered important. Candidate sequences were chosen based on these criteria for further analysis as peptides or peptibodies. Five and nine candidate peptide sequences were selected from the TN8-IX and TN12-I libraries, respectively. 
         [0367]    B. Consensus Sequence Determination 
         [0368]    The majority of sequences selected from the TN12-I library contained a very conserved DBL motif. This motif was also observed in sequences selected from the TN8-IB library as well. Another motif, PFPWE (SEQ ID NO: 110) was also observed in sequences obtained from the TN8-IB library. 
         [0369]    A consensus peptide, FHDC KWDLLTKQWV CHGL (SEQ ID NO: 58), was designed based on the DBL motif. Since peptides derived from the TN12-I library were the most active ones, the top 26 peptide sequences based on the above ranking criteria (section 5A) were aligned by the DBL motif. The underlined “core amino acid sequence” was obtained by determining the amino acid that occur the most in each position. The two cysteines adjacent to the core sequences were fixed amino acids in the TN12-I library. The rest of the amino acid sequence in the consensus peptide is taken from one of the candidate peptides, TALL-1-12-10 (Table 2, SEQ ID NO: 37). The peptide and peptibody that was derived from this consensus sequence were most active in the B cell proliferation assay. 
       Example 2 
     Peptibodies 
       [0370]    A set of 12 TALL-1 inhibitory peptibodies (Table 5) was constructed in which a monomer of each peptide was fused in-frame to the Fc region of human IgG1. Each TALL-1 inhibitory peptibody was constructed by annealing the pairs of oligonucleotides shown in Table 6 to generate a duplex encoding the peptide and a linker comprised of 5 glycine residues and one valine residue as an NdeI to SalI fragment. These duplex molecules were ligated into a vector (pAMG21-RANK-Fc, described herein) containing the human Fc gene, also digested with {right arrow over (Nde)}I and {right arrow over (Sal)}I. The resulting ligation mixtures were transformed by electroporation into  E. coli  strain 2596 cells (GM221, described herein). Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected for each of the peptibodies. The nucleotide and amino acid sequences of the fusion proteins are shown in  FIG. 4A through 4F . 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 5  
               
             
             
               
                   
               
               
                 Peptide sequences and oligonucleotides used to 
               
               
                 generate TALL-1 inhibitory peptibodies. 
               
             
          
           
               
                   
                 Pepti- 
                   
                 Sense 
                 Antisense 
               
               
                   
                 body 
                   
                 oligo- 
                 oligo- 
               
               
                   
                 SEQ ID 
                 Peptide  
                 nucleo- 
                 nucleo- 
               
               
                 Peptibody 
                 NO 
                 Sequence 
                 tide 
                 tide 
               
               
                   
               
               
                 TALL-1-8-1-a 
                 29 
                 PGTCFPFPWEC 
                 2517-24 
                 2517-25 
               
               
                   
                   
                 THA 
                   
                   
               
               
                   
               
               
                 TALL-1-8-2-a 
                 30 
                 WGACWPFPWEC 
                 2517-26 
                 2517-27 
               
               
                   
                   
                 FKE 
                   
                   
               
               
                   
               
               
                 TALL-1-8-4-a 
                 31 
                 VPFCDLLTKHC 
                 2517-28 
                 2517-29 
               
               
                   
                   
                 FEA 
                   
                   
               
               
                   
               
               
                 TALL-1-12-4- 
                 32 
                 GSRCKYKWDVL 
                 2517-30 
                 2517-31 
               
               
                 a 
                   
                 TKQCFHH 
                   
                   
               
               
                   
               
               
                 TALL-1-12-3- 
                 33 
                 LPGCKWDLLIK 
                 2517-32 
                 2517-33 
               
               
                 a 
                   
                 QWVCDPL 
                   
                   
               
               
                   
               
               
                 TALL-1-12-5- 
                 34 
                 SADCYFDILTK 
                 2517-34 
                 2517-35 
               
               
                 a 
                   
                 SDVCTSS 
                   
                   
               
               
                   
               
               
                 TALL-1-12-8- 
                 35 
                 SDDCMYDQLTR 
                 2517-36 
                 2517-37 
               
               
                 a 
                   
                 MFICSNL 
                   
                   
               
               
                   
               
               
                 TALL-1-12-9- 
                 36 
                 DLNCKYDELTY 
                 2521-92 
                 2521-93 
               
               
                 a 
                   
                 KEWCQFN 
                   
                   
               
               
                   
               
               
                 TALL-1-12- 
                 37 
                 FHDCKYDLLTR 
                 2521-94 
                 2521-95 
               
               
                 10-a 
                   
                 QMVCHGL 
                   
                   
               
               
                   
               
               
                 TALL-1-12- 
                 38 
                 RNHCFWDHLLK 
                 2521-96 
                 2521-97 
               
               
                 11-a 
                   
                 QDICPSP 
                   
                   
               
               
                   
               
               
                 TALL-1-12- 
                 39 
                 ANQCWWDSLTK 
                 2521-98 
                 2521-99 
               
               
                 14-a 
                   
                 KNVCEFF 
                   
                   
               
               
                   
               
               
                 TALL-1- 
                 58 
                 FHDCKWDLLTK 
                 2551-48 
                 2551-49 
               
               
                 consensus 
                   
                 QWVCHGL 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 5B  
               
             
             
               
                   
               
               
                 TALL-1 inhibitory peptibodies. 
               
             
          
           
               
                   
                 Peptibody 
                   
               
               
                   
                 SEQ ID 
                   
               
               
                 Peptibody 
                 NO 
                 Peptide Sequence 
               
               
                   
               
               
                 TALL-I-8-1-a 
                 111 
                 MPGTCFPFPW ECTHAGGGGG VDKTHTCPPC PAPELLGGPS 
               
               
                   
                   
                 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 
               
               
                   
                   
                 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 
               
               
                   
                   
                 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 
               
               
                   
                   
                 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 
               
               
                   
                   
                 SLSLSPGK 
               
               
                   
               
               
                 TALL-1-8-2-a 
                 112 
                 MWGACWPFPW ECFKEGGGGG VDKTHTCPPC PAPELLGGPS 
               
               
                   
                   
                 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 
               
               
                   
                   
                 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 
               
               
                   
                   
                 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 
               
               
                   
                   
                 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 
               
               
                   
                   
                 SLSLSPGK 
               
               
                   
               
               
                 TALL-1-8-4-a 
                 113 
                 MVPFCDLLTK HCFEAGGGGG VDKTHTCPPC PAPELLGGPS 
               
               
                   
                   
                 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 
               
               
                   
                   
                 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 
               
               
                   
                   
                 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 
               
               
                   
                   
                 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 
               
               
                   
                   
                 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 
               
               
                   
                   
                 SLSLSPGK 
               
               
                   
               
               
                 TALL-1-12-4-a 
                 114 
                 MGSRCKYKWD VLTKQCFHHG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-3-a 
                 115 
                 MLPGCKWDLL IKQWVCDPLG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-5-a 
                 116 
                 MSADCYFDIL TKSDVCTSSG GGGG VDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-8-a 
                 117 
                 MSDDCMYDQL TRMFICSNLG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-9-a 
                 118 
                 MDLNCKYDEL TYKEWCQFNG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-10-a 
                 119 
                 MFHDCKYDLL TRQMVCHGLG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-11-a 
                 120 
                 MRNHCFWDHL LKQDICPSPG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1-12-14-a 
                 121 
                 MANQCWWDSL TKKNVCEFFG GGGGVDKTHT CPPCPAPELL 
               
               
                   
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-1- 
                 122 
                 MFHDCKWDLL TKQWVCHGLG GGGGVDKTHT CPPCPAPELL 
               
               
                 consensus 
                   
                 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 
               
               
                   
                   
                 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 
               
               
                   
                   
                 GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 
               
               
                   
                   
                 DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 
               
               
                   
                   
                 PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 
               
               
                   
                   
                 YTQKSLSLSP GK 
               
               
                   
               
               
                 TALL-112-3  
                 123 
                 MLPGCKWDLL IKQWVCDPLG SGSATGGSGS TASSGSGSAT 
               
               
                 tandem 
                   
                 HMLPGCKWDL LIKQWVCDPL GGGGGVDKTH TCPPCPAPEL 
               
               
                 dimer 
                   
                 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK 
               
               
                   
                   
                 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 
               
               
                   
                   
                 NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS 
               
               
                   
                   
                 RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 
               
               
                   
                   
                 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 
               
               
                   
                   
                 HYTQKSLSLS PGK 
               
               
                   
               
               
                 TALL-1 
                 124 
                 MFHDCKWDLL TKQWVCHGLG SGSATGGSGS TASSGSGSAT 
               
               
                 consensus 
                   
                 HMFHDCKWDL LTKQWVCHGL GGGGGVDKTH TCPPCPAPEL 
               
               
                 tandem dimer 
                   
                 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK 
               
               
                   
                   
                 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 
               
               
                   
                   
                 NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS 
               
               
                   
                   
                 RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 
               
               
                   
                   
                 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 
               
               
                   
                   
                 HYTQKSLSLS PGK 
               
               
                   
               
             
          
         
       
     
         [0000]                                  TABLE 6                    Sequences of oligonucleotides used in peptibody construction.            Oligo-   SEQ           nucleotide   ID           ID number   NO   Sequence               2517-24   71   TAT GCC GGG TAC TTG TTT CCC GTT CCC GTG GGA ATG CAC               TCA CGC TGG TGG AGG CGG TGG GG               2517-25   72   TCG ACC CCA CCG CCT CCT GGA GCG TGA GTG CAT TCC CAC               GGG AAG CCG AAA CAA GTA CCC GGC A               2517-26   73   TAT GTG GGG TGC TTG TTG GCC GTT CCC GTG GGA ATG TTT               CAA AGA AGG TGG AGG CGG TGG GG               2517-27   74   TCG ACC CCA CCG CCT CCA CCT TCT TTG AAA CAT TCC               CACGGG AAC GGC CAA CAAGCA CCC CAC A               2517-28   75   TAT GGT TCC GTT CTG TGA CCT GCT GAC TAA ACA CTG TTT               CGA AGC TGG TGG AGG CGG TGG GG               2517-29   76   TCG ACC CCA CCG CCT CCA CCA GCT TCG AAA CAG TGT TTA               GTC AGC AGG TCA CAGAAC GGA ACC A               2517-30   77   TAT GGG TTC TCG TTG TAA ATA CAA ATG GGA CGT TCT GAC               TAA ACA GTG TTT CCA CCA CGG TGG AGG CGG TGG GG               2517-31   78   TCG ACC CCA CCG CCT CCA CCG TGG TGG AAA CAC TGT TTA               GTC AGA ACG TCC CAT TTG TAT TTA CAA CGA GAA CCC A               2517-32   79   TAT GCT GCC GGG TTG TAA ATG GGA CCT GCT GAT CAA ACA               GTG GGT TTG TGA CCC GCT GGG TGG AGG CGG TGG GG               2517-33   80   TCG ACC CCA CCG CCT CCA CCC AGC GGG TCA CAA ACC CAC               TGT TTG ATC AGC AGG TCC CAT TTA CAA CCC GGC AGC A               2517-34   81   TAT GTC TGC TGA CTG TTA CTT CGA CAT CCT GAC TAA ATC               TGA CGT TTG TAC TTC TTC TGG TGG AGG CGG TGG GG               2517-35   82   TCG ACC CCA CCG CCT CCA CCA GAA GAA GTA CAA ACG TCA               GAT TTA GTC AGG ATG TCG AAG TAA CAG TCA GCA GAC A               2517-36   83   TAT GTC TGA CGA CTG TAT GTA CGA CCA GCT GAC TCG TAT               GTT CAT CTG TTC TAA CCT GGG TGG AGG CGG TGG GG               2517-37   84   TCG ACC CCA CCG CCT CCA CCC AGG TTA GAA CAG ATG AAC               ATA CGA GTC AGC TGG TCG TAC ATA CAG TCG TCA GAC A               2521-92   85   TAT GGA CCT GAA CTG TAA ATA CGA CGA ACT GAC TTA CAA               AGA ATG GTG TCA GTT CAA CGG TGG AGG CGG TGG GG               25221-93    86   TCG ACC CCA CCG CCT CCA CCG TTG AAC TGA CAC CAT TCT               TTG TAA GTC AGTTCG TCG TAT TTA CAG TTC AGG TCC A               2521-94   87   TAT GTT CCA CGA CTG TAA ATA CGA CCT GCT GAC TCG TCA               GAT GGT TTG TCA CGG TCT GGG TGG AGG CGG TGG GG               2521-95   88   TCG ACC CCA CCG CCT CCA CCC AGA CCG TGA CAA ACC ATC               TGA CGA GTC AGC AGG TCG TAT TTA CAG TCG TGG AAC A               2521-96   89   TAT GCG TAA CCA CTG TTT CTG GGA CCA CCT GCT GAA ACA               GGA CAT CTG TCC GTC TCC GGG TGG AGG CGG TGG GG               2521-97   90   TCG ACC CCA CCG CCT CCA CCC GGA GAC GGA CAG ATG TCC               TGT TTC AGC AGG TGG TCC CAG AAA CAG TGG TTA CGC A               2521-98   91   TAT GGC TAA CCA GTG TTG GTG GGA CTC TCT GCT GAA AAA               AAA CGT TTG TGA ATT CTT CGG TGG AGG CGG TGG GG               2521-99   92   TCG ACC CCA CCG CCT CCA CCG AAG AAT TCA CAA ACG TTT               TTT TTC AGC AGA GAG TCC CAC CAA CAC TGG TTA GCC A               2551-48   93   TAT GTT CCA CGA CTG CAA ATG GGA CCT GCT GAC CAA ACA               GTG GGT TTG CCA CGG TCT GGG TGG AGG CGG TGG GG               2551-49   94   TCG ACC CCA CCG CCT CCA CCC AGA CCG TGG CAA ACC CAC               TGT TTG GTC AGC AGG TCC CAT TTG CAG TCG TGG AAC A                    
pAMG21-RANK-Fc Vector
 
         [0371]    pAMG21. The expression plasmid pAMG21 (ATCC accession no. 98113) can be derived from the Amgen expression vector pCFM1656(ATCC#69576) which in turn be derived from the Amgen expression vector system described in U.S. Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the described pCFM836 plasmid (U.S. Pat. No. 4,710,473) by:
       destroying the two endogenous NdeI restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation;   replacing the DNA sequence between the unique  Aat II and  Cla I restriction sites containing the synthetic P L  promoter with a similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473) containing the P L  promoter (see SEQ ID NO: 95 below); and   substituting the small DNA sequence between the unique  Cla I and  Kpn I restriction sites with the oligonucleotide having the sequence of SEQ ID NO: 96.       
 
         [0000]    
       
         
               
               
             
           
               
                 SEQ ID NO: 95: 
                   
               
               
                 AatII 
               
               
                 5′ CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- 
               
               
                 3′ TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTMITATTTAAGTATA- 
               
               
                    -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA- 
               
               
                    -TTTTTIGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT- 
               
               
                    -TACCACTGGCGGTGATACTGAGCACAT 3′ 
               
               
                    -ATGGTGACCGCCACTATGACTCGTGTAGC 5′ 
               
               
                                                 ClaI 
               
               
                   
               
               
                 SEQ ID NO: 96: 
               
               
                 5′ CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′ 
               
               
                 3′ TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′ 
               
               
                    ClaI                                          KpnI 
               
             
          
         
       
     
         [0375]    The expression plasmid pAMG21 can then be derived from pCFM1656 by making a series of site-directed base changes by PCR overlapping oligonucleotide mutagenesis and DNA sequence substitutions. Starting with the  Bgl II site (plasmid by #180) immediately 5′ to the plasmid replication promoter P   copB   and proceeding toward the plasmid replication genes, the base pair changes are as shown in Table 7 below. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 7  
               
             
             
               
                   
               
               
                 Base pair changes resulting in pAMG21 
               
             
          
           
               
                   
                   
                 bp in  
                 bp changed 
               
               
                   
                 pAMG21 bp # 
                 pCFM1656 
                 to in pAMG21 
               
               
                   
                   
               
               
                   
                 # 204 
                 T/A 
                 C/G 
               
               
                   
                   
               
               
                   
                 # 428 
                 A/T 
                 G/C 
               
               
                   
                   
               
               
                   
                 # 509 
                 G/C 
                 A/T 
               
               
                   
                   
               
               
                   
                 # 617 
                 — 
                 insert two G/C bp 
               
               
                   
                   
               
               
                   
                 # 679 
                 G/C 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 980 
                 T/A 
                 C/G 
               
               
                   
                   
               
               
                   
                 # 994 
                 G/C 
                 A/T 
               
               
                   
                   
               
               
                   
                 # 1004 
                 A/T 
                 C/G 
               
               
                   
                   
               
               
                   
                 # 1007 
                 C/G 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 1028 
                 A/T 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 1047 
                 C/G 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 1178 
                 G/C 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 1466 
                 G/C 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 2028 
                 G/C 
                 bp deletion 
               
               
                   
                   
               
               
                   
                 # 2187 
                 C/G 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 2480 
                 A/T 
                 T/A 
               
               
                   
                   
               
               
                   
                 # 2499-2502 
                 
                   AGTG 
                 
                 
                   GTCA 
                 
               
               
                   
                   
                 TCAC 
                 CAGT 
               
               
                   
                   
               
               
                   
                 # 2642 
                 
                   TCCGAGC 
                 
                 7 bp deletion 
               
               
                   
                   
                 AGGCTCG 
                   
               
               
                   
                   
               
               
                   
                 # 3435 
                 G/C 
                 A/T 
               
               
                   
                   
               
               
                   
                 # 3446 
                 G/C 
                 A/T 
               
               
                   
                   
               
               
                   
                 # 3643 
                 A/T 
                 T/A 
               
               
                   
                   
               
             
          
         
       
     
         [0376]    The DNA sequence between the unique  Aat II (position #4364 in pCFM1656) and  Sac II (position #4585 in pCFM1656) restriction sites is substituted with the DNA sequence below (SEQ ID NO: 97): 
         [0000]    
       
         
               
               
             
           
               
                 [AatII sticky end]               5′     GCGTAACGTATGCATGGTCTCC- 
                   
               
               
                 (position #4350 in pAMG21)       3′ TGCACGCATTGCATACGTACCAGAGG- 
               
               
                   
               
               
                 -CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT- 
               
               
                 -GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA- 
               
               
                   
               
               
                 -GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC- 
               
               
                 -CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTGTTTAGGCG- 
               
               
                   
               
               
                 -CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC- 
               
               
                 -GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG- 
               
               
                   
               
               
                 -CATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT- 
               
               
                 -GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCA- 
               
               
                   
               
               
                                                                  AatII 
               
               
                 -TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC- 
               
               
                 -AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG- 
               
               
                   
               
               
                 -TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC- 
               
               
                 -AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG- 
               
               
                   
               
               
                 -GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC- 
               
               
                 -CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG- 
               
               
                   
               
               
                 -TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC- 
               
               
                 -ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG- 
               
               
                   
               
               
                 -ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTC- 
               
               
                 -TAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG- 
               
               
                   
               
               
                 -GATAATTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA- 
               
               
                 -CTATTAATAGTTGATCTCTTCCTTGTTAATTACCATACAAGTATGTGCGTACATTTTTAT- 
               
               
                   
               
               
                 -AACTATCTATATAGTTGTCTTTCTCTGAATGTGCAAAACTAAGCATTCCGAAGCCATTAT- 
               
               
                 -TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATA- 
               
               
                   
               
               
                 -TAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTTCTTTAA- 
               
               
                 -ATCGTCATACTTATCCCTTTGATTTGGGTCACTATTCTGGACTACTAAAGCGAAGAAATT- 
               
               
                   
               
               
                 -TTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTG- 
               
               
                 -AATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC- 
               
               
                   
               
               
                 -AATGATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCGTCATCAT- 
               
               
                 -TTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAATAATTTAATCGCAGTAGTA- 
               
               
                   
               
               
                 -AATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAG- 
               
               
                 -TTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATC- 
               
               
                   
               
               
                 -AATGAGGATAAATGATCGCGAGTAAATAATATTCACAATGTACCATTTTAGTCATATCAG- 
               
               
                 -TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTC- 
               
               
                   
               
               
                 -ATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGT- 
               
               
                 -TATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACA- 
               
               
                   
               
               
                 -AAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTC- 
               
               
                 -TTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAG- 
               
               
                   
               
               
                 -GCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAA- 
               
               
                 -CGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATT- 
               
               
                   
               
               
                 -ATTGGATTTTTGTCACACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTG- 
               
               
                 -TAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGAC- 
               
               
                   
               
               
                 -TAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATT- 
               
               
                 -ATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAACTAA- 
               
               
                   
               
               
                 -CTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGA- 
               
               
                 -GATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCT- 
               
               
                   
               
               
                                                                  SacII 
               
               
                 -GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAA- 
               
               
                 -CGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTT- 
               
               
                   
               
               
                 -GAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA- 
               
               
                 -CTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTAT- 
               
               
                   
               
               
                 -ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG- 
               
               
                 -TGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC- 
               
               
                   
               
               
                 -AACCGCTCTTCACGCTCTTCACGC 3′          [SacII sticky end] 
               
               
                 -TTGGCGAGAAGTGCGAGAAGTG 5′        (position #5904 in pAMG21) 
               
             
          
         
       
     
         [0377]    During the ligation of the sticky ends of this substitution DNA sequence, the outside  Aat II and  Sac II sites are destroyed. There are unique  Aat II and  Sac II sites in the substituted DNA. 
         [0378]    A gene encoding human RANK fused to the N-terminus of Fc was ligated into pAMG21 as an  Nde I to  Bam HI fragment to generate Amgen Strain #4125. This construct was modified to insert a valine codon at the junction of RANK and Fc. The adjacent valine and aspartate codons create a unique  Sal I site. This allows for the fusion of peptides at the N-terminus of Fc3 between the unique  Nde I and  Sal I sites. The RANK sequence is deleted upon insertion of a new  Nde I- Sal I fragment. The sequence of the vector is given in  FIG. 5A  through 5M. 
         [0379]    GM221 (Amgen #2596). 
         [0380]    The Amgen host strain #2596 is an  E. coli  K-12 strain derived from Amgen strain #393, which is a derivative of  E. coli  W1485, obtained from the  E. coli  Genetic Stock Center, Yale University, New Haven, Conn. (CGSC strain 6159). It has been modified to contain both the temperature sensitive lambda repressor cI857s7 in the early  ebg  region and the lacI Q  repressor in the late  ebg  region (68 minutes). The presence of these two repressor genes allows the use of this host with a variety of expression systems, however both of these repressors are irrelevant to the expression from luxP R . The untransformed host has no antibiotic resistances. 
         [0381]    The ribosome binding site of the cI857s7 gene has been modified to include an enhanced RBS. It has been inserted into the  ebg  operon between nucleotide position 1170 and 1411 as numbered in Genbank accession number M64441Gb_Ba with deletion of the intervening  ebg  sequence. The sequence of the insert is shown below with lower case letters representing the  ebg  sequences flanking the insert shown below (SEQ ID NO: 98): 
         [0000]    
       
         
               
             
           
               
                 ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGTAAACTAGTCAA 
               
               
                   
               
               
                 CACGCACGGTGTTAGATATTTATCCCTTGCGGTGATAGATTGAGCACATC 
               
               
                   
               
               
                 GATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAACCAT 
               
               
                   
               
               
                 TAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAA 
               
               
                   
               
               
                 AAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGAT 
               
               
                   
               
               
                 GGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCAT 
               
               
                   
               
               
                 TAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTT 
               
               
                   
               
               
                 GAAGAATTTAGCCCTTCAATCGCCAGAGAATCTACGAGATGTATGAAGCG 
               
               
                   
               
               
                 GTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTC 
               
               
                   
               
               
                 TCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAG 
               
               
                   
               
               
                 GTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCA 
               
               
                   
               
               
                 TTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAA 
               
               
                   
               
               
                 GCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTG 
               
               
                   
               
               
                 TTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACC 
               
               
                   
               
               
                 TTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAA 
               
               
                   
               
               
                 CCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGA 
               
               
                   
               
               
                 AAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAGACTAGT 
               
               
                   
               
               
                 GGATCCACTAGTgtttctgccc 
               
             
          
         
       
     
         [0382]    The construct was delivered to the chromosome using a recombinant phage called MMebg-cI857s7enhanced RBS #4 into F′tet/393. After recombination and resolution only the chromosomal insert described above remains in the cell. It was renamed F′tet/GM101. F′tet/GM101 was then modified by the delivery of a lacI Q  construct into the  ebg  operon between nucleotide position 2493 and 2937 as numbered in the Genbank accession number M64441Gb_Ba with the deletion of the intervening  ebg  sequence. The sequence of the insert is shown below with the lower case letters representing the  ebg  sequences flanking the insert (SEQ ID NO: 99) shown below: 
         [0000]    
       
         
               
             
           
               
                 ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCA 
               
               
                   
               
               
                 TGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGT 
               
               
                   
               
               
                 AACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTT 
               
               
                   
               
               
                 CCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAA 
               
               
                   
               
               
                 GTCGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACA 
               
               
                   
               
               
                 ACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTC 
               
               
                   
               
               
                 TGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC 
               
               
                   
               
               
                 GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGT 
               
               
                   
               
               
                 CGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTG 
               
               
                   
               
               
                 GGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA 
               
               
                   
               
               
                 GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGAC 
               
               
                   
               
               
                 ACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCG 
               
               
                   
               
               
                 TGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGC 
               
               
                   
               
               
                 CCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATA 
               
               
                   
               
               
                 TCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGA 
               
               
                   
               
               
                 GTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATC 
               
               
                   
               
               
                 GTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAAT 
               
               
                   
               
               
                 GCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG 
               
               
                   
               
               
                 TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACC 
               
               
                   
               
               
                 ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTT 
               
               
                   
               
               
                 GCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCG 
               
               
                   
               
               
                 TCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCC 
               
               
                   
               
               
                 TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTC 
               
               
                   
               
               
                 CCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga 
               
             
          
         
       
     
         [0383]    The construct was delivered to the chromosome using a recombinant phage called AGebg-LacIQ#5 into F′tet/GM101. After recombination and resolution only the chromosomal insert described above remains in the cell. It was renamed F′tet/GM221. The F′tet episome was cured from the strain using acridine orange at a concentration of 25 μg/ml in LB. The cured strain was identified as tetracyline sensitive and was stored as GM221. 
         [0384]    Expression in  E. coli.    
         [0385]    Cultures of each of the pAMG21-Fc-fusion constructs in  E. coli  GM221 were grown at 37° C. in Luria Broth medium. Induction of gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml. Cultures were incubated at 37° C. for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-fusions were most likely produced in the insoluble fraction in  E. coli . Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 10% β-mercaptoethanol and were analyzed by SDS-PAGE. In each case, an intense Coomassie-stained band of the appropriate molecular weight was observed on an SDS-PAGE gel. 
       Example 3 
     TALL-1 Peptibody Inhibits TALL-1 Mediated B Cell Proliferation 
       [0386]    Mouse B lymphocytes were isolated from C57BL/6 spleens by negative selection. (MACS CD43 (Ly-48) Microbeads, Miltenyi Biotech, Auburn, Calif.). Purified (10 5 ) B cells were cultured in MEM, 10% heat inactivated FCS, 5×10 −5 M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin) in triplicate in 96-well flat bottom tissue culture plates with 10 μg/ml TALL-1 protein and 2 μg/ml of Goat F(ab′) 2  anti-mouse IgM (Jackson ImmunoResearch Laboratory, West Grove, Pa.) with the indicated amount of recombinant TALL-1 peptibody for a period of 4 days at 37° C., 5% CO 2 . Proliferation was measured by the uptake of radioactive  3 [H] thymidine after an 18-hour incubation period. 
       Example 4 
     TALL-1 Peptibody Blocks TALL-1 Binding to its Receptors 
       [0387]    Reacti-Gel 6× (Pierce) were pre-coated with human AGP3 (also known as TALL-1, Khare et al., Proc. Natl. Acad. Sci. 97:3370-3375, 2000) and blocked with BSA. 100 pM and 40 pM of AGP3 peptibody samples were incubated with indicated various concentrations of human AGP3 at room temperature for 8 hours before run through the human AGP3-coated beads. The amount of the bead-bound peptibody was quantified by fluorescent (Cy5) labeled goat anti-human-Fc antibody (Jackson Immuno Research). The binding signal is proportional to the concentration of free peptibody at binding equilibrium. Dissociation equilibrium constant (K D ) was obtained from nonlinear regression of the competition curves using a dual-curve one-site homogeneous binding model (KinEx™ software). K D  is about 4 pM for AGP3 peptibody (SEQ ID NO: 123) binding with human AGP3 ( FIG. 9 ). 
         [0388]    To determine if this AGP3 peptibody can neutralize murine AGP3 binding as well as human AGP3, a BlAcore neutralizing assay was utilized. All experiments were performed on a BlAcore 3000 at room temperature. Human TACI-Fc protein (Xia et al, J. Exp. Med. 192, 137-144, 2000) was immobilized to a Bl chip using 10 mM Acetate pH 4.0 to a level of 2900RU. A blank flow cell was used as a background control. Using a running buffer of PBS (without calcium or magnesium) containing 0.005% P20, 1 nM recombinant human AGP3 (in running buffer plus, 0.1 mg/ml BSA) was incubated without and with indicated various amount of AGP3 peptibody (x axis) before injected over the surface of the receptor. Regeneration was performed using 8 mM glycine pH 1.5 for 1 minute, 25 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) pH 10.5, 1 M NaCl for 1 minute. For determination of murine AGP3 binding, human his-tagged TACI was immobilized to 1000 RU in the above buffer. 5 nM recombinant murine AGP3 (in running buffer plus, 0.1 mg/ml BSA) was incubated without and with the various amounts indicated in  FIGS. 11A  and B of AGP3 peptibody (x axis) before injected over the surface of the receptor. Regeneration was performed with 10 mM HCl pH2, twice for 30 seconds. Relative binding of both human and murine AGP3 at presence vs absence of AGP3 peptibody (SEQ ID NO: 123) was measured (y axis). Relative binding response was determined as (RU-RU blank/RUo-RU blank). The AGP3 peptibody (SEQ ID NO: 123) inhibited both human and murine AGP3 binding to its receptor TACI ( FIGS. 10A and 10B ). 
         [0389]    To examine if this AGP3 peptibody blocks AGP3 binding to all three receptors (TACI, BCMA and BAFFR), recombinant soluble receptor TACI, BCMA and BAFFR proteins were immobilized to CM5 chip. Using 10 mM acetate, pH4, human TACI-Fe was immobilized to 6300 RU, human BCMA-Fc to 5000 RU, and BAFFR-Fc to 6000 RU. 1 nM of recombinant human AGP3 (in running buffer containing 0.1 mg/ml BSA and 0.1 mg/ml Heparin) or 1 nM recombinant APRIL protein (Yu, et al., Nat. Immunol., 1:252-256, 2000) were incubated with indicated amount of AGP3 peptibody before injection over each receptor surface. Regeneration for the AGP3 experiment was done with 8 mM glycine, pH 1.5, for 1 minute, followed by 25 mM CAPS, pH 10.5, 1M NaCl for 1 minute. Regeneration for the APRIL experiment was performed with 8 mM glycine, pH 2, for one minute, followed by 25 mM CAPS, pH 10.5, 1 M NaCl for one minute. Relative binding of AGP3 or APRIL was measured. AGP3 peptibody (SEQ ID NO: 123) blocked AGP3 binding to all three receptors ( FIG. 11A ). AGP3 peptibody didn&#39;t affect APRIL binding to the receptors ( FIG. 11B ). 
       Example 5 
     AGP3 Peptibody Blocks AGP3 Mediated B Cell Proliferation 
       [0390]    Mouse B lymphocytes were isolated from C57BL/6 spleens by negative selection. (MACS CD43 (Ly-48) Microbeads, Miltenyi Biotech, Auburn, Calif.). Purified (10 5 ) B cells were cultured in minimal essential medium (MEM), 10% heat inactivated fetal calf serum (FCS), 5×10 −5 M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin) in triplicate in 96-well flat bottom tissue culture plates with 10 ng/ml AGP3 (TALL-1) protein and 2 μg/ml of Goat F(ab′) 2  anti-mouse IgM (Jackson ImmunoResearch Laboratory, West Grove, Pa.) with the indicated amount of recombinant AGP3 peptibody (SEQ ID NO: 123) for a period of 4 days at 37° C., 5% CO 2 . Proliferation was measured by the uptake of radioactive  3 [H] thymidine after an 18-hour incubation period. 
       Example 6 
     AGP3 Peptibody on AGP3-Stimulated Ig Production in Mice 
       [0391]    Mice (Balb/c females of 9-14 weeks of age and 19-21 g of weight) were purchased from Charles River Laboratories, Wilmington, Mass. Mice (n=10) were treated i.p. with 1 mg/Kg of human AGP3 once a day for five consecutive days followed by 5 mg/Kg or 0.5 mg/Kg of AGP3 peptibody (SEQ ID NO: 123) or by saline or by 5 mg/Kg of human Fc. Other mice were left untreated. Mice were sacrificed on the sixth day to measure serum IgM and IgA, which were measured by ELISA. Briefly, plates were coated with capture antibodies specific for IgM or IgA (Southern Biotechnology Associates, Birmingham, Ala.), blocked, and added with dilutions of standard (IgM from Calbiochem, San Diego, Calif. and IgA from Southern Biotechnology Associates) or test samples. Captured Ig were revealed using biotinylated antibodies specific for IgM or IgA (Southern Biotechnology Associates), neutravidin-conjugated peroxidase (Pierce, Rockford, Ill.), and tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL, Gaithersburg, Md.). Optical densities were quantitated in a Thermomax ELISA reader (Molecular Devices, Menlo Park, Calif.). 
         [0392]    Human AGP3-stimulated increase in serum levels of IgM and IgA was blocked by 5 mg/Kg of the anti-AGP3 peptibody (SEQ ID NO: 123) and not by 0.5 mg/Kg ( FIGS. 12A and 12B ). 
       Example 7 
     AGP3 Peptibody Reduced Spleen B Cell Number in Mice 
       [0393]    Mice (as above, n=7) were treated i.p. for seven consecutive days with 5 mg/Kg or 1.5 mg/Kg or 0.5 mg/Kg of AGP3 peptibody (SEQ ID NO: 123) or with saline or with 5 mg/Kg of human Fc. Mice were sacrificed on the eighth day to count spleen B cell number. Spleens were collected in saline and gently disrupted by manual homogenization to yield a cell suspension. The total cell number was obtained with a HE counter (Technicon, Tarrytown, N.Y.). Percentages of B cells were derived by immunofluorescence double staining and flow cytometry using fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated Ab against CD3 and B220, respectively (PharMingen, San Diego, Calif.) and a FACScan analyser (Becton and Dickinson, Mountain View, Calif.). B cells were identified for being CD3-B220+. At all doses, the AGP3 peptibody (SEQ ID NO: 123) decreased spleen B cell number in a dose-response fashion ( FIGS. 12A and 12B ) (SEQ ID NO: 123). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 AGP3 Pb Reduces B Cell Number in Normal Mice 
               
             
          
           
               
                   
                   
                 dose  
                 spleen B cell 
                   
                   
               
               
                   
                 n = 7 
                 (1/day × 7) 
                 (1 × 10e6) 
                 SD 
                 t test 
               
               
                   
                   
               
               
                   
                 saline 
                   
                 51.3 
                 9.6 
                   
               
               
                   
                 Fc 
                   5 mg/Kg 
                 45.5 
                 7.1 
                   
               
               
                   
                 Peptibody 
                   5 mg/Kg 
                 20.1 
                 3.8 
                 1.37856E−05 
               
               
                   
                   
                 1.5 mg/Kg 
                 22.6 
                 6.9 
                 5.10194E−05 
               
               
                   
                   
                 0.5 mg/Kg 
                 25.8 
                 3.6 
                 0.000111409 
               
               
                   
                   
               
             
          
         
       
     
       Example 8 
     AGP3 Peptibody Reduced Arthritis Severity in Mouse CIA Model 
       [0394]    Eight to 12 week old DBA/1 mice (obtained from Jackson Laboratories, Bar Harbor, Me.) were immunized with bovine collagen type II (bCII) (purchased from University of Utah), emulsified in complete Freunds adjuvant (Difco) intradermally at the base of tail. Each injection was 100 μl containing 100 μg of bCII. Mice were boosted 3 weeks after the initial immunization with bCII emulsified in incomplete Freunds adjuvant. Treatment was begun from the day of booster immunization for 4 weeks. Mice were examined for the development of arthritis. As described before (Khare et al., J. Immunol, 155: 3653-9, 1995), all four paws were individually scored from 0-3. Therefore arthritis severity could vary from 0 to 12 for each animal. AGP3 (SEQ ID NO: 123) peptibody treatment significantly reduced the severity of arthritic scores ( FIG. 13 ). 
         [0395]    Serum samples were taken one week after final treatment (day 35) for the analysis of anti-collagen antibody level. High binding ELISA plates (Immulon, Nunc) were coated with 50 μl of 4 μg/ml solution of bovine CII in carbonate buffer and plated were kept in cold overnight in the refrigerator. Plates were washed three times with cold water. 75 μl of blocking solution made up of PBS/0.05% tween 20/1% BSA was used to block non-specific binding for an hour. Samples were diluted (in blocking buffer) in dilution plates at 1:25, 1:100, 1:400, and 1:1600 and 25 μl of these samples were added to each well of the ELISA plate for a final dilution of 100, 400, 1600, and 6400 with a final volume of 100 μl/well. After incubation at room temperature for 3 hours, plates were washed three times again. 100 μl of secondary antibody diluted in blocking buffer (rat anti-mouse IgM, IgG2a, IgG2b, IgG1, IgG3-HRP) was added to each well and plates were incubated for at least 2 hours. Plates were washed four times. 100 μl of TMB solution (Sigma) was added to each well and the reaction was stopped using 50 μl of 25% sulfuric acid. Plates were read using an ELISA plate reader at 450 nm. OD was compared with a standard pool representing units/ml. AGP3 peptibody (SEQ ID NO: 123) treatment reduced serum anti-collagen II IgG1, IgG3, IgG2a, and IgG2b levels compared to PBS or Fc control treatment groups ( FIG. 14 ). 
       Example 9 
     Treatment of AGP3 Peptibody in NZB/NZW Lupus Mice 
       [0396]    Five month old lupus prone NZBx NZBWF1 mice were treated i.p. 3×/week for 8 weeks with PBS or indicated doses of AGP3 peptibody or human Fc proteins. Prior to the treatment, animals were pre-screened for protein in the urine with Albustix reagents strips (Bayer AG). Mice having greater than 100 mg/dl of protein in the urine were not included in the study. Protein in the urine was evaluated monthly throughout the life of the experiment. AGP3 peptibody (SEQ ID NO: 123) treatment led to delay of proteinuria onset and improved survival ( FIGS. 15A and 15B ). 
         [0397]    AGP3 peptibody treatment reduced B cell number in mice. Balb/c mice received 7 daily intraperitoneal injections of indicated amount of AGP3 peptibody (SEQ ID NO: 123) or human Fc protein. On day 8, spleens were collected, and subject to FACS analysis for B220+B cells as set for in Table 8. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 AGP3 Pb Reduces B Cell Number in Normal Mice 
               
             
          
           
               
                   
                   
                 dose  
                 Spleen B cell 
                   
                   
               
               
                   
                 n = 7 
                 (1/day × 7) 
                 (1 × 10e6) 
                 SD 
                 t test 
               
               
                   
                   
               
               
                   
                 saline 
                   
                 51.3 
                 9.6 
                   
               
               
                   
                 Fc 
                   5 mg/Kg 
                 45.5 
                 7.1 
                   
               
               
                   
                 Peptibody 
                   5 mg/Kg 
                 20.1 
                 3.8 
                 1.37856E−05 
               
               
                   
                   
                 1.5 mg/Kg 
                 22.6 
                 6.9 
                 5.10194E−05 
               
               
                   
                   
                 0.5 mg/Kg 
                 25.8 
                 3.6 
                 0.000111409 
               
               
                   
                   
               
             
          
         
       
     
         [0398]    The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto, without departing from the spirit and scope of the invention as set forth herein.