HK1161267A - TNF-α ANTAGONIST MULTI-TARGET BINDING PROTEINS - Google Patents

Description

TNF-alpha antagonist multi-target binding proteins

Technical Field

The present invention relates generally to the field of multi-target binding molecules and their therapeutic applications, and more specifically to fusion proteins consisting of a TNF-alpha antagonist domain and another binding domain that antagonizes a heterologous target such as IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or BLyS/APRIL, or a TNF-alpha antagonist domain and another binding domain that agonizes a heterologous target such as IL10, and compositions and therapeutic applications thereof.

Background

Tumor Necrosis Factor Receptor (TNFR) is a member of the tumor necrosis factor receptor superfamily and is a receptor for tumor necrosis factor-alpha (TNF-alpha), also known as CD120 or cachectin. There are two variants of this cytokine receptor, TNFR1 and TNFR2 (the CD120a and CD120b receptors, respectively). TNFR1 has a molecular weight of approximately 55KD and is therefore sometimes referred to as p 55. TNFR2 has a molecular weight of approximately 75kD and is therefore sometimes referred to as p 75.

Most cell types and tissues appear to express both TNF receptors. Although they are all present on the cell surface in soluble form and are all active in signaling, they can mediate different cellular responses. TNFR1 appears to be responsible for transducing most TNF responses. TNFR2 has other activities such as stimulating thymocyte proliferation, activating NF-. kappa., assisting TNFR1 in signaling in a response mediated primarily by TNFR1, such as cytotoxicity.

TNF antagonists, such as anti-TNF antibodies, may have beneficial effects on a variety of inflammatory disorders. For example, infliximab is indicated in the united states for the treatment of rheumatoid arthritis, crohn's disease, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, and ulcerative colitis. According to REMICADE(infliximab) prescription information, biological activities attributed to TNF include induction of pro-inflammatory cytokines such as Interleukins (IL)1 and 6, enhancement of leukocyte migration by increasing endothelial layer permeability and adhesion molecule expression of endothelial cells and leukocytes, activation of neutrophil and eosinophil functional activities, induction of acute phase reactants and other liver proteins, and tissue degrading enzymes produced by synoviocytes and/or chondrocytes. In recent years, the perimedullary delivery of the TNF α inhibitor etanercept has been shown to alleviate symptoms in Alzheimer's disease patients (Tobinick and Gross (2008) BMC neuron.8: 27-36; Griffin (2008) J. neuroin fluoride, 5: 3-6).

Bispecific molecules comprising a specific binding site for a TNF receptor or TNF α and a specific binding site for a heterologous molecule have been previously described (see e.g. US 2008/0260757, US 2006/0073141, US 2007/0071675, WO 2006/074399 and WO 2007/146968). U.S. patent 7,300,656 describes bispecific molecules comprising an antigen-binding domain of an anti-IFN- γ antibody and a TNF- α receptor or an extracellular region thereof.

Brief description of the drawings

FIGS. 1A-1C show that multispecific (cross-receptor (Xreceptor)) fusion proteins containing one of various different Hyper (Hyper) -IL6 binding domains fused to TNFR extracellular domains were detected by ELISA to be capable of specifically binding to Hyper-IL 6, and that these multispecific fusion proteins preferentially bind to Hyper-IL 6 compared to IL6 and IL6R alone. Only two fusion proteins were tested to bind IL6, and none bound sIL 6R.

FIG. 2 shows that a multispecific fusion protein comprising an extracellular domain of TNFR fused to one of various super-IL 6 binding domains was detected to bind TNF- α by ELISA.

FIG. 3 shows that a multispecific fusion protein containing one of various super-IL 6 binding domains fused to the extracellular domain of TNFR can bind both super-IL 6 and TNF- α as detected by ELISA.

FIG. 4 shows that a multispecific fusion protein containing one of various super-IL 6 binding domains fused to the extracellular domain of TNFR was able to block gp130 binding to super-IL 6 as detected by ELISA.

FIGS. 5A and 5B show that a multispecific fusion protein containing one of various super-IL 6 binding domains fused to the extracellular domain of TNFR blocks TF-1 cell proliferation induced by (A) IL6 or (B) super-IL 6.

FIG. 6 shows that a multispecific fusion protein containing one of various super-IL 6 binding domains fused to the extracellular domain of TNFR was able to block TNF- α binding to TNFR as detected by ELISA.

FIG. 7 shows that a multi-specific fusion protein containing the extracellular domain of TNFR fused to one of the various super-IL 6 binding domains blocks TNF- α induced killing of L929 cells.

FIG. 8 shows that a multispecific fusion protein containing the extracellular domain of a TNFR fused to the extracellular domain of a human TWEAK receptor blocks TWEAK-induced killing of HT29 cells.

Figure 9 shows that a multispecific fusion protein containing a TNFR ectodomain fused to an OPG ectodomain is able to block RANKL-mediated osteoclastogenesis in RAW246.7 cells.

Figure 10 shows that multispecific fusion proteins containing TNFR extracellular domains fused to IL6 binding domains do not bind HepG2 (liver) cells.

Figure 11 shows that a multispecific fusion protein containing a TNFR ectodomain fused to an IL6 binding domain blocks the sal 6-induced SAA response in mice.

Figure 12 shows that a multispecific fusion protein containing a TNFR ectodomain fused to an IL6 binding domain blocks the HIL 6-induced sgp130 response in mice.

Figures 13A and B show the results of studies on the ability of multispecific fusion proteins containing TNFR ectodomains fused to IL6 binding domains to block TNF α -induced mouse SAA responses 2 and 24 hours post-administration.

Detailed Description

The present invention provides multi-specific fusion proteins, referred to herein as cross-receptor (xceptor) molecules. Exemplary structures of such multi-specific fusion proteins include N-BD-ID-ED-C, N-ED-ID-BD-C and N-ED1-ID-ED2-C, wherein N-and C represent amino-and carboxy-termini, respectively, BD is an immunoglobulin-like or immunoglobulin variable region binding domain, ID is an intervening domain, and ED is an extracellular domain (e.g., extracellular domain), such as a receptor ligand binding domain, a cysteine-rich domain (e.g., LDL A-like domain; see WO 02/088171 and WO 04/044011), a semaphorin or semaphorin-like domain, and the like. In some constructs, the ID may comprise an immunoglobulin constant region or sub-region disposed between the first and second binding domains. In another construct, BD and ED are each linked to the ID by the same or different linker (e.g., a linker comprising 1-50 amino acids), such as an immunoglobulin hinge region (consisting of, for example, a top region and a core region) or a functional variant thereof, or a lectin interdomain region or a functional variant thereof, or a stem region (talk region) of a differentiated population (CD) molecule or a functional variant thereof.

Before setting forth the invention in more detail, it may be helpful to provide an understanding of the invention with a definition of certain terms used herein. Other definitions are set forth throughout this specification.

In this specification, any concentration range, percentage range, proportion range or integer range should be understood to include any integer value within the range, including fractional values (such as tenths and hundredths of integers) where appropriate, unless otherwise specified. In addition, any numerical range recited herein with respect to a physical characteristic, such as a polymer subunit, size, or thickness, should be understood to include any integer within the stated range unless otherwise specified. As used herein, the term "about" or "consisting of …" is to. + -. 20% of the stated range, value or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to one or more of the listed components. The use of alternatives (such as "or") should be understood as one, two or any combination of said alternatives. The terms "comprising" and "including" may be used synonymously herein. Further, it is understood that the application discloses individual compounds or groups of compounds derived from various combinations of the structures and substituents described herein as if each compound or group of compounds were listed individually. Thus, the selection of a particular structure or particular substituents is within the scope of the present invention.

The "binding domain" or "binding region" of the invention may be, for example, any protein, polypeptide, oligopeptide or peptide capable of specifically recognizing and binding a biomolecule (e.g., TNF- α or IL6) or a complex or assembly or aggregate of more than one same or different molecules, whether stable or transient, such as the IL6/IL6R complex. Such biomolecules include proteins, polypeptides, oligopeptides, peptides, amino acids or derivatives thereof; a lipid, fatty acid or derivative thereof; carbohydrates, sugars or derivatives thereof; a nucleotide, nucleoside, peptide nucleic acid, nucleic acid molecule, or derivative thereof; glycoproteins, glycopeptides, glycolipids, lipoproteins, proteolipids or derivatives thereof; other biomolecules that may be present in the biological sample; or any combination thereof. Binding domains include any naturally occurring, synthetic, semi-synthetic or recombinantly produced binding partner of a biomolecule or other target of interest. Various experimental methods are known for identifying binding domains of the invention that specifically bind to a particular target, including Western blotting, ELISA, or BiacoreAnd (6) analyzing.

If the binding domains and fusion proteins thereof of the invention bind to the affinity or K of the target molecule_a(i.e., the equilibrium binding constant of a particular binding interaction, in units of 1/M) is, for example, greater than or equal to about 10⁵M^-1、10⁶M^-1、10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1、10¹²M^-1Or 10¹³M^-1They are capable of binding to a desired degree, including "specific or selective" binding to the target, without significantly binding to other components in the sample. "high affinity" binding domain refers to K_aIs at least 10⁷M^-1At least 10⁸M^-1At least 10⁹M^-1At least 10¹⁰M^-1At least 10¹¹M^-1At least 10¹²M^-1At least 10¹³M^-1Or a higher binding domain. Alternatively, avidity can be defined as the equilibrium dissociation constant (K) of a particular binding interaction_d) The unit is M (e.g. 10)^-5M to 10^-13M). The affinity of the binding domain polypeptides and fusion proteins of the invention can be readily determined by conventional techniques (see, e.g., Scatchard et al (1949) Ann.N.Y.Acad.Sci.51: 660; U.S. Pat. Nos. 5,283,173; 5,468,614; Biacore)Analyzing; or equivalent).

The binding domains of the invention can be produced according to the methods described herein or by various methods known in the art (see, e.g., U.S. Pat. Nos. 6,291,161; 6,291,158). Sources include those from various species, including humans, camelids (from camels, dromedary, or llamas; Hamers-Casterman et al (1993) Nature, 363: 446 and Nguyen et al (1998) J.mol.biol., 275: 413), shark (Roux et al (1998) Proc.nat' l.Acad.Sci. (USA) 95: 11804), fish (Nguyen et al (2002) Immunogenetics, 54: 39), rodent, avian, ovine antibody gene sequences (which may be in the form of antibodies, sFv, scFv or Fab, e.g., in a phage library), sequences encoding random peptide libraries or sequences encoding various amino acids engineered into loop regions of other non-antibody scaffolds, such as fibrinogen domains (see, e.g., Weisel et al (1985) Science 230: 1388), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498), lipocalin domains (see, e.g., WO 2006/095164), V-like domains (see, e.g., U.S. patent application publication No.).2007/0065431), C-type lectin domain (Zelensky and Gready (2005) FEBS j.272: 6179) mAb²Or Fcab^TM(see, e.g., PCT patent application publication Nos. WO 2007/098934; WO 2006/072620), and the like. Furthermore, synthetic single chain IL6/IL6R complexes, such as the human IL6/IL6R complex or super-IL 6(IL6 linked to IL6R via a peptide linker) may also be used as convenient systems (e.g., mouse, HuMAb mouse)TC mouse^TMKM-miceLlama, chicken, rat, hamster, rabbit, etc.) to develop the binding domains of the invention.

Unless otherwise indicated, terms understood by those of skill in the antibody arts each have the meaning available in the art. For example, the term "V_L"and" V_H"refers to the variable binding domains derived from the light and heavy chains of an antibody, respectively. The variable binding regions are composed of discrete, well-defined subregions, which are referred to as "complementarity determining" (CDRs) and "framework regions" (FRs). The term "C_L"and" C_H"refers to an" immunoglobulin constant region "and refers to a constant region derived from a light or heavy chain of an antibody, respectively, which may be further classified as C according to the isotype (IgA, IgD, IgE, IgG, IgM) of the antibody from which the region is derived_H1、C_H2、C_H3And C_H4A constant region domain. A portion of the constant region domain constitutes the Fc region (the "crystallizable fragment" region) which contains the domains responsible for immunoglobulin effector functions such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell-mediated phagocytosis), CDC (complement-dependent cytotoxicity) and complement binding, binding to Fc receptors, increased half-life in vivo relative to polypeptides lacking the Fc region, protein A binding, and possibly even placental transfer (see Capon et al (1989) Nature, 337: 525). Furthermore, polypeptides containing an Fc region are capable of dimerizing or multimerizing. In this context "The hinge region, "also referred to as a" linker, "is an amino acid sequence that is interposed between and connects the variable binding and constant regions of a chain of an antibody and is known in the art to provide flexibility to the antibody or antibody-like molecule in the form of a hinge.

The domain structure of immunoglobulins is amenable to engineering because the antigen binding regions and the domains that produce effector functions can be exchanged between different classes and subclasses of immunoglobulin molecules. For a review of immunoglobulin structure and function see, e.g., Harlow et al, eds, antibodies: a Laboratory Manual (Antibodies: A Laboratory Manual), Chapter 14 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, 1988). For detailed description and information on various aspects of recombinant antibody technology, see "recombinant antibodies" textbook (John Wiley & Sons, NY, 1999). A collection of detailed protocols for Antibody Engineering can be found in The R.Kontermann and S.D. Subel eds, The handbook of Antibody Engineering laboratories (The Antibody Engineering Lab Manual) (Schpringer Press, Springer Verlag, Heidelberg/New York, 2000, N.Y.).

As used herein, a "derivative" refers to a chemically or biologically modified compound that is structurally similar to, and (actually or theoretically) obtainable from, the parent compound. Generally, a "derivative" is different from an "analog" in that the parent compound may be the starting material for producing the "derivative", but the parent compound is not necessarily used as the starting material for producing the "analog". An analog may have different chemical or physical properties than the parent compound. For example, derivatives may be more hydrophilic or have altered reactivity compared to the parent compound, e.g., CDRs with amino acid changes that alter their target affinity.

The term "biological sample" includes blood samples, biopsy samples, tissue explants, organ cultures, biological fluids, or any other tissue or cell, or other preparation from a subject or biological source. The subject or biological source may be, for example, a human or non-human animal, a primary cell culture, or a cell line suitable for culture, including genetically engineered cell lines containing recombinant nucleic acid sequences in chromosomally integrated or episomal form, somatic cell hybrid cell lines, immortalized or immortalizable cell lines, differentiated or differentiable cell lines, transformed cell lines, and the like. In other embodiments of the invention, the subject or biological source may be suspected of having, or at risk of having, a disease, disorder or condition, including a malignant disease, disorder or condition or a B cell disease. In certain embodiments, the subject or biological source may be a hyperproliferative, inflammatory, or autoimmune disease, and in certain other embodiments of the invention the subject or biological source may be known not to suffer from, or not at risk of suffering from, such a disease, disorder, or condition.

In certain embodiments, the invention depletes or modulates cells associated with aberrant TNF- α activity via a multispecific fusion protein that binds TNF- α and a second target outside of TNF- α, including, for example, IL6, IL6R, IL6/IL6R complex, receptor activator of nuclear factor κ B ligand (RANKL, also known as TNFSF11, ODF, CD254), IL7, IL17A, IL17F, IL17A/F, tumor necrosis factor-like weak inducer apoptosis (TWEAK; also known as tumor necrosis factor (ligand) superfamily member 12, TNFSF12), colony stimulating factor 2(CSF2, also known as granulocyte-macrophage colony stimulating factor or GM-CSF); insulin-like growth factor-1 (IGF 1); insulin-like growth factor-2 (IGF 2); IL 10; or a TNFSF13 family protein (e.g., TNFSF13, also known as proliferation-inducing ligand, APRIL, CD 256; or TNFSF13B, also known as B-lymphocyte stimulator, BLyS, CD257, BAFF). In certain embodiments, the multi-specific fusion protein comprises first and second binding domains, first and second linkers, and an intervening domain, wherein the intervening domain is fused at one end to the first binding domain that is a TNF-a ectodomain (e.g., an ectodomain, one or more cysteine-rich domains (CRDs), such as CRD1, CRD2, CRD3) via a linker and at the other end to the second binding domain via a linker. In some embodiments, less than a portion of an intact TNF- α extracellular domain is employed. In particular, domains within the extracellular domain that act as TNF- α antagonists or produce ligand binding are employed. In some embodiments, the second binding domain is not an IFN γ binding domain, such as an anti-IFN γ immunoglobulin domain or an extracellular domain of an IFN γ receptor.

In certain embodiments, the second binding domain is an IL6 antagonist (e.g., an immunoglobulin variable region specific for IL6 or the IL6/IL6R α complex), a RANKL antagonist (e.g., an immunoglobulin variable region specific for RANKL, or an osteoprotegerin ectodomain (e.g., SEQ ID NO: 737) or a RANKL binding fragment thereof), an IL7 antagonist (e.g., an immunoglobulin binding domain specific for IL7 or IL7R α, or an IL7R α ectodomain (e.g., SEQ ID NO: 738 or 739) or an IL7 binding fragment thereof), an IL17A/F antagonist (e.g., an immunoglobulin binding domain specific for IL17A, IL17F, IL17A/F, or IL17RA, IL 3717 84, IL17RA/C, an IL17RA ectodomain (e.g., SEQ ID NO: 739, 816), an IL17RA/C ectodomain, an IL 4617/C ectodomain, an IL R/C (e.g., SEQ ID NO: 740, IL 24, IL 5817/F binding fragments thereof), IL 24/A, IL 5817/or IL F, TWEAK antagonists (e.g., an immunoglobulin binding domain specific for TWEAK or TWEAKR, or TWEAKR ectodomain (e.g., SEQ ID NO: 741) or a TWEAK-binding fragment thereof), CSF2 antagonists (e.g., an immunoglobulin binding domain specific for CSF2 or CSF2R α, or CSF2R α ectodomain (e.g., SEQ ID NO: 742) or a CSF 2-binding fragment thereof), IGF1 or IGF2 antagonists (e.g., an immunoglobulin binding domain specific for IGF1 or IGF2, or an IGF1R ectodomain (e.g., SEQ ID NO: 746, 818) or an IGFBP ectodomain (e.g., SEQ ID NO: 747-753), or an IGF-binding fragment thereof), or a TYBLS/APRIL antagonist (e.g., an immunoglobulin binding domain specific for BLyS/APRIL or TACI, or a TACI ectodomain (e.g., SEQ ID NO: 743) or BAFF (also known as TNFRSF 13) Bank accession No. GenyS-APRIL (e.g., amino acid No. 443177.1-NP 76 or a fragment thereof). In other embodiments, the second binding domain is an IL10 agonist, such as IL10 (as set forth in SEQ ID NO: 754) or IL10Fc, or a functional subdomain thereof, or a single chain binding protein, such as an scFv, that specifically binds IL10R1 or IL10R 2.

Herein, when referring to a complex of IL6 with membrane IL6R α or soluble IL6R α (sIL6R α), a complex of IL6 with membrane or soluble IL6 receptor (IL6R α) is referred to as IL6xR, and when referring to a complex of IL6 with sIL6R α only, it is referred to as sIL6 xR. In some embodiments, a multispecific fusion protein comprising a specific binding domain of IL6xR has one or more of the following properties: (1) the affinity for IL6xR complex is higher than or equal to the affinity for IL6 alone or IL6R α alone, or the affinity for IL6R α alone or IL6xR complex is greater than the affinity for IL6 alone; (2) competes with membrane gp130 for binding to the sIL6xR complex or enhances the binding of soluble gp130 to the sIL6xR complex; (3) preferentially inhibits IL6 trans signaling relative to IL6 cis signaling; and (4) does not inhibit signaling by gp130 family cytokines other than IL 6.

TNF-alpha antagonists

TNFR is a type I transmembrane protein whose extracellular domain contains three well-ordered cysteine-rich domains characteristic of the TNFR superfamily (CRD1, CRD2, CRD3) and a fourth, less conserved, membrane proximal CRD (Banner et al (1993) Cell 73: 431). The TNF-alpha antagonists of the present invention inhibit the inflammatory or hyperproliferative activity of TNF-alpha. The antagonist domain can block TNFR multimerization or TNF- α binding, or the domain can bind to a component of the receptor system and block activity by preventing ligand activity or preventing assembly of the receptor complex. Various TNF-alpha antagonists are known in the art, including anti-TNF antibodies such as infliximab, and soluble TNF receptor (sTNFR). Such antibody antagonists bind and inhibit TNF- α, but do not significantly inhibit TNF- β. anti-TNF antibodies, including monoclonal antibodies, can be prepared using known techniques, and are known in the art (see, e.g., U.S. patent No. 6,509,015). The TNF- α antagonists of the invention may also include one or more TNF- α binding domains present in the extracellular domain of a TNFR.

Contemplated TNF- α antagonists include a TNFR extracellular domain or subdomain, one or more TNFR CRD domains (e.g., CRD2 and CRD3), or a TNF- α -specific antibody-derived binding domain (similar to the IL6 or IL6xR complex-specific antibody-derived binding domains described herein). In some embodiments, the TNF- α antagonist can be an extracellular domain of a TNFR ("ectodomain"), such as the ectodomain of TNFR1 or TNFR 2. TNFR ectodomain as used herein refers to sTNFR, one or more CRDs or any combination of TNFR domains. In certain embodiments, the TNF- α antagonist comprises the amino-terminal portion of TNFR2 (also referred to as p75, TNFRSF1B), such as the first 257 amino acids of TNFR2, such as GenBank accession NP-001057.1 (SEQ ID NO: 671). In other embodiments, the TNF- α antagonist comprises SEQ ID NO: 671 amino acids 23-257 (i.e., without the native leader sequence). In a preferred embodiment, the TNF- α antagonist comprises a fragment of TNFR2 (e.g., the extracellular domain) as set forth in SEQ ID NO: 671 amino acids 23-163 or SEQ ID NO: 671 amino acids 23-185 of SEQ ID NO: 671 amino acids 23-235. In other preferred embodiments, the TNF- α antagonist comprises a derivative of a fragment of TNFR2, such as the amino acid sequence of SEQ ID NO: 671 or amino acids 23-163 of SEQ ID NO: 671 or amino acids 23-185 of SEQ ID NO: 671 amino acids 23-235. In certain embodiments, the TNF- α antagonist comprises the amino-terminal portion of TNFR1 (also referred to as p55, TNFRSF1A), such as the first 211 amino acids of TNFR1, such as GenBank accession NP-001056.1 (SEQ ID NO: 672). In other embodiments, the TNF- α antagonist comprises SEQ ID NO: 672 amino acids 31-211 (i.e., without the native leader sequence).

In one aspect, the TNF- α antagonists or fusion proteins thereof of the invention are specific for TNF- α and have an affinity characterized by a dissociation constant (K)_d) About 10^-5M-10^-13M or less. In certain embodiments, the TNF- α antagonist or fusion protein thereof binds TNF- α with an affinity of less than about 300 pM. Another measure, kinetic dissociation (k)_d) (also referred to herein as k)_OFF) Is a measure of the rate of dissociation of the complex and thus the 'residence time' for the binding domain of the polypeptide of the invention to bind to the target molecule. k is a radical of_d(k_OFF) The unit of (2) is 1/second. Exemplary TNF-K of alpha antagonist_OFFMay be about 10^-4Second (e.g., about one day) to about 10^-8A/sec or less. In certain embodiments, k is_OFFThe range may be about 10^-1Second, about 10^-2Second, about 10^-3Second, about 10^-4Second, about 10^-5Second, about 10^-6Second, about 10^-7Second, about 10^-8Second, about 10^-9Second, about 10^-10A/sec or less (see Graff et al (2004) Protein Eng. Des. Sel.17: 293). In some embodiments, a TNF- α antagonist or fusion protein thereof of the invention will have a higher affinity and a lower k than the binding of the cognate TNFR to TNF- α_OFFRate binding of TNF-alpha. In other embodiments, a TNF- α antagonist of the invention or a fusion protein thereof that blocks or alters TNF- α dimerization or other cell surface activity may have a relatively intermediate affinity (i.e., K) compared to the affinity and dimerization rate associated with TNFR_dIs about 10^-8M to about 10^-9M) and a relatively moderate off-rate (i.e., k)_OFFMore closely about 10^-4In seconds).

Exemplary binding domains for use as TNF-alpha antagonists of the invention can be generated according to the methods described herein or various methods known in the art (see, e.g., U.S. Pat. nos. 6,291,161, 6,291,158). Sources include antibody gene sequences from various species including humans, camelids (from camels, dromedaries or llamas; Hamers-Casterman et al (1993) Nature, 363: 446 and Nguyen et al (1998) j.mol.biol., 275: 413), sharks (Roux et al (1998) proc.nat' l.acad.sci. (USA) 95: 11804), fish (Nguyen et al (2002) Immunogenetics, 54: 39), rodents, birds, sheep (in the form of sFv, scFv or Fab, as in phage libraries), sequences encoding random peptide libraries or sequences encoding other non-antibody scaffolds engineered amino acids in loop regions, such as the prose domain (see, e.g., weissel et al (1985) Science 230: 8), Kunitz domain (see, e.g., U.S. patent No. 6,423,498), lipoprotein domain (see, e.g., WO 2006/095164), fibrin-like domain (see, e.g., patent publication No. 2007/0065431, see, No. 2007/0065431V) C-type agglutinationThe element domain (Zelensky and great (2005) FEBS J.272: 6179), and the like. Alternatively, synthetic TNF- α or single-chain TNFR ectodomains can be used as a convenient system (e.g., mouse, HuMAb mouse)TC mouse^TMKM-miceLlama, chicken, rat, hamster, rabbit, etc.) to develop the binding domains of the invention.

In one illustrative example, a library of Fab fragment phages can be used to identify TNF- α antagonists or single-chain TNFR ectodomains of the invention that are specific for TNF- α by screening for binding to synthetic or recombinant TNF- α (using the amino acid sequence shown in GenBank accession NP-000585.2 or a fragment thereof) or single-chain TNFR ectodomains (see, e.g., Hoet al (2005) Nature Biotechnol. 23: 344). TNF- α or single-chain TNFR extracellular domains described herein or known in the art can be used for such screening. In certain embodiments, the TNF- α or single-chain TNFR ectodomain used to produce the TNF- α antagonist may further comprise an intervening domain or dimerization domain as described herein, such as an immunoglobulin Fc domain or fragment thereof.

In some embodiments, the TNF- α antagonist domain of the present invention comprises V as described herein_HAnd V_LA domain. In certain embodiments, V_HAnd V_LThe domain is a rodent (e.g., mouse, rat) domain, a humanized domain, or a human domain. In other embodiments, sequences of TNF- α antagonist domains of the invention are provided with one or more light chain variable regions (V)_L) Or one or more heavy chain variable regions (V)_H) Or both, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical in amino acid sequence, wherein each CDR has at most three amino acidsChanges occur (i.e., many changes occur in the framework regions).

The term "identical" or "percent identity" in two or more polypeptide or nucleic acid molecule sequences refers to two or more sequences or subsequences that are the same or wherein a percentage of the amino acid residues or nucleotides in a specified region are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) over the window of comparison or the specified region, when compared and aligned for maximum correspondence by manual alignment and visual inspection using methods known in the art, e.g., sequence comparison algorithms. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25: 3389 and Altschul et al (1990) J.mol.biol.215: 403.

in any of these or other embodiments described herein, V_LAnd V_HThe domains may be positioned in any orientation, separated by a linker of about 5-30 amino acids as described herein or any other amino acid sequence capable of providing a spacer function compatible with the interaction of the two sub-binding domains. In certain embodiments, connection V_HAnd V_LThe linker of the domain comprises SEQ ID NO: 497-604 and 791-796, such as linker 47(SEQ ID NO: 543) or linker 80(SEQ ID NO: 576). The multispecific binding domain will have at least two specific sub-binding domains that resemble an alpaca antibody tissue; or at least four specific sub-binding domains, resembling pairs of V_HAnd V_LMore conventional mammalian antibody composition of chains.

In other embodiments, the TNF- α antagonist domains and fusion proteins thereof of the present invention may comprise a binding domain comprising one or more complementarity determining regions ("CDRs"), or multiple copies of one or more such CDRs, obtained, derived or designed from the variable region of an anti-TNF- α or anti-TNFR scFv or Fab fragment, or the heavy or light chain variable region thereof.

CDRs are defined in the art in a variety of ways, including Kabat, Chothia, AbM, and contact definitions. The Kabat definition is based on sequence variability and is the most commonly used definition for predicting CDR regions (Johnson et al (2000) Nucleic Acids Res.28: 214). Chothia definition based on the structure of the ring region of the positioning (Chothia et al (1986) J.mol.biol.196: 901; Chothia et al (1989) Nature 342: 877). AbM definition makes a compromise between Kabat and Chothia definitions, which is the complete package developed by the Oxford Molecular Group (Oxford Molecular Group) for antibody structural modeling (Martin et al (1989) Proc. Nat' l.Acad.Sci. (USA) 86: 9268; Rees et al, ABMTM, computer programs for antibody variable region modeling (a computer program for modeling variable regions of antibodies, Oxford, UK; Oxford Molecular, Ltd.). In recent years, another definition called the contact definition has been introduced (see MacCallum et al (1996) J.mol.biol.5: 732), which is based on analysis of the crystal structure of the useful complexes.

Generally, the CDR domains in the heavy chain are designated H1, H2, and H3, which are numbered sequentially from amino terminus to carboxy terminus. CDR-H1 is about 10-12 residues in length, starting from the fourth residue after Cys as defined by Chothia and AbM, or from the five following residues as defined by Kabat. H1 may be followed by Trp, Trp-Val, Trp-Ile or Trp-Ala. H1 is approximately 10-12 residues in length according to the AbM definition, while the Chothia definition excludes the last four residues. CDR-H2 begins 15 residues after the end of H1, typically preceded by the sequence Leu-Glu-Trp-Ile-Gly (but many variations are known) and typically followed by the sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala, as defined by Kabat and AbM. The length of H2 is about 16-19 residues according to the Kabat definition, while the AbM definition predicts a length of 9-12 residues. Typically, CDR-H3 begins 33 residues after the end of H2, typically preceded by the amino acid sequence Cys-Ala-Arg and followed by the amino acid Gly, and ranges in length from 3 to about 25 residues.

Generally, the CDR regions in the light chain are designated L1, L2, and L3, which are numbered sequentially from the amino terminus to the carboxy terminus. CDR-L1 generally begins at about residue 24 and is typically preceded by Cys. The residue after CDR-L1 is always Trp, and the following sequence, which is preceded by: Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu. CDR-L1 is approximately 10-17 residues in length. CDR-L2 begins at about 16 residues after the end of L1 and is typically preceded by the residues Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. CDR-L2 is about 7 residues in length. CDR-L3 generally begins 33 residues after the end of L2, is typically preceded by Cys, and is typically followed by the sequence Phe-Gly-XXX-Gly which is about 7-11 residues in length.

Thus, a binding domain of the invention may comprise a single CDR from the variable region of anti-TNF- α or anti-TNFR or may comprise a plurality of the same or different CDRs. In certain embodiments, a binding domain of the invention comprises a V specific for TNF- α or TNFR_HAnd V_LA domain comprising a framework region and CDR1, CDR2 and CDR3 regions, wherein (a) said V_HThe domain comprises the amino acid sequence of heavy chain CDR 3; or (b) said V_LThe domain comprises the amino acid sequence of light chain CDR 3; or (c) the binding domain comprises the V of (a)_HAmino acid sequence and V of (b)_LAn amino acid sequence; or the binding domain comprises a V of (a)_HAmino acid sequence and V of (b)_LAmino acid sequence, and V_HAnd V_LPresent in the same reference sequence. In other embodiments, a binding domain of the invention comprises a V specific for TNF- α or TNFR_HAnd V_LA domain comprising a framework region and CDR1, CDR2 and CDR3 regions, wherein (a) the V_HThe domain comprises the amino acid sequences of heavy chain CDR1, CDR2, and CDR 3; or (b) said V_LThe domain comprises the amino acid sequences of light chain CDR1, CDR2, and CDR 3; or (c) the binding domain comprises the V of (a)_HAmino acid sequence and V of (b)_LAn amino acid sequence; or the binding domain comprises a V of (a)_HAmino acid sequence and V of (b)_LAmino acid sequence, wherein said V_HAnd V_LThe amino acid sequences are from the same reference sequence.

In any of the embodiments described herein comprising a specific CDR, the binding domain can comprise (i) a binding domain having a CDR with V_HThe amino acid sequence of the structural domain is at least 80 percent and 85 percentV of an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical_HA domain in which at most three amino acids of each CDR are altered (i.e., many alterations occur in the framework regions); or (ii) has a structure of_LV of an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence of a domain_LA domain in which at most three amino acids of each CDR are altered (i.e., many alterations occur in the framework regions); or (iii) V of (i)_H(iii) domains and V of (ii)_LA domain; or V of (i)_H(iii) domains and V of (ii)_LDomain of which V_HAnd V_LFrom the same reference sequence.

The TNF- α antagonist domain of the fusion proteins of the invention may be an immunoglobulin-like domain, such as an immunoglobulin scaffold. Immunoglobulin scaffolds of the invention include, but are not limited to, scfvs, domain antibodies, or heavy chain-only antibodies. In scfvs, the invention contemplates that the heavy and light chain variable regions are linked by any linker peptide known in the art so as to be compatible with the binding domain or region in the binding molecule. Exemplary linkers are based on Gly₄Linkers of Ser linker motifs, e.g. (Gly)₄Ser)_nWherein n is 1-5. If the binding domain of the fusion protein of the invention is based on or comprises a non-human immunoglobulin CDR, the binding domain may be "humanized" according to methods known in the art.

Alternatively, the TNF- α antagonist domain of the fusion proteins of the invention may be a scaffold other than an immunoglobulin scaffold. Other scaffolds contemplated by the present invention are capable of presenting TNF- α -specific CDRs in a functional conformation. Other stents contemplated include, but are not limited to: an a domain molecule, a fibronectin III domain, an anti-lipocalin (anticalin), an ankyrin repeat engineered binding molecule, an adeno-associated protein (adnectin), a kunitz (kunitz) domain or a protein AZ domain affinity protein (affibody).

IL6 antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a binding region or binding domain that antagonizes IL6 (e.g., preferentially inhibits IL6 trans signaling or inhibits both IL6 cis-and trans-signaling). In certain embodiments, the invention provides a multispecific fusion protein comprising a binding region or binding domain specific to the IL6/IL6R complex, said binding region or binding domain having one or more of the following properties: (1) an affinity for the IL6xR complex that is greater than or equal to the affinity for IL6 or IL6R α alone, or an affinity for the IL6R α or IL6/IL6R complex alone that is greater than the affinity for IL6 alone, (2) competes with membrane gp130 for binding to the sIL6/IL6R complex or increases binding of soluble gp130 to the sIL6/IL6R complex, (3) preferentially inhibits IL6 trans signaling relative to IL6 cis signaling, or (4) does not inhibit signaling of gp130 family cytokines other than IL 6. In certain preferred embodiments, the binding domain specific for the IL6/IL6R complex according to the invention has the following properties: (1) affinity for IL6R α or IL6/IL6R complex alone is greater than for IL6 alone, (2) increases binding of soluble gp130 to the sIL6xR complex, (3) preferentially inhibits IL6 trans signaling over IL6 cis signaling, and (4) does not inhibit signaling by gp130 family cytokines other than IL 6. For example, the binding region or binding domain specific for the IL6/IL6R complex may be a variable region or derivative thereof of an immunoglobulin such as an antibody, Fab, scFv, or the like. For the purposes of the present invention, it is understood that the binding region or binding domain specific for the IL6/IL6R complex is not gp130 as described herein.

As used herein, "IL 6xR complex" or "IL 6 xR" refers to a complex of IL6 and IL6 receptor, wherein the IL6 receptor (also referred to as, e.g., IL6R α, IL6RA, IL6R1, and CD126) is a membrane protein (referred to herein as mIL6R or mIL6R α) or a soluble form (referred to herein as sIL6R or sIL6R α). The term "IL 6R" includes mIL6R α and sIL6R α. In one embodiment, IL6xR comprises a complex of IL6 and mIL6R α. In certain embodiments, the IL6xR complex is held together by one or more covalent bonds. For example, the carboxy terminus of IL6R can be fused to the amino terminus of IL6 via a peptide linker, which is known in the art as super-IL 6 (see, e.g., Fischer et al (1997) nat. Biotechnol 15: 142). The super-IL 6 linker may be composed of a cross-linking compound, a sequence of 1-50 amino acids, or a combination thereof. super-IL 6 may further comprise an additional peptide tag or tags (e.g., aviflag his), or further comprise a dimerization domain, such as an immunoglobulin Fc domain or immunoglobulin constant region sub-region. In certain embodiments, IL6xR complexes are held together by non-covalent interactions, such as hydrogen bonds, electrostatic interactions, van der waals forces, salt bridges, hydrophobic interactions, and the like, or combinations thereof. For example, IL6 and IL6R may be bound non-covalently in nature (as in nature, or as synthetic or recombinant proteins), or each may be fused to a domain that promotes multimerization, such as an immunoglobulin Fc domain, to further improve the stability of the complex.

As used herein, "gp 130" refers to a signaling protein that binds to the IL6xR complex. The gp130 protein may be located in the membrane (mgp130), in soluble form (sgp130), or any other functional form thereof. Exemplary gp130 proteins have the sequence set forth in GenBank accession NP-002175.2 or any soluble or derivatized form thereof (see, e.g., Narazaki et al (1993) Blood 82: 1120 or Diamant et al (1997) FEBS Lett.412: 379). To illustrate and not to be bound by theory, the mgp130 protein can bind to the IL6/mILR or IL6/sILR complex, while sgp130 binds primarily to the IL6/sILR complex (see Scheller et al (2006) Scand. J. Immunol.63: 321). Thus, certain embodiments of the binding domains or fusion proteins thereof of the invention can inhibit IL6xR complex trans-signaling by binding to IL6xR with higher affinity than IL6 or IL6R α alone, preferably by competing with the sIL6xR complex for binding to mgp 130. A binding domain of the invention is said to "compete" with gp130 for binding to sIL6xR when (1) the binding domain or fusion protein thereof prevents gp130 from binding to sIL6xR with an affinity for binding to sIL6xR that is equal to or greater than the binding affinity of gp130 to sIL6xR, or (2) the binding domain or fusion protein thereof enhances or promotes binding of sgp130 to sIL6xR, thereby shortening the amount of time that the sIL6xR complex is available for binding mgp 130.

In one aspect, the IL6 antagonist binding domain of the invention has at least 2-fold to 1000-fold greater affinity for IL6 or the IL6xR complex than for IL6R α alone; or at least 2-fold to 1000-fold greater affinity for IL6R a or IL6xR complex than for IL6 alone. By binding to the IL6, IL6R, or IL6xR complex, the IL6 antagonist binding domain of the invention preferentially inhibits IL6 cis-and trans-signaling. In certain embodiments, the affinity of the binding domain for the IL6, IL6R, or sIL6xR complex is about the same as the affinity of gp130 for the IL6xR complex-about the same means that the affinity is equal or up to about 2-fold higher. In certain embodiments, the binding domain has at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, 1000-fold or more affinity for the IL6, IL6R, or IL6xR complex than gp130 has for the IL6xR complex. For example, if gp130 has an affinity for the IL6xR complex of about 2nM (see, e.g., Gaill et al (1999) Eur. cytokine Netw.10: 337), then the dissociation constant (Kd) for the binding domain of the IL6xR complex that has an affinity for gp130 that is at least 10 fold greater is about 0.2nM or less.

In other embodiments, the IL6 antagonist binding domain of the invention comprises a polypeptide sequence having the following properties: (a) binding affinity to sIL6xR complex is at least 2-fold, 10-fold, 25-fold, 50-fold, 75-fold to 100-fold, 100-fold to 1000-fold greater than the affinity for IL6 alone or IL6R α alone, (b) competes with membrane gp130 for binding to sIL6xR complex or increases binding of soluble gp130 to sIL6xR complex. In other embodiments, a polypeptide binding domain of the invention that binds to sIL6xR complex with an affinity that is at least 2-fold, 10-fold, 25-fold, 50-fold, 75-fold to 100-fold, 100-fold to 1000-fold greater than the affinity for binding to IL6 alone or IL6R α alone may also (i) inhibit IL6 trans signaling more significantly or preferably relative to IL6 cis signaling, (ii) not inhibit signaling by gp130 family members other than IL6, (iii) preferably inhibit IL6 trans signaling relative to IL6 cis signaling, not inhibit signaling by gp130 family cytokines other than IL6 at detectable levels, (iv) may have two or more of the above properties, or (v) may have all of the above properties.

In certain embodiments, the polypeptide IL6 antagonist binding domain of the invention binds to the sIL6xR complex with at least 2-fold to 1000-fold greater affinity than to IL6 alone or IL6R α alone, and inhibits IL6 trans signaling more significantly or preferably relative to IL6 cis signaling. The term "preferentially inhibits IL6 trans-signaling relative to IL6 cis-signaling" refers to altering trans-signaling to the extent that a decrease in sIL6xR activity is detectable without significantly altering IL6 cis-signaling (i.e., little, no, or no detectable inhibition). For example, biomarkers of sIL6xR activity (e.g., acute phase expression of Antichymotrypsin (ACT) in HepG2 cells) can be detected to detect trans-signaling inhibition. Representative experiments can be seen in Jostock et al (eur.j. biochem., 2001) -briefly, HepG2 cells were stimulated to overexpress ACT in the presence of sIL6xR (trans signaling) or IL6 (cis signaling), however, addition of spg130 inhibited sIL6 xR-induced ACT overexpression without significantly affecting IL 6-induced expression. Similarly, a polypeptide binding domain of the invention that preferentially inhibits trans-signaling of IL6 relative to cis-signaling of IL6 would inhibit sIL6 xR-induced ACT overexpression (i.e., inhibit trans-signaling) without significantly affecting IL 6-induced expression (i.e., no detectable decrease in cis-signaling). This and other assays known in the art can be used to determine preferential inhibition of IL6 trans-signaling relative to IL6 cis-signaling (see, e.g., Sporri et al (1999) int. Immunol.11: 1053; Mihara et al (1995) Br.J. Rheum.34: 321; Chen et al (2004) Immun.20: 59 for other biomarkers).

In other embodiments, signaling by a gp130 family cytokine other than IL6 is not substantially inhibited by a binding domain polypeptide of the invention or a multispecific fusion protein thereof. For example, the cis and trans signaling by the IL6xR complex through gp130 is inhibited, but signaling by one or more other gp130 family cytokines, such as by Leukemia Inhibitory Factor (LIF), ciliary neurotrophic factor (CNTF), neurogenin (NPN), cardiotrophin-like cytokine (CLC), oncostatin M (OSM), IL-11, IL-27, IL-31, cardiotrophin-1 (CT-1), or any combination thereof, is minimally or non-affected.

It will be appreciated by those skilled in the art that the in vivo half-life of the binding domains of the invention is preferably of the order of days or weeks, however, although the binding domain concentration may be lower, the target may already be sufficient because the production of IL6 and sIL6 is significantly increased under disease conditions (see, e.g., Lu et al (1993) Cytokine 5: 578). Thus, in certain embodiments, the invention binds k of the domain_OFFIs about 10^-5A second (e.g., about one day) or less. In certain embodiments, k is_OFFThe range may be about 10^-1Second, about 10^-2Second, about 10^-3Second, about 10^-4Second, about 10^-5Second, about 10^-6Second, about 10^-7Second, about 10^-8Second, about 10^-9Second, about 10^-10A/sec or less.

In an illustrative example, binding domains of the invention specific for the IL6 or IL6xR complex are identified in a library of Fab phage fragments by screening for binding to a synthetic IL6xR complex (see Hoet al (2005) Nature Biotechnol.23: 344). The synthetic IL6xR complex used in such screening comprises the structure N-IL6R α (fragment (frag)) -L1-IL6(frag) -L2-ID-C, wherein N is amino-terminal and C is carboxy-terminal, IL6R α (frag) is a fragment of full-length IL6R α, IL6(frag) is a fragment of IL6, L1 and L2 are linkers, and ID is an intervening or dimerizing domain, such as an immunoglobulin Fc domain.

More specifically, the structure of IL6xR (one of the forms of super IL6) from amino-terminus to carboxy-terminus used to identify a binding domain specific for the IL6xR complex is as follows: (a) a 212 amino acid central fragment missing the first 110 amino acids of the full-length protein from IL6R α and a carboxy-terminal part depending on the isoform used (see GenBank accession No. NP _000556.1, isoform 1 or NP _852004.1, isoform 2), fused to (2) G₃S linker, fused to 175 of (3) IL6A carboxy-terminal fragment of amino acids (i.e., the first 27 amino acids of the full-length protein are missing; GenBank accession No. NP _000591.1), further fused to (4) a peptide as set forth in SEQ ID NO: 589 the linker of the IgG2A hinge, finally fused to the dimerization domain consisting of the immunoglobulin G1(IgG1) Fc domain. In certain embodiments, one or more of the following amino acids in the dimerization domain consisting of the IgG1Fc domain are mutated (i.e., have different amino acids at that position): leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamic acid at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any of the above amino acids may be changed to alanine. In another embodiment, L234, L235, G237, E318, K320 and K322 (numbering according to EU) in the IgG1Fc domain are each mutated to alanine (i.e., L234A, L235A, G237A, E318A, K320A and K322A, respectively).

In one embodiment, the IL6xR complex used to identify the antagonist binding domain of IL6 of the present invention has the amino acid sequence as set forth in SEQ ID NO: 606, or a pharmaceutically acceptable salt thereof. In certain embodiments, there is provided a polypeptide comprising a binding domain specific for an IL6xR complex, wherein the IL6xR is sIL6xR and has the amino acid sequence set forth in SEQ ID NO: 606, or a pharmaceutically acceptable salt thereof. In other embodiments, a polypeptide comprising a binding domain specific for an IL6xR complex (1) has an affinity for IL6xR complex that is greater than or equal to the affinity for IL6 alone or IL6R α alone, or that has an affinity for IL6R α alone or IL6xR complex alone that is greater than the affinity for IL6 alone, (2) competes with membrane gp130 for binding to the sIL6xR complex or increases binding of soluble gp130 to the sIL6xR complex, (3) preferentially inhibits IL6 trans signaling over IL6 cis signaling, or (4) does not inhibit signaling of gp130 family cytokines other than IL6, (5) has any combination of properties (1) - (4), or (6) has all of properties (1) - (4). Other exemplary IL6xR complexes that can be used to identify binding domains of the invention or as reference complexes to determine any of the above-described binding properties can be found in, for example, U.S. patent publication nos. 2007/0172458; 2007/0031376, respectively; and U.S. patent 7,198,781; 5,919,763.

In some embodiments, an IL6 antagonist binding domain of the invention comprises V specific for IL6, IL6R, or IL6xR complex (as described herein), preferably human IL6, human IL6R, or human IL6xR complex_HAnd V_LA domain. In certain embodiments, V_HAnd V_LThe domain is a rodent (e.g., mouse, rat) domain, a humanized domain, or a human domain. Comprising such V specific for IL6, IL6R or IL6xR_HAnd V_LExamples of binding domains for the domains are set forth in SEQ ID NOs: 435, 496, and 373, 434. In other embodiments, a polypeptide binding domain specific for an IL6xR complex is provided that binds IL6xR with greater than or equal affinity for IL6 alone or IL6R α alone, and competes with membrane gp130 for binding to the sIL6xR complex or increases binding of soluble gp130 to the sIL6xR complex, wherein the binding domain comprises one or more light chain variable regions (V)_L) Or one or more heavy chain variable regions (V)_H) Or both as shown in SEQ ID NO: 373-434 and 435-496 sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical, wherein each CDR has at most three amino acid changes (i.e., many changes occur in the framework region).

In other embodiments, the binding domain of the invention comprises a sequence specific for IL6xR as set forth in SEQ ID NO: v listed in 435-496 and 373-434_HAnd V_LDomains, each of which is associated with such a V_HDomain, V_LThe amino acid sequence of a domain or both is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical, wherein each CDR contains at most 0-3 amino acid changes. For example, V of the present invention_HDomain, V_LAmino of a Domain or bothThe amino acid sequences can be linked to V from an exemplary crossover receptor molecule (SEQ ID NO: 608) containing the binding domain TRU (XT6) -1002, respectively_HStructural domain (e.g. amino acid 512-_LThe amino acid sequence of the domain (e.g., amino acids 649 and 758) or both is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical, wherein each CDR has at most 0-3 amino acid changes.

In any of these or other embodiments described herein, V_LAnd V_HThe domains may be positioned in any orientation, separated by a linker of up to about 10 amino acids as described herein or any other amino acid sequence capable of providing a spacer function compatible with the interaction of two sub-binding domains. In certain embodiments, connection V_HAnd V_LThe linker of the domain comprises SEQ ID NO: 497-604 and 791-796, such as linker 47(SEQ ID NO: 543) or linker 80(SEQ ID NO: 576).

In other embodiments, an IL6 antagonist binding domain of the invention may comprise one or more complementarity determining regions ("CDRs"), or multiple copies of one or more such CDRs, obtained, derived or designed from an anti-IL 6, anti-IL 6R or anti-IL 6xR complex scFv or Fab fragment, or from a heavy or light chain variable region thereof. Thus, a binding domain of the invention may comprise a single CDR3 from the variable region of anti-IL 6, anti-IL 6R, or anti-IL 6xR or may comprise multiple CDRs which may be the same or different. In certain embodiments, an IL6 antagonist binding domain of the invention comprises V_HAnd V_LA domain comprising a framework region and CDR1, CDR2 and CDR3 regions, wherein (a) said V_HThe domain comprises SEQ ID NO: the amino acid sequence of the heavy chain CDR3 set forth in any one of 435-496; or (b) said V_LThe domain comprises SEQ ID NO: the amino acid sequence of CDR3 of the light chain as set forth in any one of 373-434; or (c) the binding domain comprises (a)V_HAmino acid sequence and V of (b)_LAn amino acid sequence; or the binding domain comprises a V of (a)_HAmino acid sequence and V of (b)_LAmino acid sequence, and V_HAnd V_LPresent in the same reference sequence. In other embodiments, a binding domain of the invention comprises a V specific for an IL6xR complex_HAnd V_LA domain comprising a framework region and CDR1, CDR2 and CDR3 regions, wherein (a) said V_HThe domain comprises SEQ ID NO: the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 as set forth in any one of 435- & 496; or (b) said V_LThe domain comprises SEQ ID NO: the amino acid sequences of light chain CDR1, CDR2 and CDR3 as set forth in any one of 373-434; or (c) the binding domain comprises the V of (a)_HAmino acid sequence and V of (b)_LAn amino acid sequence; or the binding domain comprises a V of (a)_HAmino acid sequence and V of (b)_LAmino acid sequence, and V_HAnd V_LThe amino acid sequences are from the same reference sequence. SEQ ID NO: 1-187 and 787-792 and SEQ ID NO: 187-, 371-, and 793-, 798-provide exemplary light and heavy chain variable region CDRs for IL6, IL6R, or IL6xR, respectively.

SEQ ID NO: 373-434 and 799-804 provide the amino acid sequence of the light chain variable region of the IL6 antagonist, SEQ ID NO: 435-, 496-and 805-, 810 provide the IL6 antagonist heavy chain variable region.

In any of the embodiments described herein comprising a specific CDR for IL6, IL6R, or IL6xR, the binding domain may comprise (i) V_HA domain having a sequence identical to SEQ ID NO: v shown in any one of 435, 496 and 805, 810_HAn amino acid sequence in which the amino acid sequence of a domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; or (ii) V_LA domain having a sequence identical to SEQ ID NO: v shown in any one of 373, 434 and 799, 804_LAn amino acid sequence in which the amino acid sequence of a domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; or (iii) contains V of (i) together_H(iii) domains and V of (ii)_LA domain; or both V from (i) and V from the same reference sequence_H(iii) domains and V of (ii)_LA domain.

In certain embodiments, a binding domain of the invention can be an immunoglobulin-like domain, such as an immunoglobulin scaffold. The immunoglobulin scaffold of the present invention includes scFv, Fab, domain antibodies or heavy chain-only antibodies. In other embodiments, anti-IL 6 or anti-IL 6xR antibodies (e.g., non-human such as mouse or rat, chimeric, humanized, human) or Fab or scFv fragments are provided that have an amino acid sequence identical to the amino acid sequence represented by SEQ ID NOs: v listed in any one of items 435, 496, 805, 801, 373, 434, 799, 804_HAnd V_LA domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the domain, and may also have one or more of the following properties: (1) the affinity for IL6xR complex is higher than or equal to the affinity for IL6 alone or IL6R α alone, or the affinity for IL6R α alone or IL6xR complex is greater than the affinity for IL6 alone; (2) competes with membrane gp130 for binding to the sIL6xR complex or enhances the binding of soluble gp130 to the sIL6xR complex; (3) preferentially inhibits IL6 trans signaling relative to IL6 cis signaling; or (4) does not inhibit signaling by a gp130 family cytokine other than IL 6. Any of the methods described herein can use such antibodies, fabs, or scfvs. In certain embodiments, the invention provides polypeptides comprising an IL6 antagonistic binding domain (i.e., that inhibit cis and trans signaling of IL 6). In other embodiments, the IL6 antagonists of the invention do not inhibit signaling by a gp130 family cytokine other than IL 6. Exemplary IL6 antagonists include IL6 or IL6xR specific binding domains, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.).

Alternatively, the binding domain of the invention may be part of a scaffold other than an immunoglobulin. Other scaffolds contemplated include a domain molecules, fibronectin III domains, anti-lipocalin, ankyrin-engineered binding molecules, adeno-associated proteins, kunitz domains or protein AZ domain affinity proteins.

RANKL antagonists

As noted above, in certain embodiments, the present invention provides polypeptides comprising a RANKL antagonist (i.e., capable of inhibiting RANK signaling) binding region or domain. Exemplary RANKL antagonists include RANKL or RANK-specific binding domains, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scfvs, etc.), or OPG extracellular domains or fragments thereof.

Osteoprotegerin (OPG, also known as OCIF) is a member of the Tumor Necrosis Factor (TNF) receptor superfamily. OPG is a secreted soluble protein that is initially expressed as a precursor protein of a signal peptide having 21 amino acid residues. The amino-terminal half of the protein contains four cysteine-rich repeats characteristic of members of the TNF receptor superfamily. The carboxy-terminal portion of the protein contains two death domain homology regions. OPG is expressed in osteoblasts and certain tissues, including heart, kidney, liver, spleen and bone marrow (see, e.g., Boyce and Xing, Arthritis res. ther. (2007)9 (supplement 1): S1). The ligands for OPG are RANKL and TRAIL (TNF-related apoptosis-inducing ligand).

The OPG/RANK/RANKL system is involved in osteoclast formation. Osteoclasts are bone-resorbing cells that are critical for bone remodeling and bone health. RANKL binds to RANK, causing downstream signaling. Activated RANK binds to TRAF (tumor necrosis factor receptor-related factor), which in turn leads to NF- κ B activation. RANK-mediated signaling activates seven pathways including inhibition of NF- κ B kinase/NF- κ B, c-Jun amino terminal kinase/activator protein-1, c-myc, calcineurin/nuclear factor of activated T cells, src, MKK6/p38/MITF and extracellular signal-associated kinases. Grb-2-associated binding protein 2 is also capable of binding RANK and mediating signaling. OPG functions by binding RANKL and preventing it from binding RANK. Thus, OPG is a negative regulator of bone resorption.

In some embodiments, the binding domain of the invention comprises V specific for RANKL or RANK_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. Containing such V specific for RANKL_HAnd V_LExamples of binding domains for domains include those described in U.S. patent 6,740,522.

In certain embodiments, the RANKL antagonist comprises an OPG protein (also known as TR1 or OCIF) having the amino acid sequence set forth in GenBank accession No. NP _002537.3 (SEQ ID NO: 737), or any fragment thereof that still functions as a RANKL antagonist. In other embodiments, the RANKL antagonist comprises SEQ ID NO: 737 amino acids 22-401 (i.e., the natural leader sequence does not contain 21 amino acids). In other embodiments, a polypeptide binding domain specific for RANKL is provided, wherein the binding domain comprises a sequence that is identical to SEQ ID NO: 737 or the amino acid sequence of SEQ ID NO: 737, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to amino acids 22-401, which polypeptide binding domain binds to RANKL and inhibits its activity.

IL7 antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a binding region or binding domain that is antagonistic to IL7 (i.e., inhibits IL7R α signaling). Exemplary IL7 antagonists include binding domains specific for IL7, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.), or the IL7R α extracellular domain or fragments thereof.

Interleukin-7 (IL7) is a cytokine produced by fibroblasts in the T cell region of lymphoid organs and binds to interleukin-7 receptor (IL7R) (palm et al (2008) cell. mol. Immun.5: 79). IL7 stimulates precursor B cells, thymocytes, T cell progenitors and mature CD4⁺And CD8⁺Proliferation of T cells. In general, IL7 has survival and proliferation promoting activity and plays an immunomodulatory role in dendritic cells. IL7 system activated signaling cascadeJak-Stat and PI3K-Akt pathways are to be included. Binding of IL7 to IL7R promotes the trans-phosphorylation of receptor-bound Jak kinases. Activated Jak kinases phosphorylate tyrosine residues on receptors, and the resulting phosphorylated tyrosine serves as a docking site for SH2 domain proteins, including Stat family transcription factors. Jak kinases then activate the recruited Stat proteins by phosphorylation.

IL7R consists of two separate polypeptides: namely the IL7R alpha chain (IL7R alpha) and the common gamma chain (IL7R gamma). These two proteins are members of the hematopoietin superfamily (Ouellette et al (2003) prot. exp. Pur.30: 156). IL7R alpha is expressed in B cells, thymocytes, T cell progenitors, mature CD4⁺And CD8⁺T cells, dendritic cells and monocytes. It is expressed as a 459 amino acid precursor protein containing a 20 amino acid signal sequence, a 219 amino acid extracellular ligand binding domain, a 25 amino acid transmembrane domain and a 195 amino acid cytoplasmic domain.

In some embodiments, a binding domain of the invention comprises a V specific for IL7_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. Comprising such a V specific for IL7_HAnd V_LExamples of binding domains for domains include, for example, those described in U.S. patent 5,714,585. In certain embodiments, the IL7 antagonist can be the extracellular domain ("ectodomain") of IL7R α. The extracellular domain of IL7R α as used herein refers to the extracellular portion of IL7R α, soluble IL7R α, IL7R α fibronectin type II domain, or any combination thereof. In certain embodiments, the IL7 antagonist comprises the amino-terminal portion of IL7R α, the first 240 amino acids of IL7R α as set forth in GenBank accession NP-002176.2 (SEQ ID NO: 738), or any fragment thereof that still functions as an IL7 antagonist. In other embodiments, the IL7 antagonist comprises SEQ ID NO: 738 has amino acids 21-240 or 120-230 (i.e., contains no native leader sequence and fibronectin type II domain, respectively). In other embodiments, a polypeptide binding domain specific for IL7 is provided, wherein the binding domain comprises a sequence that differs from SEQ ID NO: 738 or SEQ ID NO: 738 and amino acids 21-240 or 120-130 are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 100% identical to the sequence to which said polypeptide-binding domain binds IL7 and inhibits its activity.

IL17A/F antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a binding region or domain that is antagonistic to IL17A/F (i.e., inhibits IL17A/F, IL17RC or IL17RA/C signaling). Exemplary IL17A/F antagonists include binding domains specific for IL17A, IL17F or IL17A/F, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.), or the extracellular domain of IL17A, IL17F or IL17A/F or fragments thereof.

A unique subset of T helper cells, termed Th17, produces cytokines of the interleukin 17 family. Six cytokines of IL17 (IL17A-IL17F) and five receptors (IL17RA-IL17RE) have been identified (Kolls and Linden, 2004, Immunity, 21: 467). IL17A and IL17F are about 55% homologous and have similar biological functions, but IL17A is believed to be at least 10-fold more active than IL 17F. IL17A and IL17F are able to form homodimers, and recent studies indicate that IL17A and IL17F also form heterodimers with intermediate signaling capabilities (Wright et al 2007) j.biol.chem.282: 13447, respectively; chang et al (2007) Cell Res.17: 435). The IL17A/IL17F heterodimer may be the major form of this Cytokine in vivo (Shen and Gaffen (2008) Cytokine 41: 91).

Interleukin 17A (IL 17A; originally referred to as IL 17; also known as CTLA8) is a potent cytokine. Binding of IL17A to its receptor IL17RA stimulates macrophages to secrete a variety of pro-inflammatory molecules including tumor necrosis factor-alpha (TNF α), interleukin 6(IL6), interleukin 1 β (IL1 β) and prostaglandin E2(PGE2) (Jovanovic et al (1998) J. Immunol.160: 3513). IL6 is one of the earliest defined targets of the IL17A gene and was used as a standard bioassay for detecting IL17A activity. IL17A is able to synergistically activate IL6 with other cytokines, including IL1 β, IFN γ, TNF α, and IL22, but the underlying mechanism of this synergy is not fully understood (see, e.g., Teunissen et al (1998) J. invest. Dermatol. 111: 645).

Interleukin 17F (IL 17F; also known as ML-1) is a 17kD secreted protein that, like IL17A, is capable of forming disulfide-linked homodimers. IL17A and IL17F have similar biological functions, but IL17A is believed to have higher activity than IL 17F. Recent studies have indicated that IL17A and IL17F also form heterodimers with intermediate signaling capabilities (Wright et al (2007) J.biol. chem.282: 13447; Chang et al (2007) Cell Res.17: 435). It was suggested that the IL17A/IL17F heterodimer might be the major form of this Cytokine in vivo (Shen and Gaffen (2008) Cytokine 41: 91). Although, similar to IL17A, IL17F is expressed predominantly by activated T cells, IL17F is also expressed by activated monocytes, activated basophils and mast cells (Kawaguchi et al (2002) J.Immunol.167: 4430).

The receptor IL17RA is a ubiquitous type I membrane glycoprotein that has been shown to bind IL17A with an affinity of approximately 0.5nM (Yao et al (1995) Immunity 3: 811), but IL17RC also binds IL17A with high affinity, even though IL17RC is the cognate receptor for human IL17F (Keustner et al (2007) J.Immunol.179: 5462). However, defects in IL17RA and neutralization of IL17RA antibody have been observed to abolish the function of IL17A and IL17F, suggesting that IL17RC cannot transmit IL17A or IL17F signals alone in the absence of IL17RA (Toy et al (2006) J.Immunol.177: 36; McAllist et al (2005) J.Immunol.175: 404). Moreover, forced expression of IL17RC in IL17RA deficient mice failed to restore the function of IL17A or IL17F (Toy et al, 2006).

Structurally, the extracellular domain of IL17RA contains two fibronectin III-like (FN) domains (FN1 (residues 69-183) and FN2 (residue 205-282)) joined by an elastic linker. FN domains are commonly found in type I cytokine receptors, where they mediate protein-protein interactions and ligand binding. Kramer et al identified a pre-ligand assembly domain (PLAD) completely within FN2 and determined that the FN2 linker encodes the IL17A binding site (Kramer et al (2007) J.Immunol.179: 6379). In addition, the SEFIR domains are located at amino acids 378-536 of the IL17RA sequence (GenBank accession NP-055154.3; SEQ ID NO: 739) and at amino acids 473-623 of the IL17RC sequence (GenBank accession NP-598920.2; SEQ ID NO: 740).

In some embodiments, a binding domain of the invention comprises a V specific for IL17A, IL17F, or IL17A/F_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. Comprising such V specific for IL17A, IL17F or IL17A/F_HAnd V_LExamples of binding domains for a domain include, for example, PCT patent application publication Nos. WO2006/088833, WO2007/117749, WO2008/047134, WO 2008/054603; and those described in U.S. patent application publication No. 2007/0212362. IL17R-Fc fusion protein and its use in reducing disease severity in a murine model of rheumatoid arthritis are described in U.S. Pat. No. 6,973,919.

In certain embodiments, the IL17A/F antagonist can be the extracellular domain ("extracellular domain") of IL17RA, IL17RC, or IL17 RA/C. As used herein, an IL17RA, IL17RC, or IL17RA/C extracellular domain refers to an extracellular portion of IL17RA, IL17RC, or IL17RA/C, soluble IL17RA, IL17RC, or IL17RA/C, one or more fibronectin-like domains, one or more pre-ligand assembly domains (PLADs), one or more SEFIR domains, or any combination thereof. In certain embodiments, the IL17A/F antagonist comprises an amino terminal portion of IL17RA, such as the first 307 amino acids of IL17RA as set forth in GenBank accession NP-055154.3 (SEQ ID NO: 739), or any fragment thereof that still functions as an IL17A/F antagonist. In other embodiments, the IL17A/F antagonist comprises SEQ ID NO: 739 amino acids 32-307 (i.e., without a leader sequence) or SEQ ID NO: 816. in other embodiments, the IL17A/F antagonist comprises an amino terminal portion of IL17RC, the first 539 amino acids of IL17RC as set forth in GenBank accession NP-703191.1 (SEQ ID NO: 740), SEQ ID NO: 817, or any fragment thereof which still functions as an antagonist of IL 17A/F. In other embodiments, the IL17A/F antagonist comprises SEQ ID NO: 740, amino acids 21-539 (i.e., without a leader sequence). In other embodiments, a polypeptide binding domain specific for IL17A/F is provided, wherein the binding domain comprises a sequence identical to SEQ ID NO: 739, the amino acid sequence of SEQ ID NO: 739, amino acids 32-307 of SEQ ID NO: 816, SEQ ID NO: 740, the amino acid sequence of SEQ ID NO: 740, amino acids 21-539 or SEQ ID NO: 817, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to the amino acid sequence of IL17, 17A/F and inhibiting its activity.

TWEAK antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a TWEAK antagonistic (i.e., capable of inhibiting TWEAKR signaling) binding region or binding domain. Exemplary TWEAK antagonists include binding domains specific for TWEAK, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scfvs, etc.), or TWEAKR extracellular domains or fragments thereof.

TWEAK is a cytokine belonging to the family of Tumor Necrosis Factor (TNF) ligands that regulates a variety of cellular responses, including pro-inflammatory activity, angiogenesis, and cell proliferation. TWEAK is a type II transmembrane protein that, upon cleavage, produces soluble cytokines with biological activity. The location of the various domains within the TWEAK protein is described, for example, in published U.S. patent application 2007/0280940. TWEAK overlaps with TNF in its signaling function, but it is more widely distributed in tissue. TWEAK can induce apoptosis in a cell type specific manner through a variety of cell death pathways, and has also been found to promote endothelial cell proliferation and migration and thus can be used as a modulator of angiogenesis.

In some embodiments, a binding domain of the invention comprises a V specific for TWEAK_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. V containing such TWEAK specificities_HAnd V_LExamples of binding domains for a domain include, for example, those described in U.S. patent 7,169,387 and the amino acid sequence set forth in SEQ ID NO: 3-7, the sequences of which are incorporated herein by reference. Monoclonal antibodies that block TWEAK have been shown to be effective in a collagen-induced mouse arthritis (CIA) model (Kamata et al (2006) j.immunol.177: 6433; perer et al (2006) j.immunol.177: 2610).

In certain embodiments, the TWEAK antagonist may be the extracellular domain ("ectodomain") of TWEAKR (also referred to as FN 14). As used herein, the TWEAKR extracellular domain refers to the extracellular portion of TWEAKR, soluble TWEAKR, or any combination thereof. In certain embodiments, the TWEAK antagonist comprises the amino-terminal portion of a TWEAKR, such as the first 70 amino acids of TWEAKR as listed in GenBank accession NP-057723.1 (SEQ ID NO: 741), or any fragment thereof that still functions as a TWEAK antagonist. In other embodiments, the TWEAK antagonist comprises SEQ ID NO: 741 amino acids 28-70 (i.e., without the leader sequence). In other embodiments, the TWEAK antagonist comprises a polypeptide that differs from SEQ ID NO: 741 or the amino acid sequence of SEQ ID NO: a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to amino acids 28-70 of 741, wherein the antagonist binds to TWEAK and inhibits its activity.

The ability of the binding or fusion proteins described herein to reduce binding of TWEAK to TWEAKR can be determined using assays known to those skilled in the art, including those described in U.S. patent application publication nos. 2007/0280940 and 2008/0279853.

CSF2 antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a binding region or domain that is antagonistic to CSF2 (i.e., inhibits CSF2R α signaling). Exemplary CSF2 antagonists include binding domains specific for CSF2, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.), or the CSF2R α extracellular domain or fragments thereof.

CSF2 is a cytokine used as a leukocyte growth factor. It is produced by a variety of cell types, including lymphocytes, monocytes, endothelial cells, fibroblasts, and some malignant cells. In addition to stimulating the growth and differentiation of hematopoietic precursor cells, CSF2 has various effects on cells of the immune system expressing the CSF2 receptor. The most important of these functions is the activation of monocytes, macrophages and granulocytes in several immune and inflammatory processes. Mature CSF2 is a 127 amino acid monomeric protein with two glycosylation sites, which is found in its active form as an extracellular homodimer.

The action of CSF2 is mediated by its receptor CSF2R (also known as GMR, GMCSFR or differentiated population 116(CD 116)). This receptor is normally expressed on the surface of bone marrow cells and endothelial cells, but not on lymphocytes. The natural receptor is composed of at least two subunits: i.e., a heterodimer consisting of an alpha chain (CSF2R alpha) and a beta chain (β c). The alpha subunit confers ligand specificity and binds CSF2 with nanomolar affinity (Gearing et al (1989) EMBO J.12: 3667; Gasson et al (1986) Proc. nat' l. Acad. Sci. USA 83: 669). The beta subunit is also present on the interleukin-3 receptor and interleukin-5 receptor complex and is involved in signal transduction. Binding of the beta and alpha subunits to CSF2 results in the formation of a complex with picomolar binding affinity (Hayashida et al (1990) Proc. nat' l.Acad. Sci. USA 87: 9655) and in receptor activation.

The receptor-binding domain on CSF2 has been mapped (Brown et al (1994) Eur. J. biochem.225: 873; Shanafelt et al (1991) J. biol. chem.266: 13804; Shanafelt et al (1991) EMBO J.10: 4105; Lopez et al (1986) J. Clin. invest.78: 1220). Furthermore, McClure et al demonstrate that one CSF2 molecule binds to one alpha subunit and two beta subunits to form a ternary complex (McClure et al (2003) Blood 101: 1308-. Formation of the CSF2 receptor complex results in activation of the complex signaling cascade of the JAWSTAT family involving the molecules, Shc, Ras, Raf, MAP kinase, NF κ B and phosphatidylinositol-3-kinase, and finally results in transcription of c-myc, c-fos and c-jun.

In some embodiments, a binding domain of the invention comprises a V specific for CSF2_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. Containing such a V specific for CSF2_HAnd V_LExamples of binding domains for domains include, for example, those described in U.S. patent 7,381,801. Other V specific for CSF2_HAnd V_LThe domains include SEQ ID NO: 11-20, 49-52, and 31-40, 58-61, the sequences of which are incorporated herein by reference. Neutralizing anti-CSF 2 antibodies have been shown to be effective in collagen-induced murine Arthritis models (Cook et al (2001) Arthritis Res.3: 293-298) and murine asthma models (Yamashita et al (2002) Cell immunol.219: 92).

In certain embodiments, the CSF2 antagonist may be the extracellular domain ("ectodomain") of CSF2R a. The extracellular domain of CSF2R a as used herein refers to the extracellular portion of CSF2R a, soluble CSF2R a, or any combination thereof. In certain embodiments, the CSF2 antagonist comprises an amino terminal portion of CSF2R α, such as the first 323 amino acids of CSF2R α as set forth in GenBank accession NP-006131.2 (SEQ ID NO: 742), or any fragment thereof that still functions as a CSF2 antagonist. In other embodiments, the CSF2 antagonist comprises SEQ ID NO: 742 (i.e., without the native leader sequence). In other embodiments, the CSF2 antagonist comprises a sequence identical to SEQ ID NO: 742 or the amino acid sequence of SEQ ID NO: 742, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100%, wherein the antagonist binds to CSF2 and inhibits its activity.

The ability of the binding and/or fusion proteins described herein to reduce the binding of CSF2 to its receptor can be determined using assays known to those skilled in the art, including those described in PCT patent application publication No. WO2006/122797 and U.S. patent application publication No. 2009/0053213.

IGF1/2 antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising an IGF1 or IGF2 antagonistic (i.e., capable of inhibiting IGF1 or IGF2 signaling) binding region or domain. Exemplary IGF1 or IGF2 antagonists include binding domains specific for IGF1 or IGF2, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.), or IGF1R or IGFBP extracellular domains or subdomains thereof.

Insulin-like growth factors (IGFs) comprise a class of peptides that play an important role in mammalian growth and development. Insulin-like growth factor 1(IGF1) is a secreted protein with the following characteristics: disulfide bonds (amino acids 54-96, 66-109, 95-100); d peptide domain (amino acids 111-118); carboxy-terminal propeptide domain (E peptide) (amino acids 119-153); an insulin chain A-like domain (amino acids 90-110); an insulin chain B-like domain (amino acids 49-77); an insulin-linked C-peptide like domain (amino acids 78-89); a propeptide domain (amino acids 22-48); and a signal sequence domain (amino acids 1-21).

IGF1 is synthesized in a variety of tissues, including liver, skeletal muscle, bone, and cartilage. Changes in blood concentration of IGF1 reflect changes in its synthesis and secretion in the liver, with liver synthesized and secreted IGF1 accounting for 80% of total serum IGF1 in experimental animals. The remaining IGF1 is synthesized peripherally, typically by connective tissue cell types, such as stromal cells present in most tissues. Peripherally synthesized IGF1 may act to regulate cell growth through autocrine and paracrine mechanisms. In these tissues, newly synthesized and secreted IGF1 may bind to receptors present on connective tissue cells themselves and stimulate growth (autocrine), or may bind to receptors on adjacent cell types (often epithelial cell types) that are not actually synthesized IGF1 but are stimulated to grow by locally secreted IGF1 (Clemmons, 2007, Nat Rev Drug Discov.6 (10): 821-33). IGF1 synthesis is controlled by several factors, including human pituitary growth hormone (GH, also known as growth hormone). IGF2 concentration was higher during fetal growth, but was less dependent on GH than IGF1 during adult life.

IGF1 can increase cell growth and/or survival in a variety of tissues, including skeletal muscle system, liver, kidney, intestine, nervous system tissues, heart, and lung. IGF1 also plays an important role in promoting cell growth, and therefore attempts were made to inhibit IGF1 as a potential adjunct to the treatment of atherosclerosis. Inhibition of IGF1 action is proposed as a specific treatment to potentiate the effects of other forms of anti-cancer therapy or to directly inhibit tumor cell growth.

Like IGF1, IGF2 acts through IGF 1R. IGF2 is an important autocrine growth factor in tumors due to mitogenic and anti-apoptotic functions (Kaneda et al, 2005, Cancer Res 65 (24): 11236-11240). Elevated expression of IGF2 is often found in a variety of malignancies, including colorectal, liver, esophageal, adrenal cortical, and sarcoma. Paracrine signaling by IGF2 also plays a role in tumors, including breast cancer, because IGF2 is found to be expressed in high amounts in stromal fibroblasts surrounding malignant breast epithelial cells.

Insulin-like growth factor 1 receptor (IGF1R) is a tetramer of two alpha and two beta chains linked by disulfide bonds. Cleavage of the precursor produces the alpha and beta subunits. IGF1R is associated with the protein kinase superfamily, the tyrosine protein kinase family, and the insulin receptor subfamily. It contains three fibronectin type III domains and a protein kinase domain (Lawrence et al, 2007, Current Opinion in Structural Biology 17: 699). The alpha chain helps form the ligand-binding domain, while the beta chain carries the kinase domain. It is a single transmembrane type I membrane protein, expressed in a variety of tissues.

The kinase domain has tyrosine-protein kinase activity, which is required for activation of IGF 1-or IGF 2-stimulated downstream signaling cascades. Autophosphorylation activates kinase activity. IGF1R interacts with PIK3R1, as well as the PTB/PID domains of IRS1 and SHC1 in vitro, when autophosphorylation occurs at a tyrosine residue in the cytoplasmic domain of the β subunit. IGF1R plays a crucial role in the transformation event. It is highly overexpressed in most malignant tissues and acts as an anti-apoptotic agent by increasing cell survival. Cells that do not contain this receptor, except for v-Src, cannot be transformed by most oncogenes.

The insulin-like growth factor-binding protein (IGFBP) family includes six soluble proteins of about 250 residues (IGFBP1-6) that bind IGF with nanomolar affinity. Due to their sequence homology, it is speculated that these IGFBPs share a common overall fold, and they are predicted to have closely related IGF binding determinants. Each IGFBP can be divided into three distinct domains of approximately the same length: i.e., highly conserved cysteine-rich N and C domains, and a central linker domain unique to each IGFBP. The N and C domains are involved in binding IGF, but the specific role of each of these domains in IGF binding has not been clearly established. The C-terminal domain may be responsible for the preference of IGFBPs over one IGF for another; the C-terminal domain is also involved in modulating IGF-binding affinity by interacting with extracellular matrix components, most likely in an effort to mediate IGF1 independent effects. The central linker domain is the least conserved region and has never been referenced as part of the IGF-binding site of any IGFBP. This domain is the site of post-translational modification, specific proteolysis, and binding of the acid-labile subunit to the extracellular matrix of known IGFBPs. Proteolytic cleavage occurring in this domain is believed to produce lower affinity N-and C-terminal fragments that do not compete with the IGF receptor for IGF, and thus, is believed to be the primary mechanism by which IGFBPs release IGF. However, recent studies have shown that the resulting N-and C-terminal fragments still inhibit IGF activity and have functional properties different from those of the intact protein (Sitar et al (2006) Proc. nat' l.Acad.Sci.USA.103 (35): 13028).

IGF binding proteins are secreted proteins that extend the half-life of IGF and have been shown to inhibit or stimulate the growth promoting effects of IGF on cell cultures. They alter the interaction of IGFs with their cell surface receptors and also promote cell migration. They bind equally well to IGF1 and IGF 2. The C-terminal domains of all IGFBPs show sequence homology to the thyroglobulin type 1 domain and share secondary structure common elements: alpha-helices and beta-sheets of 3-to 4-beta-strands. The core of this molecule is linked by a common three disulfide bond pair and has conserved Tyr/Phe amino acids and QC, CWCV motifs. These essential features are retained in CBP1, CBP4 and CBP-6, and the structure of the C domain has been resolved, but with significant differences in detail. For example, CBP4 has an alpha 2 helix, while the corresponding residues in CBP1 form short beta-chains that can be observed in other structures of the thyroglobulin type 1 domain superfamily. This specific region of CBP has high sequence diversity and is involved in IGF complex formation and thus can act as an affinity modulator.

Inhibition of IGF/IGF-receptor binding interferes with cell growth and represents a strategy for developing IGFBPs and variants as natural IGF antagonists for use in many common diseases caused by dysregulation of the IGF system, including diabetes, atherosclerosis, and cancer.

In some embodiments, the binding domains of the invention comprise a V specific for IGF1 or IGF2_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. The binding domain of the invention may also, or additionally, comprise the extracellular domain of IGF1R of Genbank accession NP-000866.1 (SEQ ID NO: 746) or a subdomain thereof or the amino acid sequence of SEQ ID NO: 818, or the IGF1R extracellular domain of Genbank accession NP-000587.1 (IGFBP 1; SEQ ID NO: 747), SEQ ID NO: 804, amino acid 490-723, NP-000588.2 (IGFBP 2; SEQ ID NO: 748), NP-001013416.1 (IGFBP3 isoform a; SEQ ID NO: 749), NP-000589.2 (IGFBP3 isoform b; SEQ ID NO: 750), NP-001543.2 (IGFBP 4; SEQ ID NO: 751), NP-000590.1 (IGFBP 5; SEQ ID NO: 752) or NP-002169.1 (IGFBP 6; SEQ ID NO: 753), or a subdomain thereof. In other embodiments, the IGF1 or IGF2 antagonist comprises a peptide that is identical to SEQ ID NO: 746-753 or 818, a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical in amino acid sequence, wherein said antagonist inhibitsActivity of at least one of IGF1 and IGF 2.

BLyS/APRIL antagonists

As noted above, in certain embodiments, the invention provides polypeptides comprising a BLyS/APRIL antagonist (i.e., capable of inhibiting TACI signaling) binding region or domain. Exemplary BLyS/APRIL antagonists include binding domains specific for BLyS/APRIL, such as immunoglobulin variable binding domains or derivatives thereof (e.g., antibodies, fabs, scFv, etc.), or TACI ectodomains or fragments thereof.

BLyS (also known as BAFF, TALL-1, THANK, TNFSF13B or zTNF4) and proliferation-inducing ligands (APRIL or TNFSF13) are cytokines belonging to the Tumor Necrosis Factor (TNF) ligand superfamily. BLyS and APRIL stimulate B-cell maturation, proliferation and survival (Gross et al (2000) Nature 404: 995; Gross et al (2001) Immunity 15: 289), and may be involved in the persistence of autoimmune diseases involving B-cells.

BLyS acts on B cells by binding to three TNF receptor superfamily members-TACI (also known as TNFRSF13B or CD267), BCMA, and BR3 (also known as BAFF-R). BCMA binds BLyS with weaker affinity, while APRIL binds only TACI and BCMA (see, e.g., Bossen and Schneider (2006) sensines in Immunol.18: 263). TACI appears to upregulate T-cell independent immune responses and downregulate B-cell activation and expansion (Yan (2001) nat. Immunol.2: 638; MacKay and Schneider (2008) Cytokine Growth Factor Rev.9: 263).

In some embodiments, a binding domain of the invention comprises a V specific for BLyS/APRIL_HAnd V_LA domain. In certain embodiments, the V is_HAnd V_LThe domain is a human domain. Containing such V specific for BLyS/APRIL_HAnd V_LExamples of binding domains for a domain include, for example, those described in U.S. patent application publication No. 2003/0223996 or U.S. patent No. 7,189,820. Clinically, TACI-immunoglobulin fusion proteins (atacicept) have been used to treat patients with rheumatoid Arthritis (Tak et al (2001) Arthritis Rheum.58: 61) or systemic ArthritisPatients with lupus erythematosus (Dall' Era et al (2007) Arthritis Rheum.56: 4142).

In certain embodiments, the BLyS/APRIL antagonist may be the extracellular domain of TACI ("extracellular domain"). As used herein, TACI extracellular domain refers to the extracellular portion of TACI, soluble TACI, a fragment containing one or more cysteine-rich domains (CRD), or any combination thereof. In certain embodiments, the BLyS/APRIL antagonist comprises the amino-terminal portion of TACI, such as the first 166 amino acids of TACI (SEQ ID NO: 743), as set forth in GenBank accession NP-036584.1, or any fragment thereof that remains useful as a BLyS/APRIL antagonist. In other embodiments, the BLyS/APRIL antagonist comprises SEQ ID NO: 743 (i.e., does not contain the natural leader sequence). In other embodiments, the BLyS/APRIL antagonist comprises an amino acid sequence that is identical to SEQ ID NO: 743 or the amino acid sequence of SEQ ID NO: 743 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100% identical to amino acids 21-166, wherein the antagonist binds to CSF2 and inhibits its activity.

The ability of the binding and/or fusion proteins described herein to reduce the binding of BLyS/APRIL to its receptor can be determined using assays known to those skilled in the art, including those described in U.S. patent application publication nos. 2003/0223996, 2005/0043516 and U.S. patent No. 7,189,820.

IL10 agonists

As noted above, in certain embodiments, the invention provides polypeptides comprising binding regions or domains that are agonistic (i.e., increase IL10 signaling) for IL 10. In some embodiments, the IL10 agonist binding domain is IL10 or IL10Fc, or a functional subdomain thereof. In other embodiments, the IL10 agonist binding domain is a single chain binding protein, e.g., an scFv, that specifically binds IL10R1 or IL10R 2.

IL10(Genbank accession NP-000563.1; SEQ ID NO: 754) is a member of the cytokine superfamily that shares alpha-helical structures. Although there is no empirical evidence, it is suggested that all family members have six alpha-helices (Fickenscher, H., et al, (2002) Trends Immunol.23: 89). IL10 contains four cysteines, only one of which is conserved among family members. Since IL10 shows a V-shaped fold that contributes to its dimerization, it appears that disulfide bonds are not important for this structure. The amino acid identity of members of the IL10 family varies from 20% (IL-19) to 28% (IL-20) (Dumouter et al (2002) Eur. cytokine Net. 13: 5).

IL10 was originally thought to be a Th2 cytokine that inhibits IFN- α and GM-CSF cytokine production by Th1 cells in mice (Moore et al, 2001, Annu. Rev. Immunol.19: 683; Fiorentino et al (1989) J. exp. Med.170: 2081). Human IL10 is 178 amino acids long and comprises an 18 amino acid signal sequence and a 160 amino acid mature segment. Its molecular weight is about 18kDa (monomer). Human IL10 contains no potential N-linked glycosylation sites and is not glycosylated (Dumouter et al, (2002) Eur. cytokine Net.13: 5; Vieira et al (1991) Proc. Nat' l. Acad. Sci. USA 88: 1172). It contains four cysteine residues, forming two intrachain disulfide bonds. Helices A-D of one monomer interact non-covalently with helices E and F of a second monomer to form a non-covalent V-shaped homodimer. Functional regions on the IL10 molecule have been mapped. At the N-terminus, pre-helix A residues #1-9 are involved in mast cell proliferation, while at the C-terminus, helix F residue #152-160 mediates leukocyte secretion and chemotaxis.

Cells known to express IL10 include CD8+ T cells, microglia, CD14+ (but not CD16+) monocytes, Th2 CD4+ cells (mouse), keratinocytes, hepatic stellate cells, Th1 and Th2 CD4+ T cells (human), melanoma cells, activated macrophages, NK cells, dendritic cells, B cells (CD5+ and CD19+) and eosinophils. It is now believed that the initial observation that inhibition of IFN- γ production by T cells by IL10 is an indirect helper-cell mediated effect. However, other effects on T cells include: IL 10-induced chemotaxis of CD8+ T cells, inhibition of chemotaxis of CD4+ T cells towards IL-8, inhibition of activated IL-2 production, inhibition of T cell apoptosis by Bcl-2 upregulation, and blocking of T cell proliferation following low antigen exposure with co-stimulation of CD28 (Akdis et al (2001) Immunology 103: 131).

IL10 has several related, but unique functions on B cells. IL10 together with TGF β and CD40L induces IgA production by naive (IgD +) B cells. TGF β/CD40L is believed to promote type switching, while IL10 initiates differentiation and growth. In the absence of TGF β, IL10 cooperates with CD40L to induce IgG1 and IgG3 (human) and is therefore likely to be a direct conversion factor for the IgG subtype. Interestingly, IL10 diverged the effect of IL-4 induced IgE secretion. If IL-4 induces type switching in the presence of IL10, it reverses this effect; if IL10 is present after IgE commitment, it increases IgE secretion. Finally, the CD27/CD70 interaction in the presence of IL10 promotes the formation of plasma cells from memory B cells (Agematsu et al (1998) Blood 91: 173).

Mast cells and NK cells are also affected by IL 10. IL10 induces histamine release from mast cells and blocks the release of GM-CSF and TNF- α. This effect may be an autocrine effect, since rat mast cells are known to release IL 10. As evidence of its pleiotropic effects, IL10 had an opposite effect on NK cells. IL10 actually promotes the production of TNF- α and GM-CSF by NK cells, rather than blocking this function. In addition, it enhances IL-2-induced NK cell proliferation and contributes to IL-18-induced IFN- γ secretion by NK cells. Consistent with IL-12 and/or IL-18, IL10 enhances NK cell cytotoxicity (Cai et al, 1999, Eur. J. Immunol.29: 2658).

IL10 has a significant anti-inflammatory effect on neutrophils. It can inhibit secretion of chemokines MIP-1 alpha, MIP-1 beta and IL-8, and block production of proinflammatory mediators IL-1 beta and TNF-alpha. In addition, it reduces the capacity of neutrophils to produce superoxide, thereby interfering with PMN-mediated antibody-dependent cellular cytotoxicity. It also blocks IL-8 and fMLP-induced chemotaxis, probably through CXCR1 (Vicioso et al (1998) Eur. cytokine Net. 9: 247).

IL10 generally has an immunosuppressive effect on Dendritic Cells (DCs). It appears to promote CD14+ macrophage differentiation at the expense of DC. IL10 appears to reduce the ability of DCs to stimulate T cells, particularly Th1 type cells. It may be down-regulated, invariant or up-regulated relative to MHC-II expression (Sharma et al (1999) J.Immunol.163: 5020). As for CD80 and CD86, IL10 is capable of up-regulating or down-regulating its expression. B7-2/CD86 plays a central role in T cell activation. For this molecule, IL10 is involved in up-and down-regulation. However, perhaps the most significant regulatory effect occurred on CD40 (IL10 appears to reduce its expression). At a regional level, IL10 may block immune stimulation by inhibiting langerhans cell migration in response to pro-inflammatory cytokines. Alternatively, IL10 blocks the inflammation-induced DC maturation step, typically involving CCR1, CCR2 and CCR5 down-regulation as well as CCR7 up-regulation. This blockade and maintenance of CCR1, CCR2, and CCR5 resulted in the failure of DCs to migrate to regional lymph nodes. The result is the production of nonmotile DCs that do not stimulate T cells but bind to (and clear) pro-inflammatory chemokines rather than respond to them (D-Amico et al, (2000) nat. Immunol.1: 387).

IL10 has been reported to have a number of effects on monocytes. For example, IL10 can reduce cell surface MHC-II expression, and can also after stimulation to inhibit IL-12 production. Although it can promote the conversion of monocytes to macrophages in combination with M-CSF, the phenotype of macrophages is not apparent (i.e., CD16 +/cytotoxicity vs. CD 16-). IL10 also decreased GM-CSF secretion and IL-8 production by monocytes and also increased IL-1ra release (Gesser et al, (1997) Proc. Natl. Acad. Sci. USA 94: 14620). Hyaluronan is a component of connective tissue, which is known to be secreted by monocytes in response to IL 10. This may have a significant effect on cell migration, particularly tumor cell metastasis, and it is known that hyaluronan disrupts cell migration through the extracellular space (Gesser et al, (1997) Proc. nat' l. Acad. Sci. USA 94: 14620).

Fusion proteins of IL10 with the Fc region of murine or cynomolgus monkey (referred to as IL10Fc) inhibit macrophage function and prolong survival of islet xenografts (Feng et al (1999) Transplantation 68: 1775; Asidu et al (2007) Cytokine 40: 183) and reduce septic shock in murine models (Zheng et al (1995) J.Immunol.154: 5590).

Human IL10R1 is a single transmembrane type I transmembrane protein of 90-110kDa, expressed on a limited number of cell types (Liu et al, 1994, J.Immunol.152: 1821), weakly expressed in pancreas, skeletal muscle, brain, heart and kidney, moderately expressed in placenta, lung and liver. Monocytes, B cells, large granular lymphocytes and T cells express high levels of IL10R1(Liu et al, 1994, J.Immunol.152: 1821). The expressed protein is a 578 amino acid protein containing a 21 amino acid signal peptide, a 215 amino acid extracellular region, a 25 amino acid transmembrane region, and a 317 amino acid cytoplasmic region. There are two FNIII motifs in the extracellular region, and a STAT3 docking site and a JAK1 binding region in the cytoplasmic region (Kotenko et al, 2000 Oncogene 19: 2557; Kotenko et al, 1997, EMBO J.16: 5894). IL10R1 bound human IL10 with a Kd of 200 pM.

In some embodiments, a binding domain of the invention comprises a V specific for IL10R1 or IL10R2_HAnd V_LA domain as described herein. In certain embodiments, the V is_LAnd V_HThe domain is a human domain. V_LAnd V_HThe domains may be positioned in any orientation, separated by a linker of up to about 30 amino acids as described herein or any other amino acid sequence capable of providing a spacer function compatible with the interaction of two sub-binding domains. In certain embodiments, connection V_LAnd V_HThe linker of the domain comprises SEQ ID NO: 497-604 and 791-796. The multispecific binding domain may have at least two specific sub-binding domains that resemble an alpaca antibody tissue; or at least four specific sub-binding domains, resembling pairs of V_LAnd V_HMore conventional mammalian antibody composition of chains. In other embodiments, a binding domain of the invention specific for IL10R1 or IL10R2 may comprise one or more complementarity determining regions ("CDRs"), or multiple copies of one or more such CDRs, obtained, derived or designed from an scFv or Fab fragment of anti-IL 10R1 or anti-IL 10R2, or from a heavy or light chain variable region thereof. Thus, a binding domain of the invention may comprise a binding domain derived from anti-IL 10R1 orThe variable region of anti-IL 10R2 may have a single CDR or may comprise multiple CDRs which may be the same or different. In certain embodiments, a binding domain of the invention comprises a V specific for IL10R1 or IL10R2_LAnd V_HA domain comprising framework regions and CDR1, CDR2, and CDR3 regions.

In certain embodiments, an IL10 agonist can be the extracellular domain ("ectodomain") of IL 10. The extracellular domain of IL10 as used herein refers to the extracellular portion of IL10, soluble IL10, or any combination thereof. In other embodiments, the IL10 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 754 or the amino acid sequence of SEQ ID NO: 754, or an extracellular portion thereof, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 100%, wherein the agonist binds to IL10R1 or IL10R2 and is capable of increasing the activity of IL 10.

Multispecific fusion proteins

The present invention provides multispecific fusion proteins comprising a domain antagonistic to TNF-alpha ("TNF-alpha antagonist domain") and a domain that binds a ligand other than TNF-alpha ("heterologous binding domain"), such as IL6, IL6R, IL6xR complex, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, BLyS/APRIL, or IL 10R. It is contemplated that the TNF- α antagonist domain may be amino-terminal to the fusion protein and the heterologous binding domain at the carboxy-terminal of the fusion protein, or the heterologous binding domain may be amino-terminal and the TNF- α antagonist at the carboxy-terminal. As described herein, the binding domains of the invention may be fused to either end of an intervening domain (e.g., an immunoglobulin constant region or a subdomain thereof, preferably the CH2 and CH3 domains of an IgG such as IgG 1). Furthermore, two or more binding domains can each be linked to an intervening domain by linkers known in the art or described herein.

As used herein, "intervening domain" refers to an amino acid sequence that simply serves as a scaffold for one or more binding domains such that the fusion protein is present in the composition predominantly (e.g., 50% or more of the fusion protein) or substantially (e.g., 90% or more of the fusion protein) as a single chain polypeptide. For example, certain intervening domains may have structural (e.g., spacing, flexibility, rigidity) or biological (e.g., plasma, e.g., human blood half-life extension). Exemplary intervening domains that can extend the plasma half-life of the fusion proteins of the invention include albumin, transferrin, a serum protein-binding scaffold domain, and the like, or fragments thereof.

In certain preferred embodiments, the intervening domain comprised by the multispecific fusion protein of the present invention is a "dimerization domain," which refers to an amino acid sequence capable of facilitating the association of at least two single-chain polypeptides or proteins through noncovalent or covalent interactions, such as through the formation of hydrogen bonds, electrostatic interactions, van der waals forces, salt bridges, disulfide bonds, hydrophobic interactions, and the like, or any combination thereof. Exemplary dimerization domains include immunoglobulin heavy chain constant regions or subregions, such as Fc regions comprising IgG (e.g., IgG1, IgG2, IgG3, IgG4) CH2 and CH3 domains, preferably IgG1 CH2 and CH3 domains. It will be appreciated that the dimerization domain preferably promotes dimer formation, but is capable of forming higher order multimeric complexes (e.g., trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.).

A "constant sub-region" is a term defined herein to refer to a preferred peptide, polypeptide or protein sequence that corresponds to or is derived from all or a portion of one or more immunoglobulin constant region domains, but not all of the constant region domains found in the source antibody. In some embodiments, the constant region domain of the fusion protein of the invention lacks or has weak effector functions as follows: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement activation and complement-dependent cytotoxicity (CDC), while maintaining binding to certain F_CReceptors (e.g. F)_CRn binding) and maintains a longer half-life in vivo. In certain embodiments, a binding domain of the invention is fused to an IgG1 constant region or subregion, wherein one or more of the following amino acids in the IgG1 constant region or subregionThe amino acid is mutated: leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamic acid at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU).

Methods are known in the art for making mutations within or outside of the Fc domain that alter Fc interaction with Fc receptors (CD16, CD32, CD64, CD89, fcsr 1, FcRn) or complement component C1q (see, e.g., U.S. Pat. No. 5,624,821; Presta (2002) curr. Particular embodiments of the invention include compositions comprising an immunoglobulin or fusion protein with constant or subdomain regions from human IgG in which binding to FcRn and protein a is retained and Fc domains no longer interact or interact little with other Fc receptors or C1 q. For example, a binding domain of the invention may be fused to a human IgG1 constant region or subregion wherein the asparagine at position 297 (denoted as N297 according to EU numbering) is mutated to another amino acid to reduce or eliminate glycosylation at that position, thereby eliminating effective Fc binding to Fc γ R and C1 q. Another exemplary mutation is P331S, which eliminates C1q binding but does not affect Fc binding.

In other embodiments, the immunoglobulin Fc region may have altered glycosylation patterns relative to an immunoglobulin reference sequence. For example, various genetic techniques can be used to alter one or more specific amino acid residues that constitute a glycosylation site (see Co et al (1993) mol. Immunol.30: 1361; Jacquemon et al (2006) J. Thromb. Haemost.4: 1047; Schuster et al (2005) Cancer Res.65: 7934; Warnock et al (2005) Biotechnol. Bioeng.92: 831). Alternatively, host cells producing the fusion proteins of the invention can be engineered to produce altered glycosylation patterns. For example, one approach known in the art provides altered glycosylation patterns, in particular forms that are bisected nonfucosylated variant forms with increased ADCC. Such variants are expressed by host cells containing the oligosaccharide modifying enzyme. Alternatively, Potelligent from BioWa/Kyowa Hakko is also contemplatedTechniques to reduce the fucose content of the glycosylated molecules of the present invention. In one known method, a CHO host cell is provided for the production of recombinant immunoglobulins which alter the glycosylation pattern of the Fc region of the immunoglobulin by producing GDP-fucose.

Alternatively, the glycosylation pattern of the fusion protein of the invention is altered using chemical techniques. For example, various glycosidase and/or mannosidase inhibitors provide one or more of the desired properties of increased ADCC activity, increased Fc receptor binding, and altered glycosylation patterns. In certain embodiments, cells expressing a multispecific fusion protein of the invention (comprising a TNF-alpha antagonist domain linked to an IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or BLyS/APRIL antagonist or IL10 agonist) are cultured in medium containing sugar modifications at a concentration that increases ADCC of the immunoglycoprotein molecule produced by the host cell and is less than 800 μ M. In a preferred embodiment, cells expressing these multi-specific fusion proteins are cultured in a medium containing castanospermine or kifunensine, more preferably castanospermine, at a concentration of 100-800. mu.M, such as 100. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 500. mu.M, 600. mu.M, 700. mu.M or 800. mu.M. Methods for altering glycosylation using sugar modifications such as castanospermine are provided in U.S. patent application publication No. 2009/0041756 or PCT publication No. WO 2008/052030.

In another embodiment, the immunoglobulin Fc region may have amino acid modifications that affect binding to an effector cell Fc receptor. Any technique known in the art may be used, for example Presta et al (2001) biochem. 487 the methods described for making these modifications. In another approach, Xencor XBMA can be used^TMConstant sub-regions corresponding to the Fc domain were engineered to enhance cell killing effector function (see Lazar et al (2006) proc.nat' l.acad.sci. (USA) 103: 4005). For example, this approach can be used to generate constant subregions with improved specificity and binding for FC γ rs, thereby increasing the cell killing effector function.

In other embodiments, the constant region or subregion may optionally increase plasma half-life or placental transfer as compared to a corresponding fusion protein lacking such intervening domains. In certain embodiments, the extended plasma half-life of a fusion protein of the invention in a human is at least 2, at least 3, at least 4, at least 5, at least 10, at least 12, at least 18, at least 20, at least 24, at least 30, at least 36, at least 40, at least 48 hours, at least several days, at least one week, at least 2 weeks, at least one month, at least two months, to a few months, or longer.

The constant sub-region may comprise a portion or all of any of the following domains: c_H2Domains and C_H3Domain (IgA, IgD, IgG), or C_H3Domains and C_H4Domains (IgE, IgM). Thus, a constant sub-region as described herein may refer to a polypeptide corresponding to a portion of an immunoglobulin constant region. The constant sub-region may comprise C's derived from the same or different immunoglobulins, antibody isotypes or allelic variants_H2Domains and C_H3A domain. In some embodiments, C_H3The domain is truncated and comprises the sequence set forth in PCT publication WO2007/146968 as SEQ ID NO: 366-371 carboxy terminal sequences, which are incorporated herein by reference. In certain embodiments, a constant sub-region of a polypeptide of the invention has a C_H2Domains and C_H3Domains, which may optionally have an amino terminal linker, a carboxy terminal linker, or linkers at both ends.

A "linker" is a peptide that binds or connects to other peptides or polypeptides, such as a linker of about 2 to 150 amino acids. In the fusion proteins of the invention, a linker may link an intervening domain (e.g., an immunoglobulin-derived constant sub-region) to the binding domain, or a linker may link two variable regions of the binding domain. For example, the linker may be an amino acid sequence derived, generated or designed from the antibody hinge region sequence, a sequence linking the binding domain to a receptor, or a sequence linking the binding domain to a cell surface transmembrane region or membrane anchor. In some embodiments, the linker can have at least one cysteine that is capable of participating in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, peptide storage conditions). In certain embodiments, a linker corresponding to or similar to an immunoglobulin hinge peptide retains a cysteine corresponding to a hinge cysteine disposed toward the amino-terminus of the hinge. In other embodiments, the linker is from an IgG1 or IgG2A hinge and contains one cysteine or two cysteines corresponding to the hinge cysteines. In certain embodiments, one or more disulfide bonds, referred to as interchain disulfide bonds, are formed between intervening domains. In other embodiments, the fusion proteins of the invention may have an intervening domain fused directly to the binding domain (i.e., without a linker or hinge). In some embodiments, the intervening domain is a dimerization domain.

The intervening or dimerization domain of the multispecific fusion protein of the present invention may be linked to one or more terminal binding domains by a peptide linker. In addition to providing a spacer function, the linker may provide elasticity or rigidity within the fusion protein and between the fusion protein and its target suitable for properly orienting one or more binding domains of the fusion protein. In addition, the linker may support the expression of the full-length fusion protein and the stability of the purified protein in vitro and in vivo after administration to a subject in need thereof, such as a human, in which the linker is preferably non-immunogenic or less immunogenic. In certain embodiments, the linker of the intervening or dimerization domain of the multispecific fusion protein of the present invention may comprise a portion or all of a human immunoglobulin hinge.

Furthermore, the binding domain may comprise V_HAnd V_LDomains, these variable region domains may be joined by a linker. Exemplary variable region binding domain linkers include those belonging to (Gly)_nSer) family of linkers, e.g. (Gly₃Ser)_n(Gly₄Ser)₁、(Gly₃Ser)₁(Gly₄Ser)_n、(Gly₃Ser)_n(Gly₄Ser)_nOr (Gly)₄Ser)_nWherein n is an integer from 1 to 5 (see, e.g., linkers 22, 29, 46, 89, 90, and 116 corresponding to SEQ ID NOS: 518, 525, 542, 585, 586, and 603, respectively). In the preferred aspectsIn embodiments, these are based on (Gly)_nSer) is used to link the variable region and not the binding domain to the intervening domain.

Exemplary linkers that can be used to link an intervening domain (e.g., an immunoglobulin molecule-derived constant sub-region) to a binding domain or to link two variable regions of a binding domain can be found in SEQ ID NO: 497-604 and 791-796.

Linkers contemplated by the invention include, for example, peptides derived from any interdomain region of a member of the immunoglobulin superfamily, such as the antibody hinge region, or the stem region of a C-type lectin (type II membrane protein family). These linkers may be about 2-150 amino acids, or about 2-40 amino acids, or about 8-20 amino acids, preferably about 10-60 amino acids, more preferably about 10-30 amino acids, most preferably about 15-25 amino acids in length. For example, linker 1(SEQ ID NO: 497) is 2 amino acids long and linker 116(SEQ ID NO: 560) is 36 amino acids long.

In addition to the usual considerations of length, linkers suitable for use in the fusion proteins of the invention include antibody hinge regions selected from the group consisting of: an IgG hinge, an IgA hinge, an IgD hinge, an IgE hinge, or a variant thereof. In certain embodiments, the linker may be an antibody hinge region (upper and core regions) selected from human IgG1, human IgG2, human IgG3, human IgG4, fragments or variants thereof. As used herein, a linker as "immunoglobulin hinge region" refers to an amino acid found between the carboxy terminus of CH1 and the amino terminus of CH2 (for IgG, IgA, and IgD) or the amino terminus of CH3 (for IgE and IgM). As used herein, the "wild-type immunoglobulin hinge region" refers to a naturally occurring amino acid sequence found in an antibody heavy chain that is placed between and connects the CH1 and CH2 regions (for IgG, IgA, and IgD) or between and connects the CH2 and CH3 regions (for IgE and IgM). In a preferred embodiment, the wild-type immunoglobulin hinge region sequence is a human sequence.

From crystallographic image studies, the IgG hinge region can be subdivided into the following three regions based on function and structure: the upper, core or middle and lower hinge regions (Shin et al, Immunological Reviews 130: 87 (1992)). Exemplary upper hinge regions include EPKSCDKTHT (SEQ ID NO: 819) found in IgG1, ERKCCVE (SEQ ID NO: 820) found in IgG2, ELKTPLGDTT HT (SEQ ID NO: 821) or EPKSCDTPPP (SEQ ID NO: 822) found in IgG3, and ESKYGPP (SEQ ID NO: 823) found in IgG 4. Exemplary middle hinge regions include CPPCP found in IgG1 and IgG 2(SEQ ID NO: 834), CPRCP found in IgG3 (SEQ ID NO: 824), and CPSCP found in IgG4 (SEQ ID NO: 825). While the IgG1, IgG2, and IgG4 antibodies each have one upper and middle hinge, IgG3 has four tandem hinges-one ELKTPLGDTT HTCPRCP (SEQ ID NO: 826) and three EPKSCDTPPP CPRCP (SEQ ID NO: 827).

IgA and IgD antibodies appear to lack an IgG-like core region, and IgD appears to have two upper hinge regions in tandem (see SEQ ID NOS: 828 and 829). SEQ ID NO: exemplary wild type upper hinge regions found in the IgA1 and IgA2 antibodies are listed at 830 and 831.

In contrast, IgE and IgM antibodies comprise a CH2 region with hinge-like properties rather than the typical hinge region. SEQ ID NO: 832 (VCSRDFTPPT VKILQSSSDG GGHFPPTIQL LCLVSGYTPG TINITWLEDG QVMDVDLSTA STTQEGELAS TQSELTLSQK HWLSDRTYTC)

An "altered wild-type immunoglobulin hinge region" or an "altered immunoglobulin hinge region" refers to (a) a wild-type immunoglobulin hinge region that contains up to 30% amino acid alterations (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a wild-type immunoglobulin hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids), contains up to 30% amino acid alterations (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of the wild-type immunoglobulin hinge region comprising the core hinge region (which portion may be 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 or at least 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length). In certain embodiments, one or more cysteine residues in the hinge region of a wild-type immunoglobulin may be substituted with one or more other amino acid residues (e.g., one or more serine residues). Alternatively or additionally, the altered immunoglobulin hinge region may have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (e.g., a serine residue).

Alternative hinge and linker sequences that can be used as attachment regions can be prepared from portions of cell surface receptors that attach IgV-like or IgC-like domains. The regions between IgV-like domains when the cell surface receptor contains multiple IgV-like domains in tandem and between IgC-like domains when the cell surface receptor contains multiple IgC-like regions in tandem may also serve as linking regions or linker peptides. In certain embodiments, the hinge and linker sequences are 5-60 amino acids in length, may be substantially flexible, but may also provide more rigid characteristics, and may also contain primarily α -helical structures with little β -sheet structures. Preferably, the sequences are stable in plasma and serum and are resistant to proteolytic cleavage. In some embodiments, the sequence may contain a naturally occurring or added motif, such as CPPC, that can form one or more disulfide bonds to stabilize the C-terminus of the molecule. In other embodiments, the sequence may contain one or more glycosylation sites. Examples of hinge and linker sequences include the interdomain region between the IgV-like and IgC-like domains or between the IgC-like or IgV-like domains of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD96, CD150, CD166, and CD 244. Alternative hinges can also be prepared from disulfide-bond containing regions in type II receptors other than immunoglobulin superfamily members, such as CD69, CD72, and CD 161.

In some embodiments, the hinge linker has one cysteine residue for forming interchain disulfide bonds. In other embodiments, the hinge joint has two cysteine residues for forming interchain disulfide bonds. In other embodiments, the hinge linkers are derived from an inter-immunoglobulin domain region (such as an antibody hinge region) or a type II C-type lectin stem region (derived from a type II membrane protein; see, for example, the exemplary lectin stem region sequences shown in PCT application publication No. WO2007/146968, SEQ ID NOS: 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 289, 279, 281, 287, 297, 305, 307, 309-311, 313-one 331, 346, 373, 377, 380, or 381 of this disclosure, these sequences being incorporated herein by reference).

In one aspect, exemplary multi-specific fusion proteins comprising a TNF- α antagonist as described herein also comprise at least one additional binding region or domain specific for a target other than TNF- α, including, for example, IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or a BLyS/APRIL antagonist or IL10 agonist. For example, the multispecific fusion protein of the invention has a TNF-alpha antagonist domain linked by an intervening domain to an IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or BLyS/APRIL antagonist domain or an IL10 agonist domain. In certain embodiments, the multispecific fusion protein comprises first and second binding domains, first and second linkers, and an intervening domain, wherein the intervening domain is fused at one end to a TNF- α antagonistic first binding domain agent (e.g., TNFR ectodomain, anti-TNFR, anti-TNF- α) via the first linker and at the other end to a different binding domain via the second linker, e.g., IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or a BLyS/APRIL antagonist or IL10 agonist.

In certain embodiments, the first linker and the second linker of the multispecific fusion protein of the present invention are each independently selected from (e.g., SEQ ID NO: 497-604 and 791-796. For example, the first and second linkers may be linker 102(SEQ ID NO: 589), 47(SEQ ID NO: 543), 80(SEQ ID NO: 576), or any combination thereof. In other examples, one linker is linker 102(SEQ ID NO: 589) and the other linker is linker 47(SEQ ID NO: 543), or one linker is linker 102(SEQ ID NO: 589) and the other linker is linker 80(SEQ ID NO: 576). In other examples, V is included_HAnd V_LDomains, e.g.specific for IL6, IL6R, IL6xR, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, BLyS/APRIL or IL10, the binding domains of the invention of the TNFR ectodomain or TNF-alpha may also have a binding domain located at V_HAnd V_L(third) linker between domains, such as linker 46(SEQ ID NO: 542). In these embodiments, the linker may be flanked by 1-5 additional connecting amino acids, which may simply be the result of the production of such a recombinant molecule (e.g., the use of a particular restriction enzyme site to join nucleic acid molecules may result in the insertion of one or several amino acids), or may be considered part of the core sequence of any particular linker for the purposes of the present invention.

In other embodiments, the intervening domain of the multispecific fusion protein of the invention is comprised of an immunoglobulin constant region or subregion (preferably CH2CH3 for IgG, IgA, or IgD, or CH3CH4 for IgE or IgM) wherein the intervening domain is located between the TNF- α antagonist domain and the IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or BLyS/APRIL antagonist binding domain or IL10 agonist binding domain. In certain embodiments, the intervening domain of a multispecific fusion protein of the invention comprises a TNF- α antagonist at the amino terminus and an IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or a BLyS/APRIL antagonist binding domain or IL10 agonist binding domain at the carboxy terminus. In other embodiments, the intervening domain of the multispecific fusion protein of the invention comprises an IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or a BLyS/APRIL antagonist binding domain or an IL10 agonist binding domain at the amino terminus and a TNF- α antagonist at the carboxy terminus. In other embodiments, the immunoglobulin constant region or sub-region comprises the CH2 and CH3 domains of immunoglobulin G1(IgG 1). In related embodiments, the IgG1 CH2 and CH3 domains have one or more of the following amino acids mutated (i.e., have different amino acids at that position): leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), glutamic acid at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any of the above amino acids may be changed to alanine. In another embodiment, L234, L235, G237, E318, K320 and K322 in the CH2 domain are each mutated to alanine (i.e., L234A, L235A, G237A, E318, K320 and K322, respectively) according to EU numbering.

In some embodiments, the TNF- α antagonist in the multispecific fusion proteins of the present invention comprises a TNFR extracellular domain or subdomain, one or more TNFR CRD domains (e.g., CRD2 and CRD3), or a TNF- α specific antibody-derived binding domain (e.g., IL6, IL6R, or IL6xR complex-specific antibody-derived binding domain as described herein). In some embodiments, the TNF- α antagonist is the extracellular domain of TNFR1 or TNFR 2. In certain embodiments, the TNF- α antagonist comprises the amino-terminal portion of TNFR2 (also referred to as p75, TNFRSF1B), the first 257 amino acids as shown in GenBank accession NP-001057.1 (SEQ ID NO: 671). In other embodiments, the TNF- α antagonist comprises SEQ ID NO: 671 amino acids 23-257 (i.e., without the native leader sequence). In a preferred embodiment, the TNF- α antagonist comprises a fragment of TNFR2 (e.g., the extracellular domain) as set forth in SEQ ID NO: 671 amino acids 23-163 or SEQ ID NO: 671 amino acids 23-185 of SEQ ID NO: 671 amino acids 23-235. In other preferred embodiments, the TNF- α antagonist comprises a derivative of a fragment of TNFR2, such as the amino acid sequence of SEQ ID NO: 671 or amino acids 23-163 of SEQ ID NO: 671 or amino acids 23-185 of SEQ ID NO: 671 amino acids 23-235. In certain embodiments, the TNF- α antagonist comprises the amino-terminal portion of TNFR1 (also referred to as p55, TNFRSF1A), the first 211 amino acids as shown in GenBank accession NP-001056.1 (SEQ ID NO: 672). In other embodiments, the TNF- α antagonist comprises SEQ ID NO: 672 amino acids 31-211 (i.e., without the native leader sequence).

In other embodiments, the multispecific fusion protein of the invention has a TNF- α antagonist binding domain and an affinity for binding to IL6xRAn IL6 antagonist binding domain that is higher than IL6 alone or IL6R α alone and competes with the sIL6xR complex for binding to mgp130 or enhances sgp130 binding to the sIL6xR complex. In certain embodiments, the binding domain specific for the IL6xR complex comprises (i) V_HA domain having a sequence identical to SEQ ID NO: v listed in any one of items 435, 496 and 805, 810_HAn amino acid sequence in which the amino acid sequence of a domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; or (ii) V_LA domain having a sequence identical to SEQ ID NO: v listed in any one of 373, 434 and 799, 804_LAn amino acid sequence in which the amino acid sequence of a domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; or (iii) V of (i)_H(iii) domains and V of (ii)_LA domain; or both V from (i) and V from the same reference sequence_H(iii) domains and V of (ii)_LA domain. In one embodiment, such a V_HAnd V_LThe domains may form exemplary binding domains TRU6-1002 (see SEQ ID NOS: 374 and 436, respectively). In certain embodiments, a multispecific fusion protein comprising an IL6 antagonist binding domain detectably inhibits IL6 cis and trans signaling, optionally does not inhibit signaling of gp130 family cytokines other than IL 6.

In other embodiments, an IL6 antagonist binding domain that binds IL6xR with higher affinity than IL6 or IL6R α or IL6 alone or IL6R α alone and competes with gp130 for binding to the sIL6xR complex or enhances the binding of sgp130 to the sIL6xR complex comprises V_HAnd V_LDomains, both domains comprising framework regions and CDR1, CDR2 and CDR3 regions, wherein (a) V_HThe domain comprises SEQ ID NO: the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 as set forth in any one of 435-, 496-and 805-810; or (b) V_LThe domain comprises SEQ ID NO: the amino acid sequences of the light chain CDR1, CDR2 and CDR3 as set forth in any one of 373-434 and 799-804; or (c) the binding domain comprises the V of (a)_HAmino acid sequence and V of (b)_LAn amino acid sequence; or the binding domain comprises a sequence derived from the same referenceV of (a) of the sequence_HAmino acid sequence and V of (b)_LAn amino acid sequence. V of these multi-specific fusion proteins_LAnd V_HThe domains can be positioned in any orientation and can be separated by a linker of about 5-30 amino acids as described herein. In certain embodiments, connection V_HAnd V_LThe linker of the domain comprises the amino acid sequence of linker 47(SEQ ID NO: 543) or linker 80(SEQ ID NO: 576). In certain embodiments, a multispecific fusion protein comprising an IL6 antagonist binding domain detectably inhibits IL6 cis and trans signaling, preferably inhibits trans signaling, optionally does not inhibit signaling of a gp130 family cytokine other than IL 6.

Exemplary structures of such multi-specific fusion proteins, referred to herein as cross-receptor molecules, include N-BD-X-ED-C, N-ED-X-BD-C, N-ED1-X-ED2-C, wherein BD is an immunoglobulin-like or immunoglobulin variable region binding domain, X is an intervening domain, and ED is the receptor ectodomain, semaphorin domain, and the like. In some constructs, X may comprise an immunoglobulin constant region or sub-region disposed between the first and second binding domains. In some embodiments, the intervening domain (X) in the multispecific fusion protein of the present invention comprises, from amino-terminus to carboxy-terminus, the following structure: -L1-X-L2-, wherein L1 and L2 are each independently a linker comprising 2 to about 150 amino acids; x is an immunoglobulin constant domain or subdomain (preferably CH2CH3 of IgG 1). In other embodiments, the multispecific fusion protein has an intervening domain that is albumin, transferrin, or other serum protein binding protein, wherein the fusion protein substantially or predominantly retains the form of a single chain polypeptide in the composition. In another embodiment, the multispecific fusion protein of the invention has the following structure: N-BD1-X-L2-BD2-C, wherein N and C represent amino and carboxy termini, respectively; BD1 is a TNF- α antagonist that is at least about 90% identical to the TNFR ectodomain; -X-is-L1-CH 2CH3-, wherein L1 is the IgG1 hinge, optionally mutated by substitution of the first cysteine, wherein-CH 2CH 3-is the CH2CH3 region of the Fc domain of IgG1, optionally mutated to eliminate Fc γ RI-III interactions while maintaining FcRn interactions; l is2 is selected from SEQ ID NO: 497 + 604 and 791 + 796 are not based on (G)₄S) a joint; and BD2 is IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, or BLys/APRIL antagonist binding domain or IL10 agonist binding domain (as described herein).

In a specific embodiment, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide having a sequence identical to SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence that is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence set forth in SEQ ID NO, and (b) an IL6 antagonist that binds IL6xR with greater affinity than IL6, IL6R α or IL6 alone or IL6R α alone and competes with mgp130 for binding to the sIL6xR complex or enhances sgp130 binding to sIL6xR comprising a heavy chain variable region and a light chain variable region wherein the amino acid sequences of CDR1, CDR2 and CDR3, respectively, are identical to SEQ ID NO: 435-496 and 805-810 are at least 80% -100% identical, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively identical to the amino acid sequences shown in SEQ ID NO: 373-434 and 799-804 are at least 80% -100% identical, wherein either the TNF- α antagonist of (a) or the IL6 antagonist of (b) is fused to a first linker, and (ii) said first linker is fused to a second linker comprising SEQ ID NO: the immunoglobulin heavy chain constant regions of CH2 and CH3 of amino acids 275 and 489 of 608, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, and (iv) the second linker is fused to the TNF- α antagonist of (a) or the IL6 antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) or linker 80(SEQ ID NO: 576), the second linker is linker 102(SEQ ID NO: 589), IL6 antagonist V_HAnd V_LAnother (third) linker between domains is linker 46(SEQ ID NO: 542).

In other embodiments, the multispecific fusion protein of the invention has a sequence identical to SEQ ID NO: 607, 668 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in any of these sequences, with or without the leader peptide (i.e., the first 23 amino groups in these sequencesAcid) in a sample. In other embodiments, the multispecific fusion protein of the invention comprises a TNF- α antagonist comprising the amino acid sequence of SEQ ID NO: 671 amino acids 23-257, 23-163, 23-185, or 23-235, said IL6 antagonist binding to IL6xR complex with higher affinity than to IL6, IL6R α or IL6 alone or IL6R α alone and competing with mgp130 for binding to sIL6xR complex or enhancing sgp130 binding to sIL6xR, comprising a heavy chain amino acid sequence linked to SEQ ID NO: 436 shown as V_HSEQ ID NO: 374 denoted by V_LWherein the TNF- α antagonist is linked to the amino terminus of the intervening domain by linker 47(SEQ ID NO: 543) and the IL6 antagonist is linked to the carboxy terminus of the intervening domain by linker 102(SEQ ID NO: 589), the intervening domain comprising a peptide comprising SEQ ID NO: the immunoglobulin heavy chain constant region of amino acids 275 and 489 of 608 and CH2 and CH 3. In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 608.

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence which is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence set forth in SEQ ID NO: 741 an amino acid sequence that is at least 80% -100% identical from amino terminus to carboxy terminus or carboxy terminus to amino terminus, wherein (i) the TNF- α antagonist of (a) or the TWEAK antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a polypeptide comprising SEQ ID NO: 798 CH2 and CH3 of amino acids 275 and 489, (iii) said CH2CH3 constant region polypeptide is fused to a second linker, (iv) said second linker is fused to the TNF- α antagonist of (a) or the TWEAK antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 798 (the corresponding nucleic acid sequence is SEQ ID NO: 805).

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence that is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence represented by SEQ ID NO: 737 an amino acid sequence that is at least 80% to 100% identical, wherein from amino terminus to carboxy terminus or from carboxy terminus to amino terminus, (i) the TNF- α antagonist of (a) or the RANKL antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a polypeptide comprising SEQ ID NO: 799 CH2 of amino acid 253-468 and an immunoglobulin heavy chain constant region of CH3, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, (iv) the second linker is fused to the TNF- α antagonist of (a) or the RANKL antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 799 (the corresponding nucleic acid sequence is SEQ ID NO: 806).

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence which is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence shown in SEQ ID NO: 818 or 746 at least 80% -100% identical amino acid sequence wherein from amino terminus to carboxy terminus or from carboxy terminus to amino terminus, (i) the TNF- α antagonist of (a) or the IGF antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a polypeptide comprising SEQ ID NO: the immunoglobulin heavy chain constant regions of CH2 and CH3 of amino acids 253-468 in 800, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, (iv) the second linker is fused to the TNF- α antagonist of (a) or the IGF antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 800 (the corresponding nucleic acid sequence is SEQ ID NO: 807).

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence which is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence set forth in SEQ ID NO: 738, wherein from amino terminus to carboxy terminus or from carboxy terminus to amino terminus, (i) the TNF- α antagonist of (a) or the IL7 antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a second linker comprising SEQ ID NO: the immunoglobulin heavy chain constant regions of CH2 and CH3 of amino acids 253-468 in 801, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, (iv) the second linker is fused to the TNF- α antagonist of (a) or the IL7 antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 801 (the corresponding nucleic acid sequence is SEQ ID NO: 808).

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence which is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence set forth in SEQ ID NO: 739. 740, 816 or 817 are at least 80% -100% identical in amino acid sequence, wherein from amino terminus to carboxy terminus or from carboxy terminus to amino terminus, (i) the TNF- α antagonist of (a) or the IL7 antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a second linker comprising SEQ ID NO: the immunoglobulin heavy chain constant regions of CH2 and CH3 of amino acids 253-468 in 802 or 803, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, (iv) the second linker is fused to the TNF- α antagonist of (a) or the IL7 antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 802 or 803 (the corresponding nucleic acid sequences are SEQ ID NOS: 809 and 810, respectively).

In other embodiments, the multispecific cross-receptor fusion protein has (a) a TNF- α antagonist comprising a peptide that differs from SEQ ID NO: 671 or 672 or SEQ ID NO: 671 or 672 an amino acid sequence which is at least 80% -100% identical to a contiguous fragment of about 140-215 amino acids of the sequence shown in SEQ ID NO: 747 or 818 is at least 80% -100% identical to SEQ ID NO: 804 is at least 80% -100% identical to SEQ ID NO: 812 is at least 80% -100% identical to amino acids 21-922 of SEQ ID NO: 813 having an amino acid sequence that is at least 80% -100% identical to amino acids 21-726 of SEQ ID NO, wherein (i) the TNF- α antagonist of (a) or the IGF antagonist of (b) is fused to a first linker, (ii) the first linker is fused to a polypeptide comprising SEQ ID NO: the immunoglobulin heavy chain constant regions of CH2 and CH3 of amino acids 253-468 in 804, (iii) the CH2CH3 constant region polypeptide is fused to a second linker, (iv) the second linker is fused to the TNF- α antagonist of (a) or the IGF antagonist of (b). In certain embodiments, the first linker is linker 47(SEQ ID NO: 543) and the second linker is linker 175(SEQ ID NO: 791). In one embodiment, the multi-specific fusion protein has the amino acid sequence of SEQ ID NO: 804 (the corresponding nucleic acid sequence is SEQ ID NO: 811).

Preparation of a multispecific fusion protein

In order to efficiently produce any of the binding domain polypeptides or fusion proteins described herein, a leader peptide is used to facilitate secretion of the expressed polypeptide and fusion protein. It is contemplated that any conventional leader peptide (signal sequence) may be used to direct the initially expressed polypeptide or fusion protein into the secretory pathway and result in a leader peptide at or near the point of attachment of the polypeptide or fusion protein to the polypeptideThe leader peptide is cleaved from the mature polypeptide or fusion protein. Selecting a particular leader peptide according to factors known in the art, e.g., a sequence encoded using a polynucleotide that readily comprises a restriction endonuclease cleavage site at the beginning or end of the coding sequence of the leader peptide to facilitate molecular engineering manipulations, provided that the amino acids identified by such introduced sequence do not unacceptably interfere with processing of the leader peptide from the initially expressed protein; or if the leader peptide is not cleaved off during maturation of the polypeptide or fusion protein, it does not unacceptably interfere with any desired function of the polypeptide or fusion protein molecule. Exemplary leader peptides of the invention include the native leader sequence (i.e., the sequence that is expressed with the native protein) or use a heterologous leader sequence, such as H₃N-MDFQVQIFSFLLISASVIMSRG(X)_n-CO₂H (wherein X is any amino acid, n is 0-3) (SEQ ID NO: 744) or H₃N-MEAPAQLLFLLLLWLPDTTG-CO₂H(SEQ ID NO：745)。

As described herein, binding domains described herein, such as variants and derivatives of the extracellular, light and heavy chain variable regions and CDRs are also contemplated. In one example, insertional variants are provided in which one or more amino acid residues are added to the amino acid sequence of a particular binding agent. Insertions may be located at either or both ends of the protein, or may be located within the internal region of the amino acid sequence of a particular binding agent. Variant products of the invention also include mature specific binding agent products, i.e., specific binding agent products with the leader or signal sequence removed and the resulting protein having an additional amino terminal residue. The additional amino-terminal residue may be from another protein, or may comprise one or more residues that cannot be identified as from a particular protein. Polypeptides having additional methionine residues at the-1 position are contemplated, as well as polypeptides of the invention having additional methionine and lysine residues at the-2 and-1 positions. Variants having additional Met, Met-Lys or Lys residues (or collectively having one or more basic residues) are particularly useful in increasing recombinant protein production in bacterial host cells.

As used herein, "amino acid" refers to a natural (naturally occurring) amino acid, a substituted natural amino acid, an unnatural amino acid, a substituted unnatural amino acid, or any combination thereof. In this context, the standard single or three letter symbols are used to indicate natural amino acids. Natural polar amino acids include asparagine (Asp or N) and glutamine (Gln or Q); and basic amino acids such as arginine (Arg or R), lysine (Lys or K), histidine (His or H) and derivatives thereof; and acidic amino acids such as aspartic acid (Asp or D) and glutamic acid (Glu or E) and derivatives thereof. Natural hydrophobic amino acids include tryptophan (Trp or W), phenylalanine (Phe or F), isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V) and derivatives thereof; and other non-polar amino acids such as glycine (GIy or G), alanine (Ala or a), proline (Pro or P) and derivatives thereof. Natural amino acids of moderate polarity include serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y), cysteine (Cys or C), and derivatives thereof. Unless otherwise indicated, any amino acid described herein may be in the D-or L-configuration.

Substitution variants include fusion proteins in which one or more amino acid residues in the amino acid sequence are removed or substituted with another residue. In some embodiments, the nature of the substitution is a conservative substitution; however, non-conservative substitutions are also encompassed by the present invention. Amino acids can be classified according to physical properties and their contribution in the secondary and tertiary structure of proteins. Conservative substitutions are recognized in the art as substitutions with amino acids of similar nature. Exemplary conservative substitutions may be found in table 1 (see WO 97/09433, page 10, published 3/13 1997), as shown below.

TABLE 1 conservative substitutions I

Alternatively, conserved amino acids can be grouped according to Lehninger (Biochemistry, second edition; WP publishing company, Worth Publishers, Inc.) NY: NY (1975), pages 71-77, as shown in Table 2 below.

TABLE 2 conservative substitutions sII

Variants or derivatives may also have additional amino acid residues due to the use of a particular expression system. For example, the use of a commercially available vector expressing the desired polypeptide as part of a glutathione-S-transferase (GST) fusion product can provide the desired polypeptide with an additional glycine residue at the-1 position after cleavage of the GST component of the desired polypeptide. Variants produced by expression in other vector systems are also contemplated, including variants in which a histidine tag is incorporated into the amino acid sequence, typically at the carboxy and/or amino terminus of the sequence.

Deletion variants in which one or more amino acid residues in the binding domain of the invention are removed are also contemplated. Deletions may be made at one or both ends of the fusion protein, or by removal of one or more residues within the amino acid sequence.

In certain exemplary embodiments, the fusion proteins of the invention are glycosylated, with the glycosylation pattern depending on a variety of factors, including the host cell in which the protein is expressed (if produced in a recombinant host cell) and the culture conditions.

The invention also provides derivatives of the fusion proteins. Derivatives include specific binding domain polypeptides that carry modifications other than insertions, deletions or substitutions of amino acid residues. In certain embodiments, the modification is a covalent modification, including, for example, chemical bonding to polymers, lipids, and other organic or inorganic moieties. Derivatives of the invention can be prepared that extend the circulating half-life of the specific binding domain polypeptide, or can be designed to increase the targeting capacity of the polypeptide to a cell, tissue or organ of interest.

The invention also includes fusion proteins that are covalently modified or derivatized to include one or more water-soluble polymer linkers, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described in U.S. Pat. nos. 4,640,835; 4,496,689, respectively; 4,301,144, respectively; 4,670,417, respectively; 4,791,192 and 4,179,337. Other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose and other sugar-based polymers, poly (N-vinyl pyrrolidone) -polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol) and polyvinyl alcohol, and mixtures of these polymers. Particularly preferred are polyethylene glycol (PEG) derivatized proteins. The water-soluble polymer may be bonded at a specific position, for example at the amino terminus of a protein or polypeptide of the invention, or randomly attached to one or more side chains of the polypeptide. The use of PEG to improve therapeutic ability is described in us patent 6,133,426.

A particular embodiment of the invention is an immunoglobulin or Fc fusion protein. Such fusion proteins may have a long half-life, for example, hours, a day or more, or a week or more, particularly when the Fc domain is capable of interacting with FcRn, a neonatal Fc receptor. The binding site in the Fc domain to FcRn is also the site of binding of bacterial proteins a and G. The tight binding between these proteins can be used as a means of purifying the antibodies or fusion proteins of the invention, for example, using protein a or protein G affinity chromatography during protein purification.

Protein purification techniques are well known to those skilled in the art. These techniques involve, at some level, a coarse fractionation of polypeptide and non-polypeptide components. Further purification often is required using chromatographic and electrophoretic techniques in order to achieve partial or complete purification (or purification to homogeneity). Analytical methods which are particularly suitable for the preparation of pure fusion proteins are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectric focusing. Particularly effective methods for peptide purification are fast protein liquid chromatography and HPLC.

Certain aspects of the invention relate to the purification, and in particular embodiments, to the significant purification, of fusion proteins. The term "purified fusion protein" as used herein refers to a composition that is separable from other components, wherein the fusion protein is purified to any degree relative to its naturally obtainable state. Thus, a purified fusion protein also refers to a fusion protein that is separate from its naturally occurring environment.

Generally, "purified" refers to a fusion protein composition that has been fractionated to remove various other components, and which substantially retains the expressed biological activity. The term "substantially purified" as used herein refers to a fusion binding protein composition wherein the fusion protein constitutes the major component of the composition, e.g., about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or more by weight of the proteins in the composition.

Various methods for quantitatively determining the degree of purification are known to those skilled in the art from the present invention. They include, for example, determining the specific binding activity of the active component or evaluating the content of fusion protein in the component by SDS/PAGE analysis. A preferred method of assessing the purity of a protein component is to calculate the binding activity of the component and compare it to the binding activity of the initial extract to calculate the degree of purification, herein assessed by "fold purification". The actual unit used to represent the level of binding activity will, of course, depend on the particular assay technique chosen for follow-up purification and whether the expressed fusion protein has detectable binding activity.

Various techniques suitable for protein purification are well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc., or precipitation by heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase chromatography, hydroxyapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is well known in the art, it is believed that the order in which the various purification steps are performed may be altered, or that certain steps may be omitted, and still result in a process suitable for preparing a substantially purified protein.

It is not generally required that the fusion protein is always provided in the most purified state. Indeed, it is contemplated that in certain embodiments, proteins with lower levels of purification may be used. Fewer purification steps may be used in combination, or partial purifications may be performed using different versions of the same overall purification scheme. For example, it will be appreciated that the level of purification of cation exchange column chromatography carried out with an HPLC apparatus is generally higher than that of the same technique carried out with a low pressure chromatography system. A process with a relatively low degree of purification may have advantages in terms of overall recovery of the protein product, or in terms of maintaining the binding activity of the expressed protein.

It is known that polypeptide migration can be altered (sometimes significantly) with different SDS/PAGE conditions (Capaldi et al (1977) biochem. Biophys. Res. Comm.76: 425). Thus, it will be appreciated that the apparent molecular weight of the purified or partially purified fusion protein expression product may vary under different electrophoretic conditions.

Polynucleotides, expression vectors and host cells

The present invention provides polynucleotides (isolated or purified or pure polynucleotides) encoding the multispecific fusion proteins of the present invention, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells (e.g., host cells) transformed or transfected with a polynucleotide or vector of the present invention.

In certain embodiments, polynucleotides (DNA or RNA) encoding a binding domain of the invention, or a multi-specific fusion protein containing one or more such binding domains, are contemplated. Expression cassettes encoding multi-specific fusion protein constructs are provided in the accompanying examples.

The invention also relates to vectors, in particular recombinant expression constructs, comprising the polynucleotides of the invention. In one embodiment, the invention contemplates vectors comprising polynucleotides encoding multispecific fusion proteins comprising a TNF-alpha antagonist domain of the invention and IL6, RANKL, IL7, IL17A/F, eak, CSF2, IGF1, IGF2 or BLys/APRIL antagonist binding domain or IL10 agonist binding domain, as well as other polynucleotide sequences that cause or facilitate the transcription, translation, and processing of the coding sequence of such multispecific fusion proteins.

Suitable cloning and expression vectors for use in prokaryotic and eukaryotic hosts can be found, for example, in Sambrook et al, molecular cloning: a Laboratory Manual, second edition, Cold Spring Harbor (Cold Spring Harbor), NY, (1989). Exemplary cloning/expression vectors include cloning vectors, shuttle vectors, and expression constructs that may be based on plasmids, phagemids, cosmids, viruses, artificial chromosomes or any nucleic acid vectors known in the art to be suitable for amplifying, transferring, and/or expressing an included polynucleotide.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Exemplary vectors include plasmids, yeast artificial chromosomes, and viral genomes. Some vectors are capable of autonomous replication in a host cell, whereas other vectors may integrate into the host cell genome and thereby replicate together with the host genome. In addition, certain vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors") which contain a nucleic acid sequence operably linked to expression control sequences and thereby capable of directing the expression of such sequences.

In certain embodiments, the expression construct is derived from a plasmid vector. Illustrative constructs include a modified pNASS vector (Clontech, Palo Alto, CA) having a nucleic acid sequence encoding an ampicillin resistance gene, a polyadenylation signal, and a T7 promoter site; pDEF38 and pNEF38(CMC ICOS Biotechnologies, Inc.)) having the CHEF1 promoter; and pD18 (Longza corporation (Lonza)) having a CMV promoter. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al, 1995; Sambrook et al, supra; see also, e.g., the catalogues of Invitrogen, San Diego, Calif., Novagen, Madison, Wis.), and Falmax corporation of Piscataway, N.J.). Useful constructs comprising the dihydrofolate reductase (DHFR) -coding sequence under appropriate regulation can be prepared to facilitate enhanced levels of production of the fusion protein, depending on gene amplification upon application of an appropriate selection agent (e.g., methotrexate).

Typically, recombinant expression vectors comprise an origin of replication and a selectable marker that allow transformation of the host cell, and a promoter derived from a highly expressed gene that directs transcription of downstream structural sequences, as described above. The vector to which the polynucleotide of the invention is operably linked produces a cloning or expression construct. Exemplary cloning/expression constructs contain at least one expression control element, such as a promoter, operably linked to a polynucleotide of the present invention. Other expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites, are also contemplated for use in the vectors and cloning/expression constructs of the present invention. The heterologous structural sequences of the polynucleotides of the invention are assembled in the appropriate state with translation initiation and termination sequences. Thus, for example, a fusion protein-encoding nucleic acid provided herein can be included in any expression vector construct to form a recombinant expression construct for expression of such a protein in a host cell.

The appropriate DNA sequence can be inserted into the vector by various methods. Typically, the DNA sequence is inserted into an appropriate restriction endonuclease cleavage site by methods known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, standard techniques for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases and the like, as well as various isolation techniques are contemplated. Many standard techniques can be found, for example, in Ausubel et al (Current Protocols in Molecular Biology, Inc., Greene publishing Co., Boston, Mass., and John Wiley & Sons, Inc., Boston, MA, 1993); sambrook et al (Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, Plainview, NY, 1989), Molecular Cloning, Inc.; maniatis et al (molecular cloning, Cold spring harbor laboratory Press, Prain Vee, N.Y., 1982); glover (Ed.) ("DNA Cloning" volumes I and II, IRL Press of Oxford, england, 1985); hames and Higgins (eds.) ("Nucleic Acid Hybridization", IRL Press of Oxford, UK, 1985); and so on.

The DNA sequence in the expression vector is operably linked to at least one suitable expression control sequence (e.g., a constitutive promoter or a regulatory promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or viruses thereof, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTR of retrovirus, and mouse metallothionein-I. Selection of suitable vectors and promoters is well known to those of ordinary skill in the art, and the preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulatory promoter operably linked to a nucleic acid encoding a protein or polypeptide of the invention is described herein.

Variants of the polynucleotides of the invention are also contemplated. A variant polynucleotide is at least 90% identical, preferably 95%, 99% or 99.9% identical to one of the defined sequence polynucleotides described herein, or is capable of hybridizing to one of the defined sequence polynucleotides under stringent hybridization conditions (0.015M sodium chloride, 0.0015M sodium citrate, about 65-68 ℃; or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, about 42 ℃). The polynucleotide variants should retain the ability to encode a binding domain or fusion protein thereof having the functions described herein.

The term "stringent" is used to refer to stringent conditions as are commonly understood in the art. Hybridization stringency is generally dependent on temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate, about 65-68 ℃; or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, about 42 deg.C (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

More stringent conditions (e.g., higher temperature, lower ionic strength, more formamide or other denaturant) may also be employed; however, the rate of hybridization can be affected. In the case of involving deoxyoligonucleotide hybridization, other exemplary stringent hybridization conditions include at 37 degrees C (for 14 base oligonucleotides), 48 degrees C (for 17 base oligonucleotides), 55 degrees C (for 20 base oligonucleotides) and 60 degrees C (for 23 base oligonucleotides), with 6x SSC, 0.05% sodium pyrophosphate washing.

In another aspect, the invention provides a host cell transformed, transfected or containing any of the polynucleotides or vectors/expression constructs of the invention. The polynucleotides or cloning/expression constructs of the invention are introduced into suitable cells using any method known in the art, including transformation, transfection and transduction. Host cells include cells of a subject receiving ex vivo cell therapy, including, for example, ex vivo gene therapy. Eukaryotic host cells contemplated as an aspect of the present invention, when carrying a polynucleotide, vector or protein of the present invention, include VERO properties in addition to the subject's own cells (e.g., of a human patient) because it increases proteoglycan degradation and decreases proteoglycan synthesis in articular cartilage (Hymowitz et al (2001) EMBO J.20: 5332-41).

IL17RA has been shown to play a role in a variety of inflammatory conditions, including arthritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis and asthma (Li et al (2004) Huazhong Univ. Sci. technology. Med. Sci.24: 294; Fujino et al (2003) Gut 52: 65; Kauffman et al (2004) J.invest. Dermatol.123: 1037. 1044; Mannon et al (2004) N.Engl. J.Med.351: 2069; Matusevicius et al (1999) Mult.Scler.5: 101; Linden et al (2000) Eur.Respir.J.15: 973; and Molergcly et al (2001) J.AlrgClin.Immunol.108: 430).

Associated TWEAK receptor, TWEAKR or fibroblast growth factor-inducing protein 14(Fn14) is a member of the TNF receptor superfamily expressed by non-lymphocyte types (Wiley et al (2001) Immunity 15: 837). TWEAK and TWEAKR are expressed relatively low in normal tissues, but are significantly upregulated in tissue injury and disease. The TWEAK/R pathway promotes acute tissue repair functions, thereby exerting physiological effects after acute injury, but playing a pathological role in chronic inflammatory diseases. Unlike TNF, TWEAK has no significant role in development and homeostasis. For a review of the TWEAK/R pathway, see Burkly et al (2007) Cytokine 40: 1. persistently activated TWEAK promotes chronic inflammation, pathological proliferation and angiogenesis and may hinder tissue repair by inhibiting progenitor cell differentiation. TWEAK proteins have been identified on the surface of activated monocytes and T cells, on tumor cell lines, and within the cells of resting and activated monocytes, dendritic cells, and NK cells. Expression of TWEAK is markedly elevated locally in acute injury, inflammatory diseases and cancer, all of which are associated with infiltration of inflammatory cells and/or activation of persistent innate immune cell types. Circulating TWEAK levels have been shown to be significantly elevated in patients with chronic inflammatory diseases such as multiple sclerosis and systemic lupus erythematosus.

Monoclonal antibodies that block TWEAK have been shown to be effective in a mouse collagen-induced arthritis (CIA) model (Kamata et al (2006) j.immunol.177: 6433; perer et al (2006) j.immunol.177: 2610). The arthritic activities of TWEAK and TNF on human synoviocytes are often additive or synergistic and appear to be independent of each other, suggesting that TWEAK and TNF may be parallel in rheumatoid arthritis pathology. It is speculated that the variability of the clinical response of RA patients to TNF inhibitors may reflect the pathological contribution of TWEAK. Cells, HeLa cells, Chinese Hamster Ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of the expressed multivalent binding molecule, see U.S. patent application publication No. 2003/0115614), COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, a549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda (e.g., Sf9 cells), saccharomyces cerevisiae cells, and any other eukaryotic cell known in the art that can be used to express and optionally isolate a protein or peptide of the invention. Prokaryotic cells are also contemplated, including E.coli (Escherichia coli), Bacillus subtilis, Salmonella typhimurium, Streptomyces, or any prokaryotic cell known in the art to be suitable for expressing and optionally isolating the proteins or peptides of the invention. In particular, in isolating proteins or peptides from prokaryotic cells, it is contemplated that techniques known in the art for extracting proteins from inclusion bodies may be employed. Those skilled in the art will be able to understand how to select an appropriate host based on the teachings herein. Host cells capable of glycosylating the fusion proteins of the invention are contemplated.

The term "recombinant host cell" (or simply "host cell") refers to a cell that contains a recombinant expression vector. It is understood that such terms refer not only to the particular cell, but to the progeny of such a cell. Certain changes may occur during serial passages due to mutation or environmental influences, and such progeny may not, in fact, be identical to the parent cell, but are still within the scope of the term "host cell" as used herein.

Recombinant host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying specific genes. One of ordinary skill in the art will readily understand the culture conditions, e.g., temperature, pH, etc., for selecting a particular host cell for expression. Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include, for example, Gluzman (1981) Cell 23: 175, and other cell lines capable of expressing a compatible vector, such as C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors comprise an origin of replication, a suitable promoter, and optionally an enhancer, and further comprise any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' -flanking nontranscribed sequences, such as described herein with respect to the preparation of multivalent binding protein expression constructs. DNA sequences derived from SV40 splicing and polyadenylation sites may be used to provide the required nontranscribed genetic elements. The constructs can be introduced into host cells by a variety of Methods familiar to those skilled in the art, including calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (Davis et al (1986) Methods based on Molecular Biology (Basic Methods in Molecular Biology)).

In one embodiment, a host cell is transduced with a recombinant viral construct that directs a protein or polypeptide of the invention. The transduced host cell is capable of producing a viral particle containing the expressed protein or polypeptide from a portion of the host cell membrane incorporated into the viral particle during viral budding.

Compositions and methods of use thereof

For the treatment of human and non-human mammals suffering from a disease state associated with TNF- α, IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, BLys/APRIL or IL10 dysregulation, a multispecific fusion protein of the invention is administered to the subject on a one-or multiple-dose schedule in an amount effective to ameliorate the symptoms of the disease state. As polypeptides, the multispecific fusion proteins of the present invention may be suspended or dissolved in a pharmaceutically acceptable diluent, which optionally comprises a stabilizer of other pharmaceutically acceptable excipients, such diluent being useful for intravenous injection or infusion administration, as detailed below.

A pharmaceutically effective dose is a dose that prevents, inhibits the occurrence of, or treats (alleviates to some extent, preferably all of) a disease state. The pharmaceutically effective dose will depend upon the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the particular subject contemplated for treatment, concurrent administration, and other factors recognized by those skilled in the medical arts. For example, 0.1mg/kg to 100mg/kg body weight of the active ingredient may be administered (either as a single dose, or as multiple doses administered hourly, daily, weekly, monthly, or in combination at appropriate intervals) depending on the potency of the binding domain polypeptide or multispecific protein fusion of the invention.

In certain aspects, the invention provides compositions of fusion proteins. The pharmaceutical compositions of the invention generally comprise one or more types of binding domains or fusion proteins, and a pharmaceutically acceptable carrier, excipient, or diluent. Such carriers should be non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutically acceptable carriers for therapeutic purposes are well known in the art of pharmacy, see, e.g., remington's pharmaceutical sciences (Mack Publishing Co.), a.r. gennaro, 1985. For example, sterile saline at physiological pH and phosphate buffered saline may be used. The pharmaceutical compositions may contain preservatives, stabilizers, dyes, and the like. For example, sodium benzoate, sorbic acid, or parabens can be added as preservatives, supra, 1449. In addition, antioxidants and suspending agents may also be employed, as above. The compounds of the present invention may be used in the form of the free base or as a salt, both of which fall within the scope of the present invention.

The pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, sugars (such as glucose, sucrose or dextrin), chelating agents (such as EDTA), glutathione, or other stabilizers or excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary suitable diluents. Preferably, the product is formulated as a lyophilizate using a suitable excipient solution as a diluent.

In certain embodiments, cis-signaling of IL6 is poorly inhibited or not inhibited, i.e., any inhibition of cis-signaling is not significant, meaning that the inhibition is absent, asymptomatic, or undetectable. The degree of inhibition of IL6 trans-signaling may vary, but in general the degree of alteration of trans-signaling has a positive effect on such disease conditions mediated or associated with signal transduction. In certain embodiments, inhibition of IL6 trans-signaling by a binding domain polypeptide or fusion protein thereof of the invention may block, stop, or reverse disease progression.

The compositions of the invention are useful for treating disease conditions mediated by TNF- α, IL6, RANKL, IL7, IL17A/F, TWEAK, CSF2, IGF1, IGF2, BLyS/APRIL, or IL10 signaling in humans and non-human mammals.

Increased IL-6 production and IL-6 signaling levels have been implicated in a variety of disease processes, including Alzheimer's disease, autoimmune diseases (e.g., rheumatoid arthritis, SLE), inflammation, myocardial infarction, Paget's disease, osteoporosis, solid tumors (e.g., colon, RCC prostate and bladder cancers), certain cancers of the nervous system, B-cell malignancies (e.g., Karman's disease, some lymphoma subtypes, chronic lymphocytic leukemia, particularly malignant melanoma). In some cases, IL-6 is also involved in the proliferative pathway because it interacts with other factors, such as heparin-binding epithelial growth factor and hepatocyte growth factor (see, e.g., Grant et al (2002) Oncogene 21: 460; Badache and Hynes (2001) Cancer Res.61: 383; Wang et al (2002) Oncogene 21: 2584). Similarly, the TNF superfamily is known to be involved in various diseases, such as cancer (tumorigenesis, including proliferation, migration, metastasis), autoimmune disease (SLE, diabetes), chronic heart failure, bone resorption or atherosclerosis, etc. (see, e.g., Aggarwal (2003) Nature Rev.3: 745; Lin et al (2008) Clin. Immunol.126: 13).

Mutations in the RANK gene that cause increased levels of RANK-mediated signal transduction can lead to increased osteoclastogenesis, resulting in increased osteolysis observed in some familial paget disease patients (see, e.g., Boyce and Xing, Arthritis Research and Therapy 2007; 9 suppl 1: S1). RANKL is thought to play a role in inducing tumor cell proliferation because it is expressed by certain malignant tumor cells and in psoriatic arthritis (see, e.g., Ritchlin et al (2003) j. clin. invest.111: 821; Mease (2006) Psoriasis Forum 12: 4). Treatment of postmenopausal women with low bone density with denosumab (denosumab), a monoclonal antibody that inhibits RANKL, increased bone mineral density and inhibited bone turnover markers. Similarly, denosumab has been used clinically to treat subjects with rheumatoid Arthritis (see, e.g., Cohen et al (2008) Arthritis Rheum.58: 1299) and osteolytic cancer (see, e.g., Lipton et al (2007) J.Clin. Oncol.25: 4431). Direct injection of OPG can reduce bone resorption (see, e.g., Morony et al (1993) J. bone Min. Res. 14: 1478).

The IL7 pathway is associated with bone diseases such as rheumatoid arthritis, multiple myeloma and periodontitis (Colucci et al (2007) j. pathol.212: 47). The IL7 pathway is also implicated in rheumatoid arthritis, as IL7 can be produced by inflamed synovial cells and induces the production of cell-contact dependent Th1 cytokines in a co-culture of synovial T cells and monocytes (van Roon et al, (2008) ann. IL7 promotes a number of pro-inflammatory responses including T cell activation, potentially suppressing regulatory T cell function in rheumatoid arthritis. IL7 also induces bone loss in vivo by triggering T cell production as well as the key osteoclastogenic cytokines RANKL and TNF α. In addition, IL7 leads to expansion of the OC precursor pool by inducing bone marrow B220+ cell proliferation (Toraldo et al (2003) Proc. nat' l Acad. Sci. (USA) 100: 125). The level and activity of IL7 in rheumatoid arthritis patients did not respond to anti-TNF α therapy, indicating that IL7 may be a good target for treatment of these patients (van Roon et al, 2008).

High levels of IL17A are associated with several chronic inflammatory diseases, including rheumatoid arthritis, psoriasis and multiple sclerosis. For example, increased levels of IL17 in synovial fluid of patients with Rheumatoid Arthritis (RA) have been reported, and it is believed that increased levels of IL17 play a role in the bone destruction characteristic of RA. IL17 also induces NO production in chondrocytes and human osteoarthritic cartilage explants (Attur et al (1997) Arthritis Rheum. 40: 1050). Moreover, agents that neutralize IL17A have been shown to significantly improve disease severity in several mouse human disease models.

IL17F has been implicated in the development of several autoimmune diseases, including arthritis (including rheumatoid arthritis and Lyme arthritis), Systemic Lupus Erythematosus (SLE), multiple sclerosis and asthma (Betteli and Kuchroo (2005) J.exp.Med.201: 169-71; Oda et al (2006) am.J.Resp.Crit.Care Med.2006, 1/15; Numasake et al (2004) Immunol.Lett.95: 97-104). Hymowitz et al showed that IL17F has unique properties among known inflammatory cytokines

U.S. patent 7,169,387 describes the preparation of a monoclonal antibody specific for TWEAK and its use in blocking the development of Graft Versus Host Disease (GVHD) in a mouse model of chronic GVHD. U.S. patent application publication No. 2007/0280940 describes TWEAKR trap receptors and antibodies to TWEAKR and TWEAK, as well as their use in treating central nervous system diseases associated with brain edema and cell death.

Several groups have demonstrated the presence of CSF2 and its receptor in synovial joints of arthritic patients (see, e.g., Alvaro-Gracia et al (1991) J.Immunol.146: 3365). Moreover, CSF2 can cause rheumatoid arthritic flushing when CSF2 is used to treat neutropenia in patients with Fisher's syndrome (Hazenberg et al (1989) Blood 74: 2769) or post-chemotherapy (de Vries et al (1991) Lancet 338: 517). In multiple sclerosis, elevated levels of CSF2 are associated with the active phase of the disease (Carrieri et al (1998) immunopharmacol. immunotoxin.20: 373; McQualter et al (2001) J.exp. Med.194: 873). Elevated lung CSF2 levels, as well as eosinophil levels, were found in asthmatic patients (Broide and Firestin (1991) J.Clin.invest.88: 1048).

IGF1R was identified in the treatment of cancer, including sarcomas (Scotlandi and Picci (2008) curr. Opin. Oncol.20: 419-27; Yuen and Macaulay (2008) Expert Opin. the. Targets 12: 589-603).

Elevated levels of BLyS/APRIL have been found in patients with autoimmune diseases such as Systemic Lupus Erythematosus (SLE), rheumatoid Arthritis and Sjogren's syndrome, and elevated levels of BLyS have been associated with increased disease severity (chema et al (2001) Arthritis Rheum.44: 1313; Groom et al (2002) J.Clin.Invest.109: 59; Zhang et al (2001) J.Immunol.166: 6). In addition, APRIL, BLyS and TACI, as well as BCMA, produce strong survival and growth-inducing signals in vitro on malignant cells taken from Hodgkin's Lymphoma (HL) tumor tissue, suggesting that these proteins may have some role in the treatment of HL and other forms of cancer (Chiu et al (2007) Blood 109: 729).

IL10 is known to have immunosuppressive properties (Commins et al (2008) J. allergy Clin. Immunol.121: 1108-11; Ming et al (2008) Immunity 28: 468-476), and positive responses were observed following administration of IL10 to patients with psoriasis (Asadullah et al (1999) Arch. Dermatol.135: 187-92) and inflammatory bowel disease (Schreiber et al (2000) Gastroenterology 119: 1461-72).

Medicaments comprising a binding domain of the invention are useful for the treatment of autoimmune diseases and other diseases, including Alzheimer's disease, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis, Chronic Obstructive Pulmonary Disease (COPD), Crohn's disease, ulcerative colitis, severe refractory asthma, TNFRSF 1A-associated periodic syndrome (TRAPS), endometriosis, Systemic Lupus Erythematosus (SLE), Inflammatory Bowel Disease (IBD), Sjogren's syndrome, multiple sclerosis, Graves' disease, severe refractory asthma, Hashimoto's disease, Castleman's disease, central nervous system inflammation, stroke, cerebral edema, graft rejection, graft-versus-host disease (GVHD), acute and chronic inflammation, atopic dermatitis, shock, enteropathic arthritis, active arthritis, inflammatory bowel disease, rett's syndrome, SEA syndrome (seronegative (sero-tendinity), tendinopathy (enthsopathiy), Arthropathy (artropathiy) syndrome), dermatomyositis, scleroderma, vasculitis, myositis, osteoarthritis, sarcoidosis, sclerosis, dermatitis, atopic dermatitis, lupus, stigmatosis, myasthenia gravis, celiac disease, Guillain-Barre disease (Guillain-Barre disease), type I diabetes, Idison's disease, Paget's disease, degenerative joint disease, osteoporosis, and other diseases, including loss of bone mass and cancer, including hormone-independent prostate cancer, osteolytic cancer, multiple myeloma, B cell proliferative disorders such as B cell non-Hodgkin's lymphoma, and advanced cancers of the kidney, breast, colon, lung, brain, and other tissues.

It is also contemplated that the multispecific fusion protein compositions of the present invention are administered in combination with a second agent. The second agent may be one accepted in the art as a standard treatment for a particular disease state (e.g., inflammation, autoimmune disease, and cancer). Examples of contemplated second agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, chemotherapeutics, radiation therapy or other active and auxiliary agents, or any combination thereof.

"pharmaceutically acceptable salt" refers to a salt of a binding domain polypeptide or fusion protein of the invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: (1) an acid addition salt formed with: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when the acidic proton present in the parent compound is replaced with a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or a complex compound with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

In a specific exemplary embodiment, a polypeptide or fusion protein of the invention is administered intravenously, e.g., by bolus injection or infusion. In addition to intravenous administration, routes of administration include oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal, intranasal, and perispinal routes. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernosal, intrathecal, intracanal, intraurethral injection or infusion techniques. The pharmaceutical composition is formulated such that the active ingredients contained therein are bioavailable after administration of the composition to a patient. The composition to be administered to the patient may be in the form of one or more dosage units, for example, a tablet may be a single dosage unit, and a plurality of dosage units may be contained in a container of one or more compounds of the invention in aerosol form.

In oral administration, excipients and/or binders such as sucrose, kaolin, glycerol, starch dextran, cyclodextrin, sodium alginate, ethyl cellulose and carboxymethyl cellulose may be present. Optionally, sweeteners, preservatives, dyes/colorants, flavor enhancers, or any combination thereof may be present. Optionally, a coating shell may also be used.

In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizing agents, isotonic agents, or any combination thereof may optionally be included.

For nucleic acid-based formulations or formulations comprising the expression products of the invention, dosages of from about 0.01. mu.g/kg to about 100mg/kg body weight are administered, for example, by intradermal, subcutaneous, intramuscular or intravenous routes, or any route known in the art to be suitable for use in the given situation. For example, a preferred dose is from about 1. mu.g/kg to 20mg/kg, particularly preferably from about 5. mu.g/kg to 10 mg/kg. It will be apparent to those skilled in the art that the number and frequency of administration will depend on the host response.

The pharmaceutical compositions of the invention may be in any form that can be administered to a patient, for example, in solid, liquid or gaseous (aerosol) form. The compositions may be in liquid form for administration by any of the routes described herein, such as elixirs, syrups, solutions, emulsions or suspensions.

As used herein, a liquid pharmaceutical composition, whether in solution, suspension or other similar form, may comprise one or more of the following components: sterile diluents such as water for injection, saline solutions (e.g., physiological saline), ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono-or diglycerides (which may be used as a solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetate, citrate or phosphate; chelating agents such as ethylenediaminetetraacetic acid; and tonicity adjusting substances such as sodium chloride or dextrose. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred additive. The injectable pharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the formulation, such as delivery vehicles, including aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of adjuvants used in such carriers include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), Lipopolysaccharide (LPS), dextran, IL-12, GM-CSF, interferon gamma and IL-15.

Although any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier may vary depending on the mode of administration and whether sustained release is desired. For parenteral administration, the carrier may comprise water, saline, alcohol, fat, wax, buffer, or any combination thereof. For oral administration, any of the above carriers or solid carriers can be employed, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, or any combination thereof.

The present invention contemplates dosage units comprising the pharmaceutical compositions of the present invention. Such dosage units include, for example, single or multi-dose vials or syringes, including two-compartment vials or syringes, one containing the pharmaceutical composition of the invention in lyophilized form and the other containing a diluent for reconstitution. A multi-dose dosage unit may also be, for example, a (infusion) bag or tube for connection to an intravenous infusion set.

The invention also contemplates kits comprising a pharmaceutical composition of the invention in a unit-dose or multi-dose container, such as a vial, and a set of instructions for administering the composition to a patient suffering from a condition described herein.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, tables, sequences, web pages, and the like, referred to in this specification, are incorporated herein by reference, in their entirety. The following examples are intended to illustrate, but not limit, the present invention.

Examples

Cross receptor (Xceptor) sequences

The amino acid sequence of an exemplary multi-specific fusion protein with the extracellular domain of TNFRSF1B and an anti-IL 6xR binding domain is set forth in SEQ ID NO: 607-668, the corresponding nucleotide expression cassettes are respectively shown in SEQ ID NO: 673-734 (note that the mature protein lacks the signal peptide sequence shown in SEQ ID NO: 607-668). A multispecific fusion protein having TNFRSF1B extracellular domain at the amino terminus and an anti-IL 6xR binding domain at the carboxy terminus is referred to herein as TRU (XT6) -1001-TRU (XT6) -1062. Oppositely oriented fusion proteins-i.e., having an anti-IL 6xR binding domain at the amino terminus and TNFRSF1B extracellular domain at the carboxy terminus-are referred to herein as TRU (X6T) -1008 and TRU (X6T) -1019.

The amino acid sequence of an exemplary multi-specific fusion protein having the extracellular domain of TNFRSF1B and the TWEAK, RANKL, IGF1, IL7, IL17, or IGF antagonist binding domain is set forth in SEQ ID NO: 798-804, the corresponding nucleotide expression cassettes are respectively shown in SEQ ID NO: 80-811.

Essentially according to Hoet et al (2005) Nature Biotechnol.23: 344, a Fab binding domain phage library was screened for binding domains specific for IL6xR complex. Cloning of this binding domain by PCR amplification-briefly, G was generated by PCR SuperMix (Invitrogen, San Diego, Calif.) and by overlap₄Suitable primers for the S-linker were initially annealed at 56 ℃ for 9 cycles, and then at 62 ℃The temperature was run for 20 cycles to amplify the VL and VH regions from the Fab library clones. The PCR products were separated on an agarose gel and purified using Qiagen (Qiagen) (Chatsworth, CA) PCR purification columns. The second round of the stitching reaction involved mixing equimolar VL and VH products with amplification buffer and water, denaturing at 95 ℃ for 5 seconds, and then slowly cooling to room temperature. For amplification, dNTP mix and amplification enzyme were added and incubated at 72 ℃ for 10 seconds. After addition of the outer primers (5 'VH and 3' VL), the mixture was treated 35 times with an annealing temperature of 62 ℃ and an extension reaction cycle of 45 min. The resulting 750 base pair product was gel purified and digested with EcoRI and NotI and then cloned into plasmid pD28 (see U.S. patent application publication No. 2005/0136049 and PCT application publication No. WO2007/146968 for details). Binding activity was detected by ELISA as described by Hoet et al (2005).

The activity of various SMIPs and the cross-receptor fusion proteins described herein were tested as described below. Abbreviations used in the following examples include the following terms: PBS-T: PBS, pH7.2-7.4 and 0.1% Tween20; working buffer solution: PBS-T containing 1% BSA; blocking buffer: PBS-T containing 3% BSA.

Example 1

Expression of Cross-receptors

Using FreeStyle^TM293 expression System (Invitrogen, Carlsbad, Calif.) certain cross-receptor fusion proteins described herein were expressed in 293 cells according to the manufacturer's instructions.

28ml FreeStyle per 30ml transfection system was used^TM293 expression Medium 3x10 contained⁷And (4) cells. On the day of transfection, aliquots of the cell suspension were transferred to microcentrifuge tubes and the viability and cell mass of the cell pellet were determined by trypan blue exclusion. The suspension was vortexed vigorously for 45 seconds to disperse the cell pellet, using a Coulter CounterAnd a hemocytometer to determine the total number of cells. The viability of the cells was over 90%. The shake flask containing the desired cells was placed in a 37 ℃ incubator on an orbital shaker.

For each transfection sample, lipid-DNA complexes were prepared as described below. Using Opti-MEMI dilution of 30. mu.g plasmid DNA to a total volume of 1ml, gentle mixing. Using Opti-MEMI dilution of 60. mu.l 293fectin^TMTo a total volume of 1ml, gently mixed and incubated at room temperature for 5 minutes. After 5 minutes incubation, diluted DNA was added to the diluted 293fectin^TMIn the total volume of 2ml, mix gently. The resulting solution was incubated at room temperature for 20-30 minutes to form DNA-293fectin^TMAnd (c) a complex.

In incubation of DNA-293fectin^TMFor complexes, the cell suspension was removed from the incubator and an appropriate volume of cell suspension was placed into a sterile, disposable 125ml conical shake flask. For the 30ml transfection system, fresh, preheated FreeStyle was added^TM293 expression medium to a total volume of 28 ml.

DNA-293fectin^TMAfter incubation of the complexes was complete, 2ml of DNA-293fectin was added^TMThe complex was added to a shake flask. 2ml of Opti-MEMI, but not DNA-293fectin^TMThe complex was added to a negative control vial. The total volume contained in each flask was 30ml and the final cell density was about 1X10⁶Viable cells/ml. In 8% CO on an orbital shaker₂The cells were incubated in a 37 ℃ incubator under a humid atmosphere with a shaker rotation speed of 125 rpm. Cells were harvested approximately 7 days after transfection and expression of recombinant protein was determined.

As described above, the cross-receptor molecule having TNFRSF1B extracellular domain and IL6/HIL6 binding domain, TWEAKR extracellular domain, OPG extracellular domain, IL7R extracellular domain, IL17R extracellular domain or TGF β RII extracellular domain was expressed in 293 cells.

Example 2

Determination of Cross-receptor binding to IL6 and SuperIL 6 by ELISA

The binding activity of super-IL 6(HIL6 or IL6xR), recombinant human IL6(rhIL6) and human soluble IL6R to the cross-receptors TRU (XT6) -1002, 1019, 1025, 1042, 1058 and TRU (X6T) -1019(SEQ ID NOs: 608, 625, 631, 648, 664 and 670, respectively) was tested essentially as follows.

HIL6 and IL6 binding

Mu.l of goat anti-human IgG-Fc (Jackson ImmunoResearch, West Grove, Pa.) antibody was PBS, 2. mu.g/ml solution prepared at pH7.2-7.4, was added to each well of the 96-well plate. The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l blocking buffer (PBS-T containing 3% BSA or 10% normal goat serum) was added to each well, the plate was covered and incubated at room temperature for 2 hours (or overnight at 4 ℃). After washing the plates three times with PBS-T, 100. mu.l/well of cross-receptor TNFRSF1B serially diluted three times from 300ng/ml with working buffer, anti-HIL 6 sample and human gp130-Fc chimera (R of Minneapolis, Minn.) were added in duplicate to anti-human IgG-Fc coated plates&D systems Co Ltd (R)&D Systems, Minneapolis, MN)), the plates were capped and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 150pM of human super IL-6 or recombinant human IL-6 solution in working buffer was added in duplicate, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 150ng/ml anti-human IL-6-biotin (R) in working buffer at 100. mu.l/well was added&System D) solution, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase couple diluted 1: 4,000 in working buffer was addedLinked streptavidin (sterase, San Francisco, Calif.) was applied to the plates by capping and incubating at room temperature for about 30 minutes. After washing the plate six times with PBS-T, 100. mu.l/well of 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (Pierce, Rockford, IL)) was added for about 3-5 minutes, followed by 50. mu.l of stop buffer (1N H) per well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

sIL6R binding

Mu.l of goat anti-human IgG-Fc (ICN Pharmaceuticals, Costa Mesa, Calif.) was added to each well of the 96-well plate as a 2. mu.g/ml solution of PBS at pH 7.2-7.4. The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l blocking buffer (PBS-T containing 3% BSA or 10% normal goat serum) was added to each well, the plate was covered and incubated at room temperature for 2 hours (or overnight at 4 ℃). After washing the plates three times with PBS-T, 100. mu.l/well of cross-receptor TNFRSF1B serially diluted three times from 300ng/ml with working buffer, anti-HIL 6 sample, positive control anti-human IL-6R (R of Minneapolis, Minn.) were added in duplicate to anti-human IgG-Fc coated plates&System D) and negative control human IgG or human gp130-Fc chimera (R)&D systems Co.), the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 75pM recombinant human sIL-6R (R) in working buffer was added in duplicate&System D) solution, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 100ng/ml anti-human IL-6R-biotin (R) in working buffer was added&D systems Co.), the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase-coupled streptavidin (sterase, san Francisco, Calif.) diluted 1: 4,000 in working buffer was added, the plate was covered and incubated at room temperature for about 30 minutes. After washing the plate six times with PBS-T, 100. mu.l/well of 3 was added,3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (pierce, rockford, Ill.) for about 3-5 minutes, then 50. mu.l stop buffer (1N H) was added to each well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

The data in FIGS. 1A and 1B show that all cross-receptor fusion proteins, whether the TNFRSF1B ectodomain is located at the amino terminus or the carboxy terminus of the fusion protein molecule, bind to HIL 6. Moreover, these experiments demonstrated that the cross-receptor protein was specific for the IL6xR complex, as only two cross-receptors bound rhIL6 (fig. 1B) and none bound sIL6R (fig. 1C). In a related study, it was found that the cross receptors TRU (XT6) -1002 and SMIP TRU (S6) -1002 cross-react with IL6 from the non-human primate cynomolgus monkey (Mucaca mulatta).

Example 3

Determination of Cross-receptor binding to TNF-alpha by ELISA

TNF- α was tested for binding activity to the cross receptors TRU (XT6) -1002, 1042, 1058, 1019 and TRU (X6T) -1019(SEQ ID NOs: 608, 648, 664, 625 and 670, respectively) essentially as follows.

Mu.l of goat anti-human IgG-Fc (ICN pharmaceuticals, Inc. of Keslameisa, Calif.) was added to each well of the 96-well plate as a 2. mu.g/ml solution of the antibody in PBS at pH 7.2-7.4. The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l of blocking buffer was added to each well, the plate was capped and incubated at room temperature for 2 hours (or overnight at 4 ℃). After washing the plates three times with PBS-T, 100. mu.l/well of cross-receptor TNFRSF1B serially diluted three times from 300ng/ml with working buffer, an anti-HIL 6 sample, a positive control Enbrel, were added in duplicate to anti-human IgG-Fc coated plates(Eitacept) and recombinant human TNFR2(TNFRSF1B) -Fc chimera (R of Minneapolis, Minnesota)&System D), and a negative control human IgG or human gp130-Fc chimera(R&D systems Co.), the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of recombinant human TNF-. alpha.at 2ng/ml in working buffer (R) was added in duplicate&System D) solution, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 200ng/ml anti-human TNF-. alpha. -biotin (R) in working buffer was added&System D) solution, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase-coupled streptavidin (Jackson immuno research, Wisku, Pa.) diluted 1: 1,000 in running buffer was added, the plate was covered and incubated at room temperature for about 30 minutes. After washing the plate six times with PBS-T, 100. mu.l/well of 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (pierce, rockford, Ill.) was added for about 3-5 minutes, followed by 50. mu.l of stop buffer (1N H) per well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

The data in FIG. 2 indicate that all of the cross-receptor fusion proteins tested were able to bind TNF- α, regardless of whether the extracellular domain of TNFRSF1B was located at the amino terminus or the carboxy terminus of the fusion protein.

Example 4

Detection of Cross-receptor Dual ligand binding by ELISA

The simultaneous binding of the fusion protein TRU (XT6) -1006(SEQ ID NO: 612) to the TNF-. alpha.and IL6xR complexes was detected essentially as follows.

Mu.l of human HIL-6 solution (5. mu.g/ml PBS solution, pH7.2-7.4) was added to each well of the 96-well plate. The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l of blocking buffer was added to each well, the plate was capped and incubated at room temperature for 2 hours (or overnight at 4 ℃). After washing the plates three times with PBS-T, 100. mu.l/well of cross-acceptor T serially diluted three times from 300ng/ml with working buffer was added in duplicate to the HIL-6 coated platesNFRSF1B sample HIL 6. The negative control contained only human gp130-Fc chimera (R of Minneapolis, Minnesota)&D systems corporation), Enbrel(etanercept) and working buffer only. The plate was covered and incubated at room temperature for 1.5 hours. After washing the plate five times with PBS-T, 100 microliters/well of recombinant human TNF- α (R of Minneapolis, Minn.) at 2ng/ml in working buffer was added in duplicate&D systems), the plate was covered and incubated at room temperature for about 1.5 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 200ng/ml of anti-human TNF-. alpha. -biotin (R) in working buffer was added in duplicate&D systems), the plate was covered and incubated at room temperature for about 1.5 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase-coupled streptavidin (Jackson immuno research, Wisku, Pa.) diluted 1: 1,000 in running buffer was added, the plate was covered and incubated at room temperature for about 30 minutes. After washing the plate six times with PBS-T, 100. mu.l/well of 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (pierce, rockford, Ill.) was added for 3-5 minutes, followed by 50. mu.l of stop buffer (1N H) per well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

The data in FIG. 3 show that the cross-receptor protein can bind both ligands (in this case TNF-. alpha.and super IL6) simultaneously.

Example 5

Detection of Cross-receptor blocking of binding of SuperIL 6 to GP130 by ELISA

Cross-receptor fusion proteins TRU (XT6) -1004, 1006, 1007, 1008, 1013 and 1019(SEQ ID NOS: 610, 612, 613, 614, 619 and 625) were tested for blocking the binding of super IL6(IL6xR) to the soluble gp130 receptor essentially as follows.

To each well of a 96-well plate, 100. mu.l of 0.25-0.5. mu.g/ml human gp130-Fc insert was addedCombination (R of Minneapolis, Minn.) of Minn Sudada&System D) PBS solution (pH 7.2-7.4). The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l blocking buffer (PBS-T containing 3% BSA or 10% normal goat serum) was added to each well, the plate was covered and incubated at room temperature for 2 hours (or overnight at 4 ℃). Five-fold serial dilutions were made with working buffer starting from 50 μ g/ml for the following samples: cross-receptor TNFRSF1B anti-HIL 6 sample, positive control human gp130-Fc chimera (R)&D systems Co.) and anti-human IL-6R (R)&D systems Co.) and negative control anti-human IL-6 (R)&D systems Co.), human IgG or Enbrel(etanercept). Equal volumes of serially diluted cross-receptor samples were mixed with super IL-6 (final super IL-6 concentration of 2.5ng/ml) and incubated for 1 hour at room temperature. After washing the plates three times with PBS-T, 100. mu.l/well of serially diluted Cross-receptor/HIL 6 mixture, human gp130-Fc chimera, anti-human IL-6R, anti-human IL-6, human IgG and Enbrel were added in duplicate to the human IgG-Fc coated plates(etanercept), the plate was covered with a lid and incubated at room temperature for about 1.5 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase-conjugated anti-mouse IgG-Fc (Pierce, Rockford, Ill.) diluted 1: 10,000 with working buffer was added, the plate was covered and incubated at room temperature for about 1 hour. After washing the plate six times with PBS-T, 100. mu.l/well of 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (Pierss) was added for about 5-15 minutes, and then 50. mu.l of stop buffer (1N H) was added per well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

The data in FIG. 4 show that cross-receptor proteins comprising the anti-IL 6xR binding domain block the binding of soluble gp130 to HIL 6.

Example 6

Cross-receptor blockade of IL6 and SuperIL 6 induced cell proliferation

Cross-receptor fusion proteins TRU (XT6) -1011, 1014, 1025, 1026, 1002 and TRU (X6T) -1019(SEQ ID NOs: 617, 620, 631, 632, 608 and 670) were tested for blocking IL6 or super IL6(IL6xR) induced TF-1 cell proliferation essentially as follows.

The day before the proliferation assay, 0.3X10 was added to each well of a 96-well flat bottom plate⁶Fresh growth medium (10% FBS-RPMI 1640; 2mM L-glutamine; 100 units/ml penicillin; 100. mu.g/ml streptomycin; 10mM HEPES; 1mM sodium pyruvate; and 2ng/ml Hu GM-CSF) for TF-1 cells (human erythroleukemia cells). Cells were then harvested and washed twice with assay medium (no GM-CSF, no cytokines, the remainder being the same as the growth medium) and then at 1X10⁵The density of individual cells/ml was resuspended in assay medium. To block IL-6 activity, serial dilutions of TNFSFR1B of interest anti-HIL-6 cross-receptor or antibody were combined with a fixed concentration of recombinant human IL-6(rhIL-6) (R of Minneapolis, Minnesota) in a 96-well plate&D systems Co.) or super IL-6(HIL-6) 5% CO at 37 deg.C₂Preincubation was performed for 1 hour under the conditions. The controls used included human IgG; human gp130-Fc chimera (R)&System D corporation); anti-hIL-6 antibody (R)&System D corporation); and anti-hIL-6R antibody (R)&D systems corporation). After pre-incubation, 1 × 10 was added to each well⁴1 cell (100. mu.l). The final assay mixture (total volume 200. mu.l/well, containing TNFSFR1B:: HIL-6, rhIL-6 or HIL-6 and cells) was incubated at 37 ℃ with 5% CO₂And incubated under the conditions of (1) for 72 hours. Adding into the final 4-6 hr of culture³H-thymidine (20. mu. Ci/ml solution in assay medium, 25. mu.l/well). Cells were collected on UniFilter-96GF/c plates and incorporation was determined using a TopCount reader (Packer)³H-thymidine. Data are expressed as cpm mean ± SD of triplicate wells. Percent block was 100- (test cpm-control cpm/max cpm-control cpm) 100.

The data in FIGS. 5A and 5B show that all cross-receptor proteins, whether TNFRSF1B ectodomain is located at the amino-or carboxy-terminus of the fusion protein molecule, block cell proliferation induced by IL6 or super IL6 alone or simultaneously.

Example 7

Cross-receptor blocking of TNF-alpha binding to TNFR by ELISA assay

Cross-receptor fusion proteins TRU (XT6) -1004, 1006, 1007, 1008, 1013, and 1019(SEQ ID NOS: 610, 612, 613, 614, 619, and 625, respectively) were tested for blocking TNF- α binding to TNF receptors essentially as described below.

To each well of a 96-well plate 100. mu.l of 0.25-0.5. mu.g/ml recombinant human TNFR2-Fc chimera (R of Minneapolis, Minn.) was added&System D) PBS solution (pH 7.2-7.4). The plate was covered and incubated at 4 ℃ overnight. After four washes with PBS-T, 250. mu.l blocking buffer (PBS-T containing 3% BSA or 10% normal goat serum) was added to each well, the plate was covered and incubated at room temperature for 2 hours (or overnight at 4 ℃). Five-fold serial dilutions were performed with working buffer from 50-250 μ M on the following samples: cross receptor TNFRSF1B anti-HIL 6 sample, positive control Enbrel(etanercept) and anti-TNF-alpha (R)&System D) and positive control human gp130-Fc chimera (R)&System D) and human IgG. Equal volumes of serially diluted cross-receptor samples were mixed with TNF α (final concentration of TNF α was 2.5ng/ml) and incubated for 1 hour at room temperature. After washing the plates three times with PBS-T, 100. mu.l/well of serially diluted cross-receptor/TNF α mixture, Enbrel, were added in duplicate to recombinant human TNFR2-Fc coated plates(etanercept), anti-TNF α, human gp130-Fc chimera and human IgG, the plates were capped and incubated at room temperature for about 1.5 hours. After washing the plate five times with PBS-T, 100. mu.l/well of 200ng/ml anti-human TNF-. alpha. -biotin (R) in working buffer was added&System D) solution, the plate was covered and incubated at room temperature for about 1-2 hours. After washing the plate five times with PBS-T, 100. mu.l/well of horseradish peroxidase-coupled streptavidin (Jackson immuno research, Wisku, Pa.) diluted 1: 1,000 in running buffer was added, the plate was covered and incubated at room temperature for about 30 minutes. After washing the plate six times with PBS-T, 100. mu.l/well of 3, 3, 5, 5-Tetramethylbenzidine (TMB) substrate solution (pierce, rockford, Ill.) was added for about 3-5 minutes, followed by 50. mu.l of stop buffer (1N H) per well₂SO₄) To terminate the reaction. The absorbance of each well was read at 450 nm.

The data in FIG. 6 show that the cross-receptor protein blocks TNF- α binding to the TNF receptor, which is approximately the same as the blocking effect of TNFR-Fc.

Example 8

Cross-receptor blockade of TNF-alpha induced cell killing

Cross-receptor fusion proteins TRU (XT6) -1011, 1014, 1025, 1026, 1002 and TRU (X6T) -1019(SEQ ID NOS: 617, 620, 631, 632, 608 and 670, respectively) were tested for blocking TNF-. alpha.induced killing of L929 cells essentially as follows.

Preparation of a Density of 2X 10 from Medium (10% FBS-RPMI 1640; 2mM L-glutamine; 100 units/ml penicillin; 100. mu.g/ml streptomycin; and 10mM HEPES)⁵Individual cells/ml suspension of L929 mouse fibroblast cells (ATCC, Manassas, Va.) then 100. mu.l of suspension was added to each well of a flat bottom black 96-well plate at 37 ℃ with 5% CO₂Incubated overnight in a humidified incubator. Cross-receptor TNFRSF1B serially diluted in assay medium (same composition as culture medium but supplemented with 2% FBS). anti-HIL 6 samples were incubated with equal volumes of recombinant human TNF- α (rhTNF α; R of Minneapolis, Minn.Tsuda)&D systems Co.) at 37 deg.C with 5% CO₂Incubated in a humidified incubator for 1 hour. Positive control (i.e., blocking TNF. alpha. induction)Led L929 cell killing agents) include Enbrel(etanercept), rhTNFR2-Fc chimera (R of Minneapolis, Minnesota)&System D) and anti-TNF α antibodies (R of minneapolis, minnesota&D systems corporation). Negative controls included assay medium alone (no TNF-. alpha.added) and antibody hIgG (TNF. alpha.added). To analyze TNF α activity, the culture medium of L929 cells was removed and then 50 μ L TNF α/cross receptor or control mixture and 50 μ L actinomycin D (Sigma Aldrich, st. louis, MO) were added per well (4 μ g/ml freshly prepared working solution). Then, 5% CO at 37 ℃₂The cells were incubated in a humidified incubator of (1) for 24 hours. To determine cell viability, 100 μ l of ATPlite 1-step reagent (PerkinElmer, Waltham, MA) was added per well according to the manufacturer's instructions, shaken for 2 minutes, and then luminescence was measured with a TopCount reader (packer).

The data in FIG. 7 show that all cross-receptor proteins in this assay, whether the TNFRSF1B ectodomain is located at the amino terminus or the carboxy terminus of the fusion protein molecule, blocked the TNF- α -induced cell killing effect.

Example 9

Detection of Cross-receptor binding to ligand by ELISA

Cross-receptor molecules comprising the TNFRSF1B ectodomain and the TWEAKR ectodomain (SEQ ID NO: 798), the OPG ectodomain (SEQ ID NO: 799), the TGF β RII ectodomain or the IL7R ectodomain (SEQ ID NO: 801) were tested for the ability to bind to the ligand TWEAK, RANKL, TGF β or IL7, respectively, essentially as described below.

Mouse and human ligands (R & D systems, Minnesota) at a concentration of 1 μ g/ml in PBS were added to wells of a 96 well plate (100 μ l/well). The plates were incubated at 4 ℃ overnight. After five washes with PBS-T, 250. mu.l blocking buffer (PBS-T containing 3% BSA) was added to each well, the plate was capped and incubated for 2 hours at Room Temperature (RT). Cross-receptors were prepared in triplicate dilutions from 300ng/ml in working buffer (PBS-T with 1% BSA). Irrelevant cross-receptors were used as negative controls. The plates were incubated at room temperature for 1 hour. After five washes with PBS-T, 100. mu.l/well of HRP-conjugated anti-human IgG-Fc (diluted 1: 5000 with working buffer) was added, the plates were capped, and incubated for 1 hour at room temperature. After five washes with PBS-T, 100 microliters of Quant-Blu substrate (Pierce, Rockford, Ill.) was added to each well. The plate was incubated at room temperature for 10-30 minutes and fluorescence was measured at 325/420 nm.

The results are shown in Table 3 below. Tnfrxgfbetarii was not tested for binding to mouse TGF β, however it was noted that mouse and human TGF β were 99% identical.

Table 3.

ND is not detected

Example 10

Cross-receptor blockade of TWEAK-induced cell killing

Blockade of the TWEAK-induced killing of HT29 cells by a cross-receptor comprising the extracellular domain of TNFRSF1B and the extracellular domain of TWEAKR (SEQ ID NO: 798) was detected using the method described by Nakayama et al (J.Immunol.168: 734, 2002).

Briefly, in a 96 well flat bottom plate, a plate containing 200ng/ml human TWEAK (R of Minneapolis, Minn.) was used&System D) medium (RPMI with 10% FCS and 1mM sodium pyruvate) 100 μ l per well, 5% CO at 37 ℃₂Incubated in a humidified incubator for 1.5 hours. Negative controls included irrelevant cross-receptor protein (TWEAK added) and assay medium alone (with and without TWEAK added). After incubation, each time100ul of a 40ng/ml solution of human IFN-. gamma.containing the protein (R of Minneapolis, Minn.) was added to the wells&System D) medium containing 5x10³HT29 cells (ATCC, Marnasas, Va.). Then 5% CO at 37 ℃₂The plates were incubated in a humidified incubator for 96 hours. To analyze TWEAK activity by measuring cell viability, 100 μ L of medium was removed from HT29 cells, and 10 μ L of LWST-8 reagent (Dojindo Molecular Technologies, Rockville, MD) was added to each well. At 37 deg.C, 5% CO₂The plate was incubated for 2 hours and the absorbance of each well was read at 450 nm.

FIG. 8 is a graph showing that a cross-receptor fusion protein containing the extracellular domain of human TWEAK receptor blocks TWEAK-induced cell killing in this assay.

Example 11

Cross-receptor blockade of RANKL-mediated osteoclastogenesis

The cross-receptor comprising the extracellular domain of TNFRSF1B and the extracellular domain of OPG (SEQ ID NO: 799) was tested for blocking RANKL-mediated osteoclastogenesis in RAW246.7 cells using the method described by Lee et al (J.biol.chem.280 (33): 29929, 2005).

Briefly, a 96-well flat bottom plate was prepared with a plate containing 30ng/ml mRANKL (R of Minneapolis, Minn.)&System D) medium (DMEM with 10% FCS) was serially diluted with cross-receptors (50 μ l/well). At 37 deg.C, 5% CO₂The plates were incubated in a humidified incubator of (1.5) hours. After incubation, 5 × 10 in each well was added³50 microliters of culture medium of RAW246.7 cells (ATCC of Marnasas, Va.). At 37 deg.C, 5% CO₂The plates were incubated in a humidified incubator for 6 days. Negative controls included irrelevant cross-receptor protein (with RANKL) and medium alone (with or without RANKL).

After 6 days, osteoclast-produced tartrate-resistant acid phosphatase (TRAP) activity was measured by ELISA (IDS, Fountain HillsAZ) of aromatic hill, arizona. Briefly, 25ul of each well was removed and added to a prepared microtiter plate coated with anti-mouse TRAP antibody. Add 75ul of 0.9% NaCl to each well, followed by 25. mu.L of release agent. The kit contains ELISA positive controls for different amounts of recombinant mouse TRAP and protein. The plates were incubated at room temperature for 1 hour. After washing the plate three times with PBS-T, 100ul of pNPP substrate solution was added to all wells and the plate was incubated at 37 ℃ for 2 hours. The reaction in each well was stopped with 25. mu.L of 0.32M NaOH and the absorbance read at 405 nm.

The data in figure 9 show that cross-receptor fusion proteins containing human OPG block osteoclastogenesis as measured by TRAP activity in RANKL treated RAW246.7 cells.

Example 12

By BIACOREDetermination of binding affinity to TNF alpha

As described below, using BiacoreT100 device (GE Healthcare, Piscataway, NJ) for determination of Cross-receptor fusion proteins TRU (XT6) -1002(SEQ ID NO: 608) and EnbrelThe ability to bind TNF α.

TNF α conjugates were captured with a monoclonal mouse anti-human Fc, which was covalently coupled to a carboxymethyldextran surface via an amine with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride and N-hydroxysuccinimide (CM 4). Unoccupied sites on the activated surface are blocked by ethanolamine. The capture antibody (called anti-hFc) is able to bind C of IgG Fc of all subclasses_H2 domain and no dissociation from the captured TNF α conjugate was detected during the assay. For each cycle, at the streamA low density (< 100RU) of a given TNF α conjugate is captured on flow cell2 and a high density (> 300RU) of a given TNF α conjugate is captured on flow cell 4, while flow cells 1 and 3 serve as reference cells. A single concentration (0-8nM) of TNF α was injected at 40 μ l/min for 525 seconds per cycle. The dissociation times for 0-4nM TNF α were 1 min and for 0 and 8nM TNF α 1 h. At the end of the cycle, 3M MgCl was used₂The surface was gently regenerated and this reagent was able to dissociate the proteins bound to the anti-hFc capture antibody. Calculation of the dissociation rate constant k Using data from 8nM TNF α injection on high density surfaces_d. The value of this parameter is then fixed and the binding rate constant k is calculated using data from the low density surface_aAnd R_{Maximum of}. The scheme can improve the signal-to-noise ratio of the dissociation phase data to the maximum extent, and reduce the mass transport limit (mass transport limit) of the association phase data. These analyses were performed with BIA evaluation software. The results of this study are listed in table 4 below.

These data indicate the affinity of the TNF α R portion of the bispecific molecule TRU (XT6) -1002 for binding to TNF α and EnbrelSimilarly.

Table 4.

Example 13

Specificity of binding to super IL6 but not to other GP130 cytokines

The effect of the cross-receptor fusion protein on the induction of TF-1 cell proliferation by IL6 and gp130 cytokine IL-11, Leukemia Inhibitory Factor (LIF), oncostatin M (OSM) and cardiotrophin-1 (CT-1) was examined essentially as follows.

One day before use in proliferation experiments, the contents were added to each well of a 96-well flat-bottom plate0.3x10⁶Fresh growth medium (10% FBS-RPMI 1640; 2mM L-glutamine; 100 units/ml penicillin; 100. mu.g/ml streptomycin; 10mM HEPES; 1mM sodium pyruvate; and 2ng/ml Hu GM-CSF) for TF-1 cells (human erythroleukemia cells). Cells were harvested and washed twice with assay medium (no GM-CSF, no cytokines, the remainder being the same as growth medium) and then at 1X10⁵The density of individual cells/ml was resuspended in assay medium. To detect blockade of LIF, OSM and CT-1 activity, serial dilutions of TNFSFR 1B:anti-HIL-6 crossover receptor TRU (XT6) -1002(SEQ ID NO: 608), TRU (XT6) -1019(SEQ ID NO: 625), TRU (XT6) -1022(SEQ ID NO: 628) and TRU (XT6) -1025(SEQ ID NO: 631) were mixed with a fixed concentration of the respective gp130 cytokine or super IL-6(HIL-6) at 37 ℃ in a 96 well plate at 5% CO₂Preincubation was performed for 1 hour alone under the conditions. After pre-incubation, 1 × 10 was added to each well⁴Individual cells (100. mu.l). The final assay mixture (total volume 200. mu.l/well, containing TNFSFR1B:: HIL-6, gp130 cytokine or HIL-6 and cells) was incubated at 37 ℃ with 5% CO₂And incubated under the conditions of (1) for 72 hours. Adding into the final 4-6 hr of culture³H-thymidine (20. mu. Ci/ml solution in assay medium, 25. mu.l/well). Cells were collected on UniFilter-96GF/c plates and assayed for incorporation using a TopCount reader (Paker Co.)³H-thymidine. Percent block was 100- (test cpm-control cpm/max cpm-control cpm) 100.

The results show that the cross-receptors block IL6 activity but not IL-11, LIF, OSM or CT-1 activity (data not shown), and therefore bind to super IL6, but have no effect on the other gp130 cytokines tested.

Example 14

SMIP and Cross-receptors bind to IL6R on hepatocytes

TRU (S6) -1002, TRU (XT6) -1019 and anti-IL 6 antibodies hu-PM1 were tested for their ability to bind IL6R on liver-derived HepG2 cells as described below.

Using FACS bufferHepG2 cells were washed and adjusted to 2X 10 with FACS buffer (PBS + 3% FBS)⁶Individual cells/ml. Add 50. mu.L of this solution to 96 well plate wells (10)⁵Individual cells/well). The plate was incubated at 37 ℃ until diluted test molecules were added. Serial dilutions of test molecules were prepared with FACS buffer to give 2X working stock, which was diluted to 1X when cells were added. Diluted test molecules were added to the cells (50 μ l/well) and the cells were incubated on ice for 20 minutes. Whole IgG was used as control. The cells were then washed twice with FACS buffer and resuspended in goat anti-human antibody conjugated to phycoerythrin (Jackson laboratories, Inc.; diluted 1: 200 with FACS buffer). After incubation for 20 min on ice in the dark, cells were washed twice with FACS buffer, resuspended in 200ul PBS and plated on LSRII^TMRead on a flow cytometer (BD Biosciences, San Jose, CA, San Jose, CA).

As shown in fig. 10, TRU (S6) -1002 and TRU (XT6) -1029 did not substantially bind HepG2 cells.

Example 15

SMIP and Cross-receptors block IL-6 and TNF activity in mice

SMIP and cross-receptor fusion proteins described herein were tested for their ability to block IL-6 or TNF-induced production of Serum Amyloid A (SAA) protein in mice as described below. SAA is one of the major acute phase proteins in humans and mice. Plasma SAA levels are found to rise over time in chronic inflammation, leading to amyloidosis, affecting the liver, kidney and spleen (Rienhoff et al (1990) mol.biol.med.7: 287). Both IL-6 and TNF induce SAA when administered alone (Benigni et al (1996) Blood 87: 1851; Ramadori et al (1988) Eur.J.Immunol.18: 1259).

(a) Blocking super IL-6 Activity

Female BALB/C mice were injected with 0.2ml PBS after orbital (retroorbital), or Enbrel(200. mu.g), TRU (S6) -1002 (200. mu.g) or TRU (XT6) -1002 (300. mu.g or 500. mu.g) in PBS. One hour later, mice were IP injected with 0.2ml PBS or 2 μ g PBS solution of human super-IL 6. Mouse sera were collected 2 and 24 hours after IP injection. Serum concentrations of SAA were determined by ELISA and sgp130 concentrations were determined by Luminex-based mouse soluble receptor assay. As shown in fig. 11 and 12, TRUs (S6) -1002 and TRUs (XT6) -1002 blocked the expression of sgp130 and SAA induced by super IL 6.

(b) Blocking TNF activity

Female BALB/C mice were injected with 0.2ml PBS after orbital (retroorbital), or Enbrel(200. mu.g), TRU (S6) -1002 (200. mu.g) or TRU (XT6) -1002 (300. mu.g) in PBS. One hour later, mice were IP injected with 0.2ml PBS or 0.5 μ g PBS solution of mouse TNF α. Mouse sera were collected 2 and 24 hours after IP injection. Serum concentrations of SAA were determined by ELISA and sgp130 concentrations were determined by Luminex-based mouse soluble receptor assay. As shown in FIGS. 13A and B, the cross-receptor TRU (XT6) -1002 blocked TNF α -induced SAA expression and the levels of SAA observed 2 hours after injection were similar to those observed with EnbrelObserved level.

Example 16

Cross receptor in vivo Activity

The therapeutic efficacy of the cross-receptor molecules described herein is tested in animal models of disease, as described below.

(a) Multiple myeloma

The activity of the cross-receptor molecules was tested using at least one of two well-characterized mouse models of multiple myeloma, namely the 5T2 multiple myeloma (5T2MM) model and the 5T33 multiple myeloma (5T33MM) model. In the 5T33 model, mice were treated with cross-receptors starting at the time of tumor cell injection (prevention mode). In the 5T2MM model, mice were treated from the onset of disease (treatment mode). The effect of treatment on tumorigenesis and angiogenesis was evaluated in two models, and bone studies were also performed using the 5T2MM model.

A5 TMM murine model of myeloma was originally developed by Radl et al (J.Immunol. (1979) 122: 609; see also Radl et al, am.J.Pathol. (1988) 132: 593; Radl, J.Immunol.today (1990) 11: 234). Its clinical features are very similar to human diseases: the tumor cells are located in the bone marrow, serum accessory protein concentrations are a measure of disease development, and increased neovascularization occurs in models 5T2MM and 5T33MM (Van Valckenborgh et al, am. J. Pathol. (1988) 132: 593), with definite presence of osteolytic bone disease in certain lines. The 5T2MM model includes moderate tumor growth and the formation of osteolytic bone lesions. These lesions are associated with decreased volume of cancerous bone, decreased bone mineral density and increased osteoclast number (Croucher et al, Blood (2001) 98: 3534). The 5T33MM model has a more rapid tumor capture (tumor take), and tumor cells grow in the liver in addition to bone marrow (Vanderkerken et al, Br. J. cancer (1997) 76: 451).

The 5T2 and 5T33MM models have been identified in detail. Monoclonal antibodies specific for the idiotypes of 5T2 and 5T33MM were generated for detection of serum accessory proteins by ELISA for maximum sensitivity, and tumor cells were specifically stained by FACS analysis and immunostaining of tissue sections (Vanderkerken et al, Br. J. cancer (1997) 76: 451). Sequence analysis of the VH genes enabled detection of cells by RT-PCR and Northern blot analysis (Zhu et al, Immunol. (1998) 93: 162). The 5TMM model, which can be used for in vitro and in vivo experiments, yields typical MM disease, and different methods can be used to assess tumor burden, serum accessory protein concentration, bone marrow angiogenesis (by measuring microvessel density), and osteolytic bone injury (using a combination of X-ray photography, densitometry, and histomorphometry) in bone marrow. These latter parameters were studied in order to use the 5TMM model in preclinical studies and to study the growth and biological properties of myeloma cells in an intact syngeneic microenvironment. Molecules targeting the MM cells themselves and molecules targeting the bone marrow microenvironment can be studied. In particular, although the 5T33MM model can be used to study the microenvironment and MM cells themselves, the 5T2MM model can also be used to study myeloma-related bone disease.

To investigate the prophylactic effect of the cross-receptor molecules described herein, C57BL/KaLwRij mice were injected with 2x 10 injection on day 0⁶5T33MM cells and cross-receptors. Mice were sacrificed on day 28 and tumor development was assessed by determining serum accessory egg concentration and percentage of tumor cells in isolated bone marrow cells (by flow cytometry or cell smear assay using anti-idiotypic antibodies). Spleen and liver were weighed and these organs were fixed with 4% formaldehyde for further analysis. Bone samples were fixed for further processing, including CD31 immunostaining on paraffin sections and quantitative determination of microvessel density.

To investigate the therapeutic efficacy of the cross-receptor molecules described herein, mice were injected with 5T2MM cells on day 0, and the cross-receptors were administered post-morbidity as determined by the presence or absence of detectable levels of serum side-protein. Mice were sacrificed approximately five weeks after cross-receptor administration and tumor development was assessed as described in the prevention study above. In addition, bone analysis was performed with X-rays to determine the number of bone lesions and trabecular bone area, TRAP staining was performed to assess osteoclast number.

(b) Rheumatoid arthritis

The therapeutic efficacy of any of the cross-receptor molecules described herein was evaluated using at least one of two murine models of Rheumatoid Arthritis (RA), the collagen-induced arthritis (CIA) and glucose-6-phosphate isomerase (G6PI) models. Both of these models have been shown to be useful in predicting the efficacy of certain classes of therapeutic drugs in RA (see Holmdahl (2000) Arthritis Res.2: 169; Holmdahl (2006) immunol.Lett.103: 86; Holmdahl (2007) Methods mol.Med.136: 185; McDeitt, H. (2000) Arthritis Res.2: 85; Kamradt and Schubert (2005) Arthritis Res.Ther.7: 20).

(i) CIA model

The CIA model is the best characterized mouse arthritis model on its pathogenic and immunological basis. Furthermore, it is the most widely used model of RA, and although not perfect in predicting the ability of drugs to inhibit patient disease, it is still considered by many to be a good model for the study of a potential new therapeutic approach to RA (Jirohort et al (2001) Arthritis Res.3: 87; Van den Berg, W.B. (2002) Current Rheumatol. Rep.4: 232; Roslonec (2003) Collagen-Induced Arthritis (Collagen-Induced Arthritis) as described in "New Ed. Immunol research methods" (Current Protocols in Immunology), ed. Coligan et al, John Wiley & Sons, Inc, Hoboken, NJ) by Hopkiken, N.J.).

In the CIA model, male DBA/1 mice were immunized with collagen II (CII) formulated with Complete Freund's Adjuvant (CFA) to induce arthritis. Specifically, mice were injected intradermally/subcutaneously with CII formulated in CFA on day-21 and boosted with CII formulated in Incomplete Freund's Adjuvant (IFA) on day 0. Within days after boosting with CII/IFA, mice developed clinical signs of arthritis. A small group of CII/IFA immunized mice (0% -10%) had no booster and showed signs of arthritis at or around day 0, and these animals were excluded from the experiment. In some CIA experiments, booster immunizations were omitted and mice were treated with cross-receptors or controls starting 21 days after CII/CFA immunizations (i.e. day 0 of the first treatment).

Mice were treated with cross-receptors, vehicle (PBS) or negative or positive controls in a prophylactic and/or therapeutic regimen. Prophylactic treatment was continued from day 0 until after disease peak in control (untreated) mice. Therapeutic treatment began when the majority of mice showed signs of mild arthritis. Enbrel demonstrated good efficacy in CIA and G6 PI-induced arthritis modelUsed as a positive control. Data collected in each experiment included clinical scores and cumulative incidence of arthritis. In 0-4 minutesClinical signs of arthritis were measured for the CIA model as shown in table 5 below.

TABLE 5

(ii) Model G6PI

In the G6PI model, DBA/1 mice were immunized with adjuvant-formulated G6PI to induce Arthritis (Kamradt and Schubert (2005) Arthritis Res. ther.7: 20; Schubert et al (2004) J.Immunol.172: 4503; Bockermann, R.et al (2005) Arthritis Res. ther.7: R1316; Iwanami et al (2008) Arthritis Rheum.58: 754; Matsumoto et al (2008) Arthritis Res.ther.10: R66). G6PI is an enzyme present in essentially all cells in vivo, but it is not clear why immunity induces disease in specific joints. In the G6PI model, a number of agents, such as CTLA4-Ig, TNF antagonists (e.g., Enbrel @)) And an anti-IL 6 receptor monoclonal antibody can inhibit the development of arthritis.

Male DBA/1 mice were immunized with G6PI formulated in Complete Freund's Adjuvant (CFA) to induce arthritis. Specifically, mice were injected intradermally/subcutaneously with G6PI formulated with CFA on day 0 and developed clinical signs of arthritis within a few days of immunization. Mice were treated with either cross-receptors, vehicle (PBS) or negative or positive controls as in the CIA model described above, with prophylactic and/or therapeutic regimens. Prophylactic treatment was continued from day 0 until after the peak disease in control mice. Therapeutic treatment began when the majority of mice showed signs of mild arthritis. Enbrel demonstrated good efficacy in CIA and G6 PI-induced arthritis modelUsed as a positive control. The data collected in each experiment included the clinical signs of arthritisScore and cumulative incidence. Scores similar to those used for the CIA model were used to assess clinical signs of arthritis for the G6PI model.

(c) Polycystic kidney disease

Using Gattone et al, nat. med. (2003) 9: 1323; torres et al, nat. med. (2004) 10: 363; wang et al, j.am.soc.nephrol. (2005) 16: 846; and Wilson (2008) curr. top. dev. biol.84: 311 to test the efficacy of the TNF antagonist-containing cross-receptor fusion proteins described herein in the treatment of polycystic kidney disease.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the embodiments of the invention set forth above are intended to be illustrative, not limiting. Various changes may be made therein without departing from the spirit and scope of the invention as defined in the appended claims. All publications cited herein are incorporated by reference as if fully set forth herein.

SEQ ID NO: 1-834 see the attached sequence Listing. The coding (including the symbol "n") in the nucleotide sequence used for the appended sequence complies with the WIPO standard ST.25(1998), appendix 2, Table 1.

Claims (14)

1. A multi-specific fusion protein having one of the following structures from amino-terminus to carboxy-terminus:

(a)BD-ID-ED；

(b) ED-ID-BD; or

(c)ED1-ID-ED2

Wherein:

ED is a TNF antagonist, ED1 and ED2 are distinct binding or extracellular domains, wherein ED1 or ED2 is a TNF antagonist;

ID is an intervening domain; and

BD is an IL6 antagonist, a RANKL antagonist, an IL7 antagonist, an IL17A/F antagonist, a TWEAK antagonist, a CSF2 antagonist, an IGF antagonist, a BLyS/APRIL antagonist or an IL10 agonist.

2. The multi-specific fusion protein of claim 1 wherein the BD is an immunoglobulin variable binding domain.

3. The multi-specific fusion protein of claim 1 or 2 wherein the ED1 and ED2 are receptor ligand-binding extracellular domains.

4. The multi-specific fusion protein of any one of the preceding claims, wherein the intervening domain has the structure:

-L1-CH2CH3-，

wherein:

l1 is an immunoglobulin hinge linker, optionally an IgG1 hinge with the first cysteine replaced by a different amino acid;

-CH2CH 3-is the CH2CH3 region of the IgG1Fc domain, optionally mutated to abolish Fc γ RI-III binding capacity while retaining FcRn binding capacity.

5. The multi-specific fusion protein of any one of the preceding claims, wherein the BD is linked to the intervening domain by a first linker and the ED is linked to the intervening domain by a second linker, wherein the first and second linkers can be the same or different.

6. The multi-specific fusion protein of claim 5 wherein the first and second linkers are selected from the group consisting of: SEQ ID NO: 497-604 and 791-796, optionally the first linker is SEQ ID NO: 576 and said second linker is SEQ ID NO: 791.

7. the multi-specific fusion protein of any one of the preceding claims, comprising the amino acid sequence of SEQ ID NO: 607-670 and 798-804.

8. A composition comprising one or more multi-specific fusion proteins of any one of the preceding claims, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. The composition of claim 8, wherein the multi-specific fusion protein is present in the composition as a dimer or multimer.

10. A polynucleotide encoding the multi-specific fusion protein of any one of claims 1-7.

11. An expression vector comprising the polynucleotide of claim 10 operably linked to an expression control sequence.

12. A host cell comprising the expression vector of claim 11.

13. A method of treating a subject having an inflammatory disease, an autoimmune disease, or a hyperproliferative disease comprising administering a therapeutically effective amount of the multi-specific fusion protein of any of the preceding claims or a composition thereof.

14. The method of claim 13, wherein the disease is rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, psoriasis, Chronic Obstructive Pulmonary Disease (COPD), crohn's disease, ulcerative colitis, severe refractory asthma, TNFRSF 1A-associated periodic syndrome (TRAPS), endometriosis, systemic lupus erythematosus, or alzheimer's disease.