JP2008504356A - Compositions and methods for treating inflammatory diseases - Google Patents

Abstract

  The present invention relates to compositions and methods for treating inflammatory diseases. More specifically, the present invention relates to antibody compositions and their use in the treatment of inflammatory diseases.

Description

  The present invention relates to methods of treating diseases including rheumatoid arthritis using antibody polypeptide constructs comprising single domain antibody ligands and bispecific ligands, compositions of the ligands, and methods of making and using the ligands. About. In particular, the present invention provides methods for preparing single domain antibodies that bind inflammatory cytokines including TNF-α and VEGF. Also disclosed is a bispecific ligand comprising a first single immunoglobulin variable region that binds to a first antigen or epitope and a second single immunoglobulin variable region that binds to a second antigen or epitope. . More particularly, the present invention relates to bispecific ligands that act to increase the half-life of a ligand in vivo by binding to at least one of a first and second antigen or epitope. Open and closed structures containing more than one binding specificity are described. For treating rheumatoid arthritis using single domain antibody constructs and bispecific ligands that bind first and second antigens that can include, for example, any combination of TNF-α, VEGF and HSA The method is disclosed.

TNF-α
As the name implies, tumor necrosis factor-α (TNF-α) was originally described as a molecule with anti-tumor properties, but the molecule is prominent in mediating inflammation and autoimmune diseases. It was later confirmed to play a major role in other processes, including roles. TNF-α is a major pro-inflammatory agent in inflammatory conditions including, for example, rheumatoid arthritis (RA), Crohn's disease, ulcerative colitis and other bowel diseases, psoriasis, toxic shock, graft-versus-host disease and multiple sclerosis. Sex cytokine.

  The pro-inflammatory effect of TNF-α induces procoagulant activity on vascular endothelial cells (Pober et al., J. Immunol. 136: 1680 (1986)) and enhances neutrophil and lymphocyte adhesion (Pober J. Immunol. 138: 3319 (1987)), stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi et al., J. Exp. Med. 166: 1390 (1987). )) And other tissue damage.

  TNF-α is synthesized as a 26 kD transmembrane precursor protein by the intracellular tail cleaved by the TNF-α converting metalloproteinase enzyme and then secreted as a 17 kD soluble protein. The active form consists of a homotrimer of 17 kD monomer that interacts with two different cell surface receptors, p55TNFR1 and p75TNFR2. There is also evidence that the cell surface bound precursor form of TNF-α can mediate several biological effects of the factor. Most cells express both the p55 and p75 receptors that mediate the different biological functions of the ligand. The p75 receptor is involved in inducing lymphocyte proliferation, and the p55 receptor is TNF-mediated cytotoxic, apoptosis, antiviral activity, fibroblast proliferation and NF-κB activation (Locksley et al., 2001, Cell 104: 487). -501).

  The TNF receptor is a member of a group of membrane proteins including the NGF receptor, Fas antigen, CD27, CD30, CD40, Ox40, and lymphotoxin α / β hexamer. Receptor binding by homotrimers induces receptor aggregation into small assemblies of two or three molecules of p75 or p55. TNF-α is mainly produced by activated macrophages and T lymphocytes, but is also produced by neutrophils, endothelial cells, keratinocytes and fibroblasts during an acute inflammatory reaction.

  TNF-α is at the apex of the cascade of pro-inflammatory cytokines (discussed in Feldmann & Maini, 2001, Ann. Rev. Immunol. 19: 163). This cytokine induces the expression or release of additional pro-inflammatory cytokines, particularly IL-1 and IL-6 (see, eg, Rutgeerts et al., 2004, Gastroenterology 126: 1593-1610). Inhibition of TNF-α inhibits the production of inflammatory cytokines including IL-1, IL-6, IL-8 and GM-CSF (Brennan et al., 1989, Lancet 2: 244).

  Because TNF-α plays a role in inflammation, it has become an important inhibitory target in efforts to reduce the symptoms of inflammatory diseases. Techniques for the inhibition of TNF-α have been sought for clinical treatment of disease, including the use of soluble TNF-α receptor and antibodies specific for TNF-α in particular. Commercial products approved for clinical use include, for example, the antibody product Remicade ™ (Infliximab (Centocor, Malvern, Pa.)); A chimeric monoclonal IgG carrying a human IgG4 constant region mouse variable region Antibody), Humira ™ (adalimumab or D2E7 (Abbott Laboratories) and soluble receptor product Enbrel ™ (etanercept, soluble p75TNFR2Fc fusion protein described in US Pat. No. 6,090,382); Imnex ).

  The role of TNF-α in inflammatory arthritis is described in, for example, Li & Schwartz, 2003, Ringer Semin. Immunopathol. 25: 19-33. In RA, TNF-α is highly expressed in inflammatory synovium, especially in the cartilage-pannus joint (DiGiovine et al., 1988, Ann. Rheum. Dis. 47: 768; Firestein et al., 1990, J. Immunol. 144: 3347. And Saxne et al., 1988, Arthritis Rheum. 31: 1041). In addition to the evidence that TNF-α increases the levels of inflammatory cytokines IL-1, IL-6, IL-8 and GM-CSF, TNF-α alone, joint inflammation, and fibroblast-like Induces synovial cell proliferation (Gitter et al., 1989, Immunology 66: 196) and induces cartilage destruction by inducing collagenase (Dayer et al., 1985, J. Exp. Med. 162: 2163; Dayer et al., 1986, J. Clin. Invest. 77: 645) and inhibits proteoglycan synthesis by articular chondrocytes (Saklatvala, 1986, Nature 322: 547; Saklatvala et al., 1985, J. Exp. Med. 162: 1208), osteoclast. Stimulates cell tumor formation and bone resorption (Abu-Ame Et al, 2000, J.Biol.Chem.275: 27307; Bertolini et al., 1986, Nature 319: 516) can be. TNF-α induces increased release of CD14 + monocytes by the bone marrow. The monocytes penetrate the joint and amplify the inflammatory response via the RANK (receptor activator or NF-κB) -RANKL signaling pathway, resulting in osteoclastic system during joint inflammation (Anandarajah & Richlin, 2004, Curr. Opin. Rheumatol. 16: 338-343).

  TNF-α is an acute phase protein that introduces macrophages and neutrophils at the site of infection by increasing vascular permeability through induction of IL-8. Once present, activated macrophages maintain and amplify the inflammatory response by continuing to produce TNF-α.

  Titration of TNF-α with the soluble receptor construct etanercept is effective for the treatment of RA but not for the treatment of Crohn's disease. In contrast, the antibody TNF-α antagonist infliximab is effective in the treatment of both RA and Crohn's disease. Thus, mere neutralization of soluble TNF-α is not the only mechanism involved in anti-TNF based therapeutic effects. Rather, blocking other pro-inflammatory signals or molecules induced by TNF-α also plays a role (Rutgeerts et al., Supra). For example, administration of infliximab reduces the penetration of neutrophils into the inflammatory site, apparently by reducing the expression of adhesion molecules. Infliximab treatment also eliminates inflammatory cells from the already inflamed intestinal mucosa in Crohn's disease. This loss of T cells in the basement membrane is mediated by apoptosis of membrane-bound TNF-α bearing cells following Fas-dependent activation of caspases 8, 9, and 3 (Luggering et al., 2001, Gastroenterology 121: 1145-1157). reference). Therefore, membrane or receptor-bound TNF-α is an important target for anti-TNF-α therapeutic approaches. Others have shown that infliximab binds to activated peripheral blood cells and basement membrane cells and induces apoptosis through activation of caspase 3 (Van den Brande et al., 2003, Gastroenterology 124: 1774- 1785).

  In the cell, the binding of trimeric TNF-α to its receptor is due to displacement of inhibitory molecules such as SODD (silencer of the dead region), and adapter factors FADD, TRADD, TRAF2, c-IAP, RAIDD and TRIP + kinase. Triggers a cascade of transduction events involving RIP1 and specific caspase signals (Goeddel, 2002, Science 296: 1634-1635 and Muzio & Saccani, Methods in Molecular Medicine: Edited by Tumor Necrostic Factor, Methos Factor Human Press, New Jersey), pp. 81-99). The assembled signaling complex can activate the cell survival pathway through NF-κB activation and subsequent downstream gene activation, and can activate the apoptotic pathway through caspase activation.

  In other diseases, similar extracellular downstream cytokine cascades and intracellular signaling pathways can be triggered by TNF-α. Thus, for other diseases or disorders where TNF-α molecules contribute to the pathology, inhibition of TNF-α provides a therapeutic approach.

VEGF
Angiogenesis plays an important role in the active proliferation of inflamed synovial tissue. Highly vascularized RA synovial tissue invades periarticular cartilage and bone tissue, leading to joint destruction.

  Vascular endothelial growth factor (VEGF) is the most potent angiogenic cytokine known. VEGF is an excreted heparin-binding homodimeric glycoprotein that exists in several alternating forms because its primary transcript is alternatively spliced (Leung et al., 1989, Science 246: 1306). VEGF is also known as vascular penetration factor (VPF) because it has the ability to induce vascular leakage, an important process in inflammation. The identification of VEGF in the synovial tissue of RA patients has highlighted the potential role of VEGF in the pathology of RA (Fava et al., 1994, J. Exp. Med. 180: 341: 346; Nagashima et al., 1995, J. Am. Rheumatol.22: 1644-1630). The role of VEGF in the pathology of RA has strengthened the study after administration of anti-VEGF antibody to the mouse collagen-induced arthritis (CIA) model. In these studies, induction of disease enhanced VEGF expression in the joint, and administration of anti-VEGF antiserum suppressed the development of arthritic disease and improved established disease (Sone et al., 2001, Biochem). Biophys.Res.Comm.281: 562-568; Lu et al., 2000, J. Immunol.164: 5922-5927).

Antibody Polypeptides Antibodies are highly specific for binding of targets and are derived from natural unique defense mechanisms, but face several challenges when applied to the treatment of disease in human patients. Conventional antibodies are large multi-subunit protein molecules that contain at least four polypeptide chains. For example, human IgG has two heavy chains and two light chains that disulfide bond to form a functional antibody. The size of conventional IgG is about 150 kD. Complete antibodies (eg, IgG, IgA, IgM, etc.) are relatively large in size and thus have limited therapeutic utility due to, for example, problems with tissue penetration. A great deal of effort has been devoted to identifying and generating smaller antibody fragments that retain antibody binding function and solubility.

The heavy and light polypeptide chains of an antibody have a variable (V) region that is directly involved in antigen interactions and a constant (C) region that provides structural support and function in non-antigen-specific interactions with immune effectors. Including. The antigen-binding region of a conventional antibody is composed of two separate regions, a heavy chain variable region (V _H ) and a light chain variable region (which can be V _L : V _κ or V _λ ). The antigen binding site itself is formed by six polypeptide loops: three polypeptide loops from the _VH region (H1, H2 and H3) and three polypeptide loops from the _VL region (L1, L2 and L3). In vivo, diverse primary repertoire V genes encoding _VH and _VL regions are generated by combinatorial rearrangement of gene segments. C regions include the light chain C regions (referred to as _{C L} regions) and _(referred to as C H 1, _{C H} 2 and _{C H} 3 regions) heavy chain C region.

Following protease digestion, several smaller antigen-binding fragments of natural antibodies were identified. These include, for example, “Fab fragment” (V _L -C _L -C _H 1-V _H ), “Fab ′ fragment” (Fab with heavy chain hinge region) and “F (ab ′) ₂ fragment” ( including a dimer) of the connected Fab 'fragments by the heavy chain hinge region. synthetic peptides consisting of the connected V _L and V _H with a linker to as "single chain F _V" (variable fragment) or "scF _V" Recombinant methods have been used to generate smaller antigen-binding fragments.

Although it is generally known that the antigen-binding unit of a natural antibody (eg in humans and most other mammals) is composed of a pair of V regions (V _L / V _H ), camelids Organisms express large quantities of fully functional and highly specific antibodies lacking light chain sequences. Camelid heavy chain antibodies are found as single heavy chain homodimers dimerized through their constant regions. The variable regions of these camelid heavy chain antibodies, referred to as V _H H regions, retain the ability to bind antigens with high specificity when isolated as V _H chain fragments (Hamers-Casterman). 1993, Nature 363: 446-448; Gahrodi et al., 1997, FEBS Lett. 414: 521-526). An antigen-binding single _VH region has also been identified from a library of mouse _VH genes amplified from, for example, genomic DNA from spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341). : 544-546). Ward et al. Named the isolated single V _H region “dAb” for “region antibody”. The term “dAb” is referred to herein as a single immunoglobulin variable region (V _H , V _HH or V _L ) polypeptide that specifically binds an antigen. “DAb” binds an antigen independent of other V regions, but when the term is used herein, “dAb” is used when no other region is required for antigen binding by a dAb; That is, a dAb can be present in a homo- or heteromultimer with other _VH or _VL regions when it binds an antigen independent of additional _VH , _VHH or _VL regions.

A single immunoglobulin variable region, such as V _H H, is the smallest known antigen binding antibody. For use in therapy, human antibodies are preferred primarily because they are unlikely to cause an immune response when administered to a patient. As noted above, isolated non-camelid _VH tends to be relatively insoluble and is often poorly expressed. Comparing the camelid V _H H with the V _H region of the human antibody, there are some important differences in the framework region of the camelid V _H H region corresponding to the V _H / V _L interface of the human V _H region. It becomes clear that it exists. In order to generate “camelized” human _VH regions with improved expression and solubility while retaining antigen binding activity (Davies & Riechmann, 1994, FEBS Lett. 339: 285-290) Mutations were made to bring the residues of V _H ³ closer to the V _H H sequences (specifically Gly44 Glu, Leu 45 Arg and Trp47 Gly). (The variable region amino acid numbering used herein is consistent with the Kabat numbering convention (Kabat et al., 1991, Sequence of Immunological Interest, 5th edition, US Department of Health and Human Services (Washington, DC)). WO 03/035694 (Muyldermans) reports that the Trp103 Arg mutation improves the solubility of non-camelid V _H regions, and Davis & Riechman (1995, Biotechnology NY 13). : 473-479), it was reported that a phage display repertoire of camelized human _VH region was generated and clones that bind haptens with an affinity of 100-400 nM were selected. Against Clones selected for binding had weaker affinities.

The antigenic region of an antibody includes two distinct regions, a heavy chain variable region (V _H ) and a light chain variable region (which can be V _L : V _κ or V _λ ). The antigen binding site itself is formed by six polypeptide loops: three polypeptide loops from the _VH region (H1, H2 and H3) and three polypeptide loops from the _VL region (L1, L2 and L3). Various primary repertoire V genes encoding V _H and V _L regions are generated by combinatorial rearrangement of gene segments. The _VH gene is produced by recombination of three gene segments, _VH , D and _JH . In humans, approximately 51 functional V _H segments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25 functional D segments (Corbett et al. (1997) J. Mol. Biol. 268: 69) and six functional _JH segments (Ravetch et al. (1981) Cell, 27: 583). The V _H segment encodes the region of the polypeptide chain that forms the first and second antigen binding loops of the V _H region (H1 and H2), whereas the V _H , D and J _H segments are VH Binding to form a third antigen binding loop of region (H3). V _L gene is produced by the recombination of only two gene segments, _{V L} and _{J L.} In humans, there are approximately 40 functional V _kappa segments (Schable and Zachau (1993) Biol.Chem.Hoppe- Seyler, 374 : 1001), 31 functional V _lambda segments (Williams et al., (1996) J.Mol.so Biol, 264:.. 220 ; Kawasaki et al., (1997) Genome Res, 7 : 250), 5 single functional J _kappa region (Hieter et al., (1982) J.Biol.Chef, 257: 1516 ) And four functional J _λ segments (Vasenik and Leder (1990) J. Exp. Med., 172: 609). The _VL segment encodes the region of the polypeptide chain that forms the first and second antigen binding loops of the _VL region (L1 and L2), whereas the _VL and _JL segments are the _VL region. Binding to form the third antigen-binding loop of (L3). Antibodies selected from this primary repertoire are believed to have sufficient diversity to bind almost all antigens with at least moderate affinity. High affinity antibodies are produced by “affinity maturation” of a rearranged gene in which point mutations are generated and selected by the immune system based on improved binding.

  Analysis of antibody structure and sequence showed that 5 out of 6 antigen binding loops (H1, H2, L1, L2, L3) have a limited number of backbone or standard structures ( Chothia and Lesk (1987) d: Mol. Biol., 196: 901; Chothia et al. (1989) Nature, 342: 877). The backbone structure is determined by (i) the length of the antigen binding loop and (ii) a particular residue or residue type at a particular major position within the antigen binding loop and antibody framework. Analysis of the loop length and major residues made it possible to predict the main chain structure of H1, H2, L1, L2 and L3 encoded by the majority of human antibody sequences (Chothia et al., (1992). J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). The H3 region is much more diverse in terms of sequence, length and structure (through the use of D segments), but the length and presence of certain residues at key positions within the loop and antibody framework, or It also forms a limited number of backbone structures for short loop lengths depending on the type of residue (Martin et al. (1996) J. Mol. Biol. 263: 800; Shirai et al. (1996) FEBS Letters 399: 1).

Bispecific Antibodies Bispecific antibodies comprising a pair of complementary _VH and _VL regions are known in the art. These bispecific antibodies should contain two pairs of V _H and V _L with each V _H / V _L pair binding to a single antigen or epitope. The described methods include hybrid hybrid cells (Milstein & Cuello, Nature 305: 537-40), minibodies (Hu et al. (1996) Cancer Res 30 56: 3055-3061;), diabodies (Holliger et al., (1993). Proc. Natl. Acad. Sci. USA 90, 6444 6448; WO 94/13804), chelated recombinant antibodies (CRAbs; (Neri et al. (1995) J. Mol. Biol. 246, 367-373), biscFv (eg Atwell et al., (1996) Mol. Immunol. 33, 1301 1312), “Nobuin-hole” stabilizing antibodies (Carter et al., (1997) Protein Sci. 6, 781 788). Body species, each containing two antigen-binding sites made by a pair of complementary V _H and V _L regions. Thus, each antibody may be capable of simultaneously binding to two different antigens or epitopes , Binding to the EACH antigen or epitope is mediated by V _H and its complementary _VL region, each of these techniques exhibit their own disadvantages, eg, inactive V _{H in} the case of hybrid hybrid cells. The / V _L pair can significantly reduce the portion of the bispecific IgG.

Also, most bispecific approaches rely on different V _H / V _L pair associations or V _H and V _L chain associations to reproduce two different V _H / V _L binding sites. Thus, because it is impossible to control the ratio of binding sites for each antigen or epitope in the assembly molecule, many of the assembly molecules bind to one antigen or epitope and not to other antigens or epitopes. In some cases it was possible to modify the heavy or light chain at the subunit interface by protein engineering to increase the number of molecules with binding sites for both antigens or epitopes (Carter et al., 1997). , Not all molecules will have binding to both antigens or epitopes.

Although there is evidence that two different antibody binding specificities can be incorporated into the same binding site, these are generally two or more corresponding to structurally related antigens or epitopes, or broadly cross-reactive antibodies. Represents specificity. For example, a cross-reacting antibody is usually bound to a free hapten and a carrier when two antigens such as chicken egg white lysozyme and turkey lysozyme are related in sequence and structure (McCafferty et al., WO 92/01047). It has been described as such when related to haptens (Griffiths AD et al., EMBO J 1994 13:14 3245-60). In a further example, WO 02/02773 (Abbott Laboratories) describes antibody molecules having “bispecificity”. The antibody molecules referred to are antibodies that are generated or selected against multiple antigens such that their specificity spans two or more single antigens. Each complementary V _H / V _L pair in the antibody of WO 02/02773 defines a single binding specificity for two or more structurally related antigens, and V _H and V in such complementary pair. Each _L region has no individual specificity.

Thus, these antibodies have a broad single specificity encompassing two structurally related antigens. Also described are multi-reactive natural autoantibodies that react with at least two (usually more) different antigens or epitopes that are not structurally related (Casali & Notlins, Ann. Rev. Immunol. 7, 515-531). It has also been shown that random peptide repertoire selection using phage display technology against monoclonal antibodies will identify a series of peptide sequences comparable to the antigen binding site. Some sequences are highly related and comparable to consensus sequences, while others are quite different and have been named mimotopes (Lane & Stephen, Current Opinion in Immunology, 1993, 5, 268- 271). Thus, it is clear that natural four-chain antibodies that associate and contain complementary V _H and V _L regions have the potential to bind many different antigens from a large number of known antigens. It is less obvious how to create binding sites for two predetermined antigens in the same antibody, especially antigens that are not necessarily structurally related.

Protein engineering methods that may be related to this were suggested. For example, it has also been proposed that it is possible to create catalytic antibodies that have binding activity to metal ions through one variable region, and contact activity to metal ions and binding activity to haptens (substrates) through complementary variable regions. (Barbas et al., 5 1993 Proc. Natl. Acad. Sci USA 90, 6385-6389). However, in this case, it has been proposed that the binding of the substrate (first antigen) and the catalyst require the binding of a metal ion (second antibody). Thus, the binding to the V _H / V _L pair is single but related to multicomponent antigens.

A bispecific antibody is generated from a camel antibody heavy chain single region where a binding contact for one antigen is made in one variable region and a binding contact for a second antigen is made in the second variable region A method for is described. However, the variable regions were not complementary. Thus, the first heavy chain variable region is selected for the first antigen, the second heavy chain variable region is selected for the second antigen, and then both regions are joined together on the same chain To give bispecific antibody fragments (Conrath et al., J. Biol. Chem. 270, 27589-27594). However, the camel heavy chain single region is specific in that it is derived from a natural camel antibody that does not have a light chain; in fact, the heavy chain single region associates with the camel light chain and is complementary Cannot form a typical V _H and V _L pair.

  A single heavy chain variable region derived from a natural antibody that normally accompanies a light chain has also been described (from a repertoire of monoclonal antibodies or regions; see EP-A-0368684). Although these heavy chain variable regions have been shown to interact specifically with one or more related antigens, they bind to other heavy or light chain variable regions and are directed against two or more different antigens. It did not create a ligand with specificity. These single regions have also been shown to have a very short in vivo half-life. Therefore, this area has limited therapeutic value.

  It has been suggested that bispecific fragments are made by linking heavy chain variable regions of different specificities to each other (as described above). The disadvantage of this approach is that the isolated antibody variable region usually interacts with the light chain, has a hydrophobic interface that is contacted by the solvent, is “sticky”, and the single region is on the hydrophobic surface. It can be possible to combine. Also, in the absence of a partner light chain, a combination of two or more different heavy chain variable regions, and possibly an association through their hydrophobic interface, to one or both of the ligands that they can separate and bind to. Bonding can be prevented. Furthermore, in this case, the heavy chain variable region is not associated with the complementary light chain variable region, so it has poor stability and can be easily denatured (Worn & Puckthun, 1998 Biochemistry 37, 13120-7).

  In our co-pending international patent application WO 03/002609 and in our co-pending unpublished UK patent application 0230203.2, we have immunoglobulin single variables that can each have different specificities. A bispecific immunoglobulin ligand containing region was described. These regions can compete with each other or act independently to bind an antigen or epitope on the target molecule.

  The present invention describes a method of treating an individual suffering from a TNF-α associated inflammatory disease. The method comprises administering to the individual a therapeutically effective amount of a single region antibody polypeptide construct, preferably a human single region antibody construct, wherein the single region antibody polypeptide construct binds human TNFα, thereby TNF-α disease is treated.

  In one aspect, the inflammatory disease is rheumatoid arthritis and the method comprises the use of one or more single domain antibody polypeptide constructs, any of which antagonize binding to human TNFα receptors. The present invention relates to one or more single domain antibody polypeptide constructs that antagonize binding of human TNFα to a receptor, a first specificity of the ligand directed to TNFα, and a second specificity of VEGF or HSA. A composition comprising a dual specific ligand directed to The present invention further describes bispecific ligands in which the first specificity of the ligand is directed to VEGF and the second specificity is directed to HSA.

  Also included herein is the use of the polypeptide constructs described herein in the preparation of a medicament for treating a disease, particularly in the preparation of a medicament for treating rheumatoid arthritis.

  In one aspect, the invention is a method of treating rheumatoid arthritis, comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human TNFα for an individual in need thereof. Including administering an amount of the composition, whereby rheumatoid arthritis is treated.

  In one embodiment, the composition prevents an increase in arthritic score when administered to a mouse of the Tg197 transgenic mouse model of arthritis.

  In other embodiments, administration of the composition to Tg197 transgenic mice comprises: a) administering the composition weekly by intraperitoneal injection to heterozygous Tg197 transgenic mice; and b) mice of step a). C) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = Scoring mice weekly for macrophenotypic signs of arthritis according to a system of severe arthritis (significant movement disorder).

  In other embodiments, the composition is administered before an onset of joint inflammation is indicated. In other embodiments, the composition is administered first when the mouse is 3 weeks old. In other embodiments, the composition is administered first when the mouse is 6 weeks of age.

  In other embodiments, the composition is Tg197 transgenic mouse that is within a statistically significant range that is equal to or greater than an equivalent dose (in mg / kg) of a drug selected from the group consisting of etanercept, infliximab, and D2E7. Has an effect in arthritis test.

  In other embodiments, the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score of 0 to 0.5. In other embodiments, the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritis score of 0 to 1.0. In other embodiments, the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score between 0 and 1.5. In other embodiments, the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score between 0 and 2.0.

  In other embodiments, the treatment comprises inhibiting the progression of rheumatoid arthritis. In other embodiments, the treatment comprises preventing or delaying the onset of rheumatoid arthritis.

  In other embodiments, administration results in a statistically significant change in one or more indicators of RA. In other embodiments, the one or more indicators of RA are: blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, X of one or more joints Includes either line imaging as well as histopathological analysis of the fixed part of one or more joints.

  In other embodiments, the one or more indicators of RA comprise a reduction in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, and Tg197 transgenic mice Are scored for the macrophenotypic signs of arthritis, where the macrophenotypic signs of arthritis are 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (Swelling, joint deformation), 3 = scored according to system of severe arthritis (significant movement disorder).

  In other embodiments, the one or more indicators of RA comprise a decrease in macrophenotypic signs of histopathological signs of arthritis in Tg197 transgenic mice and the composition is administered to Tg197 transgenic mice Tg197 transgenic mice are scored for histopathological signs of arthritis, which are realized in the joints, 0 = no detectable pathology, 1 = synovial hyperplasia and multiple Presence of morphonuclear infiltrates, 2 = pannus and fibrous tissue formation and localized subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = scored using extensive articular cartilage destruction and bone erosion system The

In other embodiments, the single region antibody polypeptide construct comprises a human single region antibody polypeptide. In other embodiments, the human single region antibody polypeptide binds TNFα. In other embodiments, the single domain polypeptide construct binds human TNFα with a Kd of less than 100 nM. In other embodiments, the single domain antibody polypeptide construct binds human TNFα with a K _d ranging from 100 nM to 50 pM. In other embodiments, the single region antibody polypeptide construct binds human TNFα with a K _d ranging from 30 nM to 50 pM. In other embodiments, the single region antibody polypeptide construct binds human TNFα with a K _d ranging from 10 nM to 50 pM. In other embodiments, the single region antibody polypeptide construct binds human TNFα with a K _d ranging from 1 nM to 50 pM.

  In other embodiments, the single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cytotoxic cell test.

  The present invention relates to a method of treating rheumatoid arthritis comprising, for an individual in need thereof, a therapeutically effective amount of a composition comprising a single domain antibody polypeptide that antagonizes binding to a receptor for human TNFα. The single domain antibody polypeptide construct further comprises a method of inhibiting the binding of human TNFα to the TNFα receptor, thereby treating rheumatoid arthritis.

  In one embodiment, the single region antibody polypeptide construct specifically binds to human TNF-α bound to a cell surface receptor.

  In other embodiments, the single region antibody polypeptide construct has an in vivo tα half-life ranging from 15 minutes to 12 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life in the range of 1 to 6 hours. In other embodiments, the single domain antibody polypeptide construct has an in vivo tβ half-life in the range of 2 to 5 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life in the range of 3 to 4 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 60 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 48 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 26 hours.

  In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 150 mg / ml. In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 100 mg / ml. In other embodiments, the single region antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 75 mg / ml. In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 50 mg / ml.

  In other embodiments, the single region antibody polypeptide construct is conjugated to a PEG molecule. In other embodiments, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa. In other embodiments, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa. In other embodiments, the PEGylated protein of the invention is averaged to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Can be combined.

  In other embodiments, the antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human TNFα. In other embodiments, the antibody construct comprises a homodimer of a single immunoglobulin variable region polypeptide that binds human TNFα. In other embodiments, the antibody construct comprises a homotrimer of a single immunoglobulin variable region polypeptide that binds human TNFα. In other embodiments, the antibody construct comprises a homotetramer of a single immunoglobulin variable region polypeptide that binds human TNFα.

  In other embodiments, it further comprises an antibody polypeptide specific for an antigen other than TNFα. In other embodiments, the antibody polypeptide specific for an antigen other than TNFα comprises a single region antibody polypeptide. In other embodiments, binding of an antigen other than TNFα by an antibody polypeptide specific for an antigen other than TNFα increases the in vivo half-life of the antibody polypeptide construct. In other embodiments, the antigen other than TNFα comprises a serum protein. In other embodiments, the serum protein is a group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. Selected from. In other embodiments, the antigen other than TNFα comprises HSA.

  In other embodiments, the treatment further comprises administration of at least one additional therapeutic agent.

からなる群から選択される抗体ポリペプチドのＣＤＲ３のアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

Comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of:

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９０％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９２％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 92% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９４％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 94% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９６％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９８％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９９％の同一性を有するアミノ酸配列を含む。 In other embodiments, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 99% identity thereto.

  The present invention is a method of treating rheumatoid arthritis comprising a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a human TNFα receptor for an individual in need thereof. Wherein the composition prevents an increase in arthritis score when administered to a mouse of the Tg197 transgenic mouse model of arthritis, and the single domain antibody polypeptide construct has human TNFα with a Kd of less than 100 nM. Binding and single domain antibody polypeptide constructs further include methods of neutralizing human TNFα as measured in a standard L929 cell test, thereby treating rheumatoid arthritis.

  The present invention includes a single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor, and prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis, Polypeptide constructs further include compositions that neutralize human TNFα as measured in a standard L929 cell test.

  The present invention comprises a single domain antibody polypeptide construct that antagonizes the binding of human TNFα to a receptor, and is a composition that prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis. Thus, single domain antibody polypeptide constructs further include compositions that inhibit the progression of rheumatoid arthritis.

  The present invention comprises a single domain antibody polypeptide construct that antagonizes the binding of human TNFα to a receptor, and is a composition that prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis. Thus, single region antibody polypeptide constructs further include compositions that bind human TNFα with a Kd of less than 100 nM.

  The present invention comprises a single domain antibody polypeptide construct that antagonizes the binding of human TNFα to a receptor, and is a composition that prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis. Single region antibody polypeptide constructs neutralize human TNFα as measured in a standard L929 cell test, and single region antibody polypeptide constructs inhibit the progression of rheumatoid arthritis, and single region antibody polypeptide constructs The peptide construct further includes a composition that binds human TNFα with a Kd of less than 100 nM.

からなる群から選択される抗体ポリペプチドのＣＤＲ３のアミノ酸配列を含む。 In a further embodiment of the previous three embodiments, the single region antibody polypeptide construct is a clone

Comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of:

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９０％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９２％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 92% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９４％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 94% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９６％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９８％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９８％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９９％の同一性を有するアミノ酸配列を含む。 In a further embodiment, the single region antibody polypeptide construct is a clone.

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 99% identity thereto.

  The present invention is a method of treating rheumatoid arthritis, comprising a therapeutically effective amount of a single domain antibody polypeptide construct that antagonizes binding to a receptor for human VEGF for an individual in need thereof. And a method whereby rheumatoid arthritis is treated.

  In one embodiment, the composition prevents an increase in arthritic score when administered to mice in a collagen-induced arthritis (CIA) mouse model. Immunization of DBA / 1 mice with mouse type II collagen induces chronic relapsing polyarthritis that provides a powerful model for human autoimmune arthritis. The model is described, for example, by Courtenay et al., 1980, Nature 282: 666-668; Kato et al., 1996, Ann., Each incorporated herein by reference. Rheum. Dis. 55: 535-539; and Myers et al., 1997, Life Sci. 61: 1861-1878.

  In one embodiment, administration of the composition to the mouse comprises: a) administering the composition weekly by intraperitoneal injection to CIA mice; b) measuring the weight of the mouse of step a) weekly; c ) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) Scoring mice for macrophenotypic signs of arthritis according to the system.

  In one embodiment, the treatment includes inhibiting the progression of rheumatoid arthritis.

  In one embodiment, the treatment includes preventing or delaying the onset of rheumatoid arthritis.

  In one embodiment, administration results in a statistically significant change in one or more indicators of RA. The change is preferably at least 10% or more.

  In one embodiment, one or more indicators of RA include blood sedimentation (ESR), rich joint index (Ritchie et al., 1968, QJ Med. 37: 393-406) and morning stiffness duration, joints Includes any of mobility, joint swelling, analysis by X-ray imaging of one or more joints, and histopathological indicators of the fixed part of one or more joints. Disease activity scores (DAS) and / or chronic arthritis systematic index (CASI) can be used to assess disease activity and changes caused by treatment (Carotti et al., 2002, Ann. Rheum. Dis. 61: 877-882, and Salaffi et al., 2000, Rheumatology 39: 90-96).

  In one embodiment, the one or more indicators of RA comprise a reduction in arthritic macrophenotypic symptoms in a mouse of a collagen-induced arthritis mouse model, the composition is administered to the mouse, and the mouse Marked for phenotypic signs, the macrophenotypic signs of arthritis are 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joints) (Deformation), 3 = scored according to system of severe arthritis (significant movement disorder).

  In one embodiment, the one or more indicators of RA comprise a decrease in macrophenotypic signs of histopathological signs of arthritis in mice of a collagen-induced arthritis mouse model, the composition is administered to the mice, Mice are scored for histopathological signs of arthritis, which are realized in the joint, 0 = no detectable pathology, 1 = synovial hyperplasia and polymorphonuclear infiltrates. Presence, 2 = pannus and fibrous tissue formation and localized subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = scored using extensive articular cartilage destruction and bone erosion system.

  In one embodiment, the single region antibody polypeptide construct comprises a human single region antibody polypeptide.

  In one embodiment, the human single region antibody polypeptide binds VEGF.

  In one embodiment, the single domain antibody polypeptide construct binds human VEGF with a Kd of less than 100 nM.

  In one embodiment, the single domain antibody polypeptide construct binds human VEGF with a Kd ranging from 100 nM to 50 pM.

  In one embodiment, the single domain antibody polypeptide construct binds human VEGF with a Kd ranging from 30 nM to 50 pM.

  In one embodiment, the single domain antibody polypeptide construct binds human VEGF with a Kd ranging from 10 nM to 50 pM.

  In one embodiment, the single domain antibody polypeptide construct binds human VEGF with a Kd ranging from 1 nm to 50 pM.

  In one embodiment, the single domain antibody polypeptide construct neutralizes human VEGF as measured in a VEGF receptor 1 test or a VEGF receptor 2 test.

  The present invention is a method of treating rheumatoid arthritis, comprising a therapeutically effective amount of a single domain antibody polypeptide construct that antagonizes binding to a receptor for human VEGF for an individual in need thereof. Wherein the single domain antibody polypeptide construct further comprises a method of inhibiting the binding of human VEGF to the VEGF receptor, thereby treating rheumatoid arthritis.

  In one embodiment, the single region antibody polypeptide construct specifically binds to human VEGF bound to a cell surface receptor.

  In one embodiment, the single region antibody polypeptide construct is conjugated to a PEG molecule.

  In one embodiment, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa.

  In one embodiment, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa.

  In one embodiment, the PEGylated protein of the invention is conjugated to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. can do.

  In one embodiment, the antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human VEGF.

  In one embodiment, the antibody construct comprises a homodimer of a single immunoglobulin variable region polypeptide that binds human VEGF.

  In one embodiment, the antibody construct comprises a homotrimer of a single immunoglobulin variable region polypeptide that binds human VEGF.

  In one embodiment, the antibody construct comprises a homotetramer of a single immunoglobulin variable region polypeptide that binds human VEGF.

  In one embodiment, the construct further comprises an antibody polypeptide specific for an antigen other than VEGF.

  In one embodiment, the antibody polypeptide specific for an antigen other than VEGF comprises a single region antibody polypeptide.

  In one embodiment, binding of an antigen other than VEGF by an antibody polypeptide specific for an antigen other than VEGF increases the in vivo half-life of the antibody polypeptide construct.

  In one embodiment, the antigen other than VEGF comprises a serum protein.

  In one embodiment, the serum protein is from the group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. Selected.

  In one embodiment, the antigen other than VEGF comprises HSA.

  In one embodiment, the single domain antibody polypeptide construct has an in vivo tα half-life ranging from 15 minutes to 12 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life in the range of 1 to 6 hours.

  In other embodiments, the single domain antibody polypeptide construct has an in vivo tβ half-life in the range of 2 to 5 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life in the range of 3 to 4 hours.

  In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 60 hours. In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 48 hours.

  In other embodiments, the single region antibody polypeptide construct has an in vivo tβ half-life ranging from 12 to 26 hours. In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 150 mg / ml. In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 100 mg / ml. In other embodiments, the single region antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 75 mg / ml. In other embodiments, the single domain antibody polypeptide construct has an in vivo AUC half-life value of 15 mg / ml to 50 mg / ml.

  In one embodiment, the treatment further comprises administration of at least one additional therapeutic agent.

  In one embodiment, the therapeutic agent is selected from the group consisting of etanercept, infliximab, and D2E7.

  In one embodiment, the therapeutic agent is corticosteroid, proteolytic enzyme, non-steroidal anti-inflammatory drug (NTHES), acetylsalicylic acid, pyrazolone, phenamic acid, diflunisal, acetic acid derivative, propionic acid derivative, oxicam, mefenamic acid, ponster , Meclofenamate, mechromene, phenylbutazone, butazolidine, diflunisal, drobido, diclofenac, voltaren, indomethacin, indosin, sulindac, clinolinyl, etodolac, rosin, ketorolac, tradol, nabumetone, lerafen, tolmethine, lectin, ibuprofen, motuline, phenoline Profen, Narfone, Flurbiprofen, Ante, Carprofen, Rimadil, Ketoprofen, Orgis, Naproxen, Anaprox, Napro Emissions, is selected from the group consisting of piroxicam and Verden.

からなる群から選択される抗体ポリペプチドのＣＤＲ３のアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

Comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of:

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９０％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９２％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 92% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９４％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 94% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９６％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９８％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又はそれに対して少なくとも９９％の同一性を有するアミノ酸配列を含む。 In one embodiment, the single domain antibody polypeptide construct is a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or an amino acid sequence having at least 99% identity thereto.

からなる群から選択されるＣＤＲ３配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further comprising a composition comprising a CDR3 sequence selected from the group consisting of:

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 85% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９０％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 90% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９２％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 92% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９４％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 94% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９６％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 96% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９８％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 98% identity thereto.

からなる群から選択されるアミノ酸配列、又はそれに対して少なくとも９９％の同一性を有する配列を含む組成物をさらに包含する。 The present invention includes single region antibody polypeptide constructs that antagonize binding of human VEGF to the receptor, wherein the single region antibody polypeptide construct comprises:

Further included are compositions comprising an amino acid sequence selected from the group consisting of, or a sequence having at least 99% identity thereto.

  The present invention relates to a method for treating rheumatoid arthritis, which antagonizes binding to a human TNFα receptor and antagonizes binding to a human VEGF receptor for an individual in need thereof. It further includes administering a therapeutically effective amount of the composition comprising the polypeptide construct, whereby rheumatoid arthritis is treated.

  In one embodiment, the composition prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis.

  In other embodiments, administration of the composition to Tg197 transgenic mice comprises: a) administering the composition weekly by intraperitoneal injection to heterozygous Tg197 transgenic mice; and b) mice of step a). C) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = Scoring the mice for macrophenotypic signs of arthritis according to a system of severe arthritis (significant movement disorder).

  In other embodiments, the composition has an effect in a Tg197 transgenic mouse arthritis test that is greater than or equal to the effect of a drug selected from the group consisting of etanercept, infliximab and D2E7 within statistical significance.

  In other embodiments, the treatment comprises inhibiting the progression of rheumatoid arthritis.

  In other embodiments, the treatment comprises preventing or delaying the onset of rheumatoid arthritis.

  In other embodiments, administration results in a statistically significant change in one or more indicators of RA.

  In other embodiments, one or more indicators of RA include: blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, x-ray imaging of one or more joints, As well as any histopathological analysis of the fixation of one or more joints.

  In other embodiments, the one or more indicators of RA comprise a reduction in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, and Tg197 transgenic mice Are scored for the macrophenotypic signs of arthritis, where the macrophenotypic signs of arthritis are 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (Swelling, joint deformation), 3 = scored according to system of severe arthritis (significant movement disorder).

  In other embodiments, the one or more indicators of RA comprise a decrease in macrophenotypic signs of histopathological signs of arthritis in Tg197 transgenic mice and the composition is administered to Tg197 transgenic mice Tg197 transgenic mice are scored for histopathological signs of arthritis, which are realized in the joints, 0 = no detectable pathology, 1 = synovial hyperplasia and multiple Presence of morphonuclear infiltrates, 2 = pannus and fibrous tissue formation and localized subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = scored using extensive articular cartilage destruction and bone erosion system The

  In other embodiments, the single region antibody polypeptide construct comprises a human single region antibody polypeptide.

  In other embodiments, the human single region antibody polypeptide binds TNFα and VEGF.

  In other embodiments, the single region antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test.

  In other embodiments, the single region antibody polypeptide construct is conjugated to a PEG molecule.

  In other embodiments, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa.

  In other embodiments, the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa.

  In other embodiments, the antibody polypeptide construct binds on average to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Is done.

  In other embodiments, the antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human TNFα and / or two or more single immunoglobulin variable region polypeptides that bind human VEGF. .

  In other embodiments, the antibody construct is a homodimer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homodimer of a single immunoglobulin variable region polypeptide that binds human VEGF. including.

  In other embodiments, the antibody construct is a homotrimer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homotrimer of a single immunoglobulin variable region polypeptide that binds human VEGF. including.

  In other embodiments, the antibody construct is a homotetramer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homotetramer of a single immunoglobulin variable region polypeptide that binds human VEGF. including.

  In other embodiments, the construct further comprises an antibody polypeptide specific for an antigen other than TNFα or VEGF.

  In other embodiments, the antibody polypeptide specific for an antigen other than TNFα or VEGF comprises a single region antibody polypeptide.

  In other embodiments, binding of an antigen other than TNFα or VEGF by an antibody polypeptide specific for an antigen other than TNFα or VEGF increases the in vivo half-life of the antibody polypeptide construct.

  In other embodiments, the antigen other than TNFα or VEGF comprises a serum protein.

  In other embodiments, the serum protein is a group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. Selected from.

  In other embodiments, the antigen other than TNFα comprises HSA.

  In other embodiments, the treatment further comprises administration of at least one additional therapeutic agent.

  The present invention includes single domain antibody polypeptide constructs that antagonize binding to human TNFα receptor and antagonize binding to human VEGF receptor when administered to mice in a Tg197 transgenic mouse model of arthritis. A composition that prevents an increase in arthritis score, wherein the single domain antibody polypeptide construct further includes a composition that inhibits the progression of rheumatoid arthritis.

  The present invention includes single domain antibody polypeptide constructs that antagonize binding to human TNFα receptor and antagonize binding to human VEGF receptor when administered to mice in a Tg197 transgenic mouse model of arthritis. A composition that prevents an increase in arthritic score, wherein the single domain antibody polypeptide construct further comprises a composition that binds human TNFα with a Kd of less than 100 nM.

  The present invention includes single domain antibody polypeptide constructs that antagonize binding to human TNFα receptor and antagonize binding to human VEGF receptor when administered to mice in a Tg197 transgenic mouse model of arthritis. A composition that prevents an increase in arthritic score, wherein the single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test, and the single domain antibody polypeptide construct is a rheumatoid arthritis The single region antibody polypeptide construct further includes a composition that binds human TNFα with a Kd of less than 100 nM.

  Another aspect is a single domain antibody polypeptide construct that antagonizes binding to the receptor for human TNFα and prevents an increase in arthritis score when administered to mice in a Tg197 transgenic mouse model of arthritis, comprising: A method for selecting a single domain antibody polypeptide construct that neutralizes human TNFα as measured in a typical L929 cell test, inhibits progression of rheumatoid arthritis, and binds human TNFα with a Kd of less than 100 nM. (1) mutating nucleic acids encoding several hypervariable region sites of the single region antibody polypeptide such that all possible amino substituents are generated at each site; and (2) step The mutant hypervariable region site generated in (1) is introduced into the phagemid display vector and displayed on the phagemid surface display protein. Forming a large population of display vectors each capable of expressing one of said mutant hypervariable region sites, and (3) the mutant hypervariable region site thus generated as a fusion Expressing the mutant hypervariable region site on the surface of the filamentous phage particle so as to be monovalently displayed from the filamentous phage particle to the gene III product of M13 encapsulated in each particle; (4 ) Screening for the ability to bind surface expressed phage particles to TNFα; (5) isolating surface expressed phage particles capable of binding to TNFα; and (6) binding TNFα. Also, when administered to mice in the Tg197 transgenic mouse model of arthritis, it prevents an increase in arthritic score and is measured in a standard L929 cell test. Neutralizes human TNFα, inhibits the progression of rheumatoid arthritis, and antagonizes the binding of human TNFα to the receptor by selecting surface expressed phage particles in step (5) that bind human TNFα with a Kd of less than 100 nM A method comprising selecting one or more phagemid species containing a display protein containing a single region antibody polypeptide construct.

  Another aspect is a method of treating rheumatoid arthritis comprising a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human VEGF for an individual in need thereof. The rheumatoid arthritis is treated, wherein the single domain antibody polypeptide construct is in the range of 15 minutes to 12 hours, 1 to 6 hours, 2 to 5 hours, or 3 to 4 hours. A method having an in vivo tα half-life.

  Another embodiment is a method of treating rheumatoid arthritis, wherein an individual in need of such treatment comprises a therapeutically effective amount of a single domain antibody polypeptide construct that antagonizes binding to a receptor for human VEGF. Administering a composition, whereby the single domain antibody polypeptide construct is a method having an in vivo tβ half-life in the range of 12 to 60 hours, 12 to 48 hours, or 12 to 26 hours.

  Another embodiment is a method of treating rheumatoid arthritis, wherein an individual in need of such treatment comprises a therapeutically effective amount of a single domain antibody polypeptide construct that antagonizes binding to a receptor for human VEGF. Administering the composition, whereby the single domain antibody polypeptide construct comprises 15 mg / ml to 150 mg / ml, 15 mg / ml to 100 mg / ml, 15 mg / ml to 75 mg / ml Or a method having an in vivo AUC half-life value of 15 mg / ml to 50 mg / ml.

Another embodiment is a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of a composition to an individual in need thereof, said composition against a receptor for human TNFα. A single domain antibody polypeptide construct that antagonizes binding, thereby treating the rheumatoid joint, wherein the composition prevents an increase in arthritic score when administered to a Tg197 transgenic mouse model of arthritis The single region antibody polypeptide construct binds human TNFα and VEGF with a Kd of less than 100 nM each, the single region antibody polypeptide construct binds human TNFα and VEGF with a Kd of 100 nM to 50 pM, respectively, wherein said single domain antibody polypeptide construct, human TNFα and VE, respectively from 30nM at 50pM a _{K d} Combining F, the single domain antibody polypeptide construct, 10 nM binds human TNFα and VEGF in 50pM of Kd from each or said single domain antibody polypeptide construct, _{K d} in the range of 50pM of each 1nm In this method, human TNFα and VEGF are bound together.

  Another embodiment is a method of treating rheumatoid arthritis, wherein an individual in need of such treatment antagonizes binding to a receptor for human TNFα and a single antagonizing binding to a receptor for human VEGF. Administering a therapeutically effective amount of the composition comprising a domain antibody polypeptide construct, wherein the single domain antibody polypeptide construct inhibits binding of human TNFα to the TNFα receptor and binding of human VEGF to the VEGF receptor. And thereby treating the rheumatoid arthritis.

  Another embodiment is a method of treating rheumatoid arthritis, wherein an individual in need of such treatment antagonizes binding to a receptor for human TNFα and a single antagonizing binding to a receptor for human VEGF. Administering a therapeutically effective amount of the composition comprising a domain antibody polypeptide construct, wherein the single domain antibody polypeptide construct inhibits binding of human TNFα to the TNFα receptor and binding of human VEGF to the VEGF receptor. And thereby treating the rheumatoid arthritis, wherein the single domain antibody polypeptide construct specifically binds to human TNFα bound to a cell surface receptor.

  Another embodiment is a method of treating rheumatoid arthritis comprising administering a therapeutically effective amount of a composition to an individual in need thereof, said composition against a receptor for human TNFα. A single domain antibody polypeptide construct that antagonizes binding and antagonizes binding of a human VEGF to a receptor thereby treating the rheumatoid arthritis, wherein the single domain antibody polypeptide construct binds to a cell surface receptor This method specifically binds to human TNFα.

  Another embodiment of the invention is a method of treating rheumatoid arthritis comprising administering an antibody construct specific for TNFα, wherein the sequence of the antibody construct is an anti-TNF-α listed herein. A method for treating rheumatoid arthritis comprising or consisting of a sequence having 85, 90, 95, 96, 97, 98, 99% or more or 100% identity to the sequence of any one of the clones .

  Another embodiment of the invention is a composition comprising an antibody construct specific for TNFα, wherein the sequence of the antibody construct is any one of the anti-TNF-α clones listed herein. It is a composition comprising or consisting of a sequence having 85, 90, 95, 96, 97, 98 or 99% identity or 100% identity to the sequence of the clone.

  Another embodiment of the present invention is a method of treating rheumatoid arthritis comprising administering an antibody construct specific for VEGF, wherein the sequence of the antibody construct is of the anti-VEGF clones listed herein. It is a method for treating rheumatoid arthritis comprising or consisting of a sequence having 85, 90, 95, 96, 97, 98, 99% or more, or 100% identity to the sequence of any one clone.

  Another embodiment of the invention is a composition comprising an antibody construct specific for VEGF, wherein the sequence of the antibody construct is that of any one of the anti-VEGF clones listed herein. It is a composition comprising or consisting of a sequence having 85, 90, 95, 96, 97, 98 or 99% identity or 100% identity to the sequence.

In other embodiments, two copies of the V _H or V _L single domain antibody that binds a first antigen or epitope, of the V _H or V _L single domain antibody that binds a second antigen or epitope A tetravalent bispecific antigen binding polypeptide construct comprising two replicates is provided. The first and second epitopes can be present on the same antigen or on different antigens. Each of the two replicas of a single region antibody that binds the first antigen or epitope is fused to the respective IgG heavy chain constant region, and two replicas of the single region antibody that binds the second antigen or epitope Each of the objects is fused to a respective light chain constant region. These tetravalent bispecific polypeptide constructs are IgG-like in that they have two antigen-binding arms connected by a heavy and light chain constant region. They have two different antigen-specific single domain antibody polypeptides in each arm, so that each arm binds two different antigens or epitopes, making the construct tetravalent and bispecific. It differs from natural IgG in that it is possible. In one embodiment, the first and second epitopes are identical such that there are four specific binding sites for that epitope present in the polypeptide construct. In other embodiments, the first and second epitopes are different and are present on the same or different antigens.

The bispecific tetravalent polypeptide constructs described herein are directed against single region antibody sequences specific for any two antigens or epitopes, specifically human TNF-α and VEGF. Specific sequences can be included, more particularly any of the single region antibody sequences described herein. In other embodiments, a _Cκ or _Cλ light chain constant region can be used, and IgG heavy chain constant regions other than IgG1 can be used.

A single-region anti-TNF-α antibody clone that prevents an increase in arthritis score when administered as a monomer to a Tg197 transgenic mouse model of arthritis, and as a monomer to a mouse of a collagen-induced arthritis mouse model Also included are constructs of this type that include single domain anti-VEGF antibody clones that prevent an increase in arthritic score. In a further embodiment, the single region antibody TNF-α antibody clone used, when used as a monomer, neutralizes TNF-α in the L929 cytotoxicity test described herein, The single domain anti-VEGF antibody clone used, when used as a monomer, antagonizes VEGF receptor binding in a test for VEGF receptor 2 binding as described herein. In a further embodiment, the single region antibody clones used bind each antigen or epitope with a _Kd of less than 100 nM. In a further embodiment, the bispecific bivalent construct binds the respective antigen or epitope with a K _d of less than 100 nM and has an arthritic score in either or both of the Tg197 and CIA models described herein. Prevent increase.

  Such tetravalent bispecific constructs can be used in the treatment of rheumatoid arthritis in the same manner as other constructs described herein in terms of administration, dose and efficacy monitoring. For example, as described above, by adding a PEG component or by further fusing a binding component specific to a serum protein such as HSA, such as an additional single region antibody, such as by increasing circulating half-life. It is possible to change the half-life of the construct.

  Then, in one aspect, a bispecific antigen binding polypeptide is described comprising a first antibody single region polypeptide that binds TNF-α and a second antibody single region polypeptide that binds VEGF. The The polypeptide can be used, for example, for the treatment of rheumatoid arthritis.

  Then, in another embodiment, a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region, and b) said first A second replica of one fusion protein and c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region; d) a second replica of the second fusion protein, wherein the first and second replicas of the first fusion protein are disulfide bonded to each other via their respective IgG heavy chain constant regions The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein, and the second Fusion Tampa The light chain constant region of the second replica of the quality is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein, and the polypeptide construct comprises the first and A tetravalent bispecific antigen binding polypeptide construct that binds the second epitope is described.

In one embodiment of the tetravalent bispecific antigen binding polypeptide construct, the single region antibody that binds the first epitope is a V region selected from V _H and V _L.

In other embodiments, the single region antibody that binds the second epitope is a V region selected from V _H and V _L.

  In other embodiments, the IgG heavy chain constant region is an IgG1 heavy chain constant region.

In other embodiments, the light chain constant region is a _Cκ or _Cλ light chain constant region. In other embodiments, the light chain constant region is a _Cκ light chain constant region.

  In other embodiments, the first and second epitopes are present on the same antigen.

  In other embodiments, the first and second epitopes are present on different first and second antigens.

  In another embodiment, one or both of the single region antibody that binds a first epitope and the single region antibody that binds a second epitope are human single region antibodies.

  In other embodiments, the single region antibody polypeptide that binds the first epitope and / or the single region antibody polypeptide that binds the second epitope is encoded by a human germline VH gene sequence. FW1, FW2 encoded by the human germline VH gene sequence, FW3 encoded by the human germline VH gene sequence, and FW4 encoded by the human germline VH gene sequence.

  In other embodiments, both the single region antibody polypeptide that binds the first epitope and the single region antibody that binds the second epitope are human single region antibodies.

  In other embodiments, the IgG heavy chain constant region is a human IgG heavy chain constant region.

In other embodiments, IgG heavy chain constant region includes _{C H} 1 region.

In other embodiments, the light chain constant region is a _Cκ light chain constant region.

In other embodiments, the construct binds TNF-α and VEGF. In other embodiments, a single region antibody that binds VEGF is fused to an IgG1 heavy chain constant region, and a single region antibody that binds TNF-α is fused to a _Cκ light chain constant region.

  In other embodiments, a single domain antibody that binds VEGF prevents an increase in arthritic score when administered to mice in a collagen-induced arthritis mouse model.

In other embodiments, the single domain antibody polypeptide component that antagonizes the activity of human VEGF binds human VEGF with a _Kd of less than 100 nM.

  In other embodiments, the single domain antibody polypeptide component that antagonizes the activity of human VEGF neutralizes human VEGF as measured in a VEGF receptor 1 test or a VEGF receptor 2 test.

  In other embodiments, the single domain antibody polypeptide component that antagonizes the activity of human VEGF specifically binds to human VEGF bound to a cell surface receptor.

In other embodiments of this aspect, single domain antibodies that bind TNF-α or antagonize the activity of human TNF-α have an arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis. Prevent increase. In other embodiments, the single domain antibody neutralizes human TNF-α as measured in a standard L929 cytotoxicity test. In other embodiments, the single domain antibody binds human TNF-α with a K _d of less than 100 nM.

からなる群から選択される抗体ポリペプチドのＣＤＲ３のアミノ酸配列、又は前記配列に対して例えば少なくとも８５％、或いは少なくとも９０％、９２％、９４％、９６％、９８％、９９％又は１００％の同一性を有するアミノ酸配列を含むことが可能である。 In any embodiment of this aspect, the single region antibody polypeptide component that antagonizes the activity of human TNF-α is, for example, a clone

CDR3 amino acid sequence of an antibody polypeptide selected from the group consisting of, or for example, at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or 100% relative to said sequence It is possible to include amino acid sequences having identity.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又は前記配列に対して例えば少なくとも８５％、或いは少なくとも９０％、９２％、９４％、９６％、９８％、９９％又は１００％の同一性を有するアミノ酸配列を含むことが可能である。 In other embodiments of this aspect, the single region antibody polypeptide component that antagonizes the activity of human TNF-α is, for example, a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identity to said sequence, for example It is possible to include an amino acid sequence having

からなる群から選択される抗体ポリペプチドのＣＤＲ３のアミノ酸配列、又は前記配列に対して例えば少なくとも８５％、或いは少なくとも９０％、９２％、９４％、９６％、９８％、９９％又は１００％の同一性を有するアミノ酸配列を含むことが可能である。 In any embodiment of this aspect, the single domain antibody polypeptide component that antagonizes binding of human VEGF to the VEGF receptor is, for example, a clone

CDR3 amino acid sequence of an antibody polypeptide selected from the group consisting of, or for example, at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or 100% relative to said sequence It is possible to include amino acid sequences having identity.

からなる群から選択される抗体ポリペプチドのアミノ酸配列、又は前記配列に対して例えば少なくとも８５％、或いは少なくとも９０％、９２％、９４％、９６％、９８％、９９％又は１００％の同一性を有するアミノ酸配列を含むことが可能である。 In other embodiments of this aspect, the single region antibody polypeptide component that antagonizes binding of human VEGF to VEGF is, for example, a clone

The amino acid sequence of an antibody polypeptide selected from the group consisting of: or at least 85%, or at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identity to said sequence, for example It is possible to include an amino acid sequence having

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, cell culture, molecular genetics, nucleic acid chemistry, high Hybridization techniques and biochemistry). Molecular, genetic and biochemical methods (generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, incorporated herein by reference. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Cold Spring Harbour. Y .; and Ausubel et al., Short protocols in Molecular Biology (1999), 4th edition, John Willy & Sons, Inc.), and chemical methods are used standard techniques.

  As used herein, the term “region” means a folded protein that retains its tertiary structure independently of the rest of the protein. In general, regions are associated with individual functional properties of the protein and can often be added, removed or transcribed to other proteins without losing the function of the rest of the protein and / or region.

“Single immunoglobulin variable region” or “single region antibody polypeptide” includes a sequence characteristic of an immunoglobulin variable region and specifically binds an antigen (ie, has a dissociation constant of 500 nM or less). Means polypeptide region. Thus, a “single region antibody polypeptide” is a complete antibody variable region as well as, for example, one or more loops that are not characteristic of the antibody variable region, or have been cleaved, or N- or C- A modified variable region substituted with an antibody variable region comprising a terminal extension, and a dissociation constant of 500 nm or less (eg, 450 nM or less, 400 nM or less, 350 nM or less, 300 nM or less, 250 nM or less, 200 nM or less, 150 nM or less, 100 nM or less), and Includes variable region folding fragments that retain the target antigen specificity of the full-length region. Preferably, the antibody single variable region is selected from the group consisting of V _H and V _L including V _kappa and V _lambda .

The phrase “single domain antibody polypeptide construct” refers to a larger polypeptide comprising one or more monomers of a single immunoglobulin variable region polypeptide sequence as well as an isolated single domain antibody polypeptide. Also encompassed are peptide constructs. It is emphasized that single domain antibody polypeptides that are part of larger constructs are uniquely capable of specifically binding target antigens. Thus, a single domain antibody polypeptide construct comprising two or more single domain antibody polypeptides, for example, V _H and V to form the binding sites required to bind specifically to a single antigen molecule _It does not include constructs where both _L regions are required. The binding of the single region antibody polypeptide ring in the single region antibody polypeptide construct can be a peptide or polypeptide bond, or other chemical bond, such as through the binding of a polypeptide monomer to a multivalent PEG. The bound single region antibody polypeptides can be the same or different, and the target specificities of the constituent polypeptides can be the same or different as well.

Complementary: Two immunoglobulin regions are “complementary” if they belong to or are derived from a family of constructs that form cognate pairs or groups and retain this characteristic. For example, the VH and VL regions of an antibody are complementary and the two VH regions are not complementary and the two V regions are not complementary. Complementary region can be found in other members of such immunoglobulin superfamily V _alpha and V _beta (or V _gamma and V _[delta]) region of the T cell receptor. Within the scope of the second configuration of the invention, the complementary regions do not bind the target molecule cooperatively but act independently on different target epitopes that may be present on the same or different molecules. Artificial regions such as regions based on protein backbones that do not bind epitopes unless designed to bind epitopes are non-complementary. Similarly, two regions based on (for example) an immunoglobulin region and a fibronectin region are not complementary.

  Immunoglobulin: This refers to a family of polypeptides that retain the immunoglobulin folding properties of antibody molecules, including two β-sheets and usually a conserved disulfide bond. Members of the immunoglobulin superfamily have broad roles in the immune system (eg, antibodies and T cell receptor molecules), involvement in cell adhesion (eg, ICAM molecules) and intracellular signaling (eg, receptor molecules such as PDGF receptors). ) Involved in many aspects of in vivo cellular and non-cellular interactions.

  The present invention is applicable to any immunoglobulin superfamily molecule that possesses a binding region. Preferably, the present invention relates to antibodies.

Joining: The variable regions according to the present invention are joined to form a group of regions. For example, complementary regions can be bound, such as binding a _VL region to a VH region. Non-complementary regions can also be combined. The regions can be joined in several ways, including joining the regions by covalent or non-covalent binding means.

  Closed structure multispecific ligand: This phrase describes a multispecific ligand as defined herein comprising at least two epitope binding regions contemplated herein. The term “closed structure” (multispecific ligand) means that the epitope binding region of a ligand is configured such that epitope binding by one epitope binding region competes with epitope binding by another epitope binding region. To do. That is, cognate epitopes can be individually bound by each epitope binding region, but not simultaneously. The closed structure of the ligand can be achieved using the methods described herein.

  Antibody: An antibody (eg, IgG) derived from any species that naturally produces antibodies, or made by recombinant DNA technology, or isolated from serum, B cells, hybrid cells, transfected cells, yeast or bacteria. IgM, IgA, IgD or IgE) or fragment (Fab, F (ab ′) 2, Fv, disulfide bond Fv, scFv, closed structure multispecific antibody, disulfide bond scFv, bispecific antibody, etc.) .

  Bispecific ligand: a ligand comprising a first immunoglobulin single variable region and a second immunoglobulin single variable region as defined herein, wherein the variable regions are monospecific immunity A ligand capable of binding to two different antigens or two epitopes on the same antigen not normally bound by globulin. For example, two epitopes can be in the same hapten, but not in the same epitope and not close enough to be bound by a monospecific ligand. Bispecific ligands according to the present invention are composed of variable regions having different specificities and do not include variable region pairs that are complementary to each other and have the same specificity.

  Antigen: A molecule that is bound by a ligand according to the invention. Typically, the antigen is bound by an antibody ligand and can enhance the antibody response in vivo. It may be a polypeptide, protein, nucleic acid or other molecule. In general, bispecific ligands according to the present invention are selected for target specificity for a particular antigen. In the case of conventional antibodies and fragments thereof, the antibody binding sites (L1, L2, L3 and H1, H2, H3) defined by the variable loop can bind to the antibody.

  Epitope: A unit of structure conventionally bound by an immunoglobulin VH / VL pair. An epitope defines the minimal binding site for an antibody and represents the target for antibody specificity. In the case of a single region antibody, an epitope represents a unit of structure that is individually bound by variable regions.

  Universal ligand: A ligand that binds to all members of the repertoire. Generally, it does not bind through the antigen binding site defined above. Non-limiting examples include protein A, protein L, and protein G.

  Selection: Induced by screening or by a Darwin selection process in which binding interactions are performed between a region and an antigen or epitope, or between an antibody and an antigen or epitope. Thus, the first variable region can be selected for binding to an antigen or epitope in the presence or absence of a complementary variable region.

  Universal framework: corresponding to the region of an antibody conserved in a sequence defined by Kabat ("Sequences of Proteins of Immunological Interest, US Department of Health and Human Services", or Chothia and Lesk, (1987) J. Mol. Biol. 196. A single antibody framework sequence corresponding to the human germline immunoglobulin repertoire or structure defined by 910-917.The present invention allows the induction of virtually any binding specificity through changes in only the high frequency region. Provide the use of a single framework or a collection of such frameworks.

  Homogeneous immunity test: An immunity test that detects a specimen without the need to separate the bound and unbound reagents.

  Substantially identical (or “substantially homologous”): a sufficient number of identical or equivalent to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleic acid sequences have similar activity A first amino acid or nucleotide sequence comprising an amino acid residue or nucleotide (eg having a similar side chain, eg having a conserved amino acid substituent). In the case of an antibody, the second antibody has the same binding specificity and at least 50% of its affinity.

  A “region antibody” or “dAb” is equivalent to a “single immunoglobulin variable region polypeptide” or “single region antibody polypeptide”, as that term is used herein.

As used herein, the phrase “specifically binds” refers to, for example, the BIAcore ™ surface plasmon resonance system and the BIAcore ™ kinetic evaluation software (eg, version 2.1). It means the binding of an antigen by an immunoglobulin variable region with a dissociation constant (K _d ) of 1 μM or less measured by surface plasmon resonance analysis used. The affinity of K _d for specific binding interactions is preferably about 500 nM or less, more preferably about 300 nM or less.

As used herein, the term “high affinity binding” means a binding of a _{Kd of} 100 nM or less.

  As used herein, the phrase “human single region antibody polypeptide” means a polypeptide having a sequence derived from a human germline immunoglobulin V region. The sequence can be one or more (if isolated from a library of human individuals or cloned human antibody gene sequences (or human antibody V region gene sequences), or then selected for binding to the desired target antigen ( “Derived from human germline V region” when a cloned human germline V region sequence is used to purify one or more diversified sequences (either randomly or by subject mutagenesis) . At a minimum, a human immunoglobulin variable region has at least 85% amino acid similarity (eg, 87%, 90%, 93%, 95%, 97%, or 99% similarity or more) to a native human immunoglobulin variable region sequence. Have sex).

  Alternatively or in addition, a “human immunoglobulin variable region” is described by four human immunoglobulin variable region framework regions (W1-FW4) (the framework regions are described by Kabat et al. (1991, supra). Is a variable region. A “human immunoglobulin variable region framework region” includes a) an amino acid sequence of a human framework region and b) a framework region comprising at least 8 contiguous amino acids of the amino acid sequence of the human framework region. The human immunoglobulin variable region can comprise an amino acid sequence of FW1-FW4 that is identical to the amino acid sequence of the corresponding framework region decoded by a human germline antibody gene segment, or the FW1-FW4 sequence is human reproductive. 10 or less amino acid sequence difference, 9 or less amino acid sequence difference, 8 or less amino acid sequence difference, 7 or less amino acid sequence difference, 6 or less with respect to the amino acid sequence of the corresponding framework region decoded by the series antibody gene segment A variable region that collectively includes 5 amino acid sequence differences, 5 amino acid sequence differences, 4 amino acid sequence differences, 3 amino acid sequence differences, 2 amino acid sequence differences, or 1 amino acid sequence differences. You can also.

  As defined herein, a “human immunoglobulin variable region” refers to a variable region that exists alone as a single immunoglobulin variable region, or that is combined with one or more additional polypeptide sequences. It has the ability to specifically bind antigen independently, regardless of whether it exists as an immunoglobulin variable region. A “human immunoglobulin variable region”, as the term is used herein, is a “humanized” immunoglobulin polypeptide, ie, a human whose constant region has been modified to be less immunogenic in humans. Does not include immunoglobulins other than (eg mice, camels, etc.).

  As used herein, the phrase “sequence characteristics of an immunoglobulin variable region” refers to 20 or more, 25 or more, 30 or more, 35 or more, 40 As mentioned above, it means an amino acid sequence that is homologous over 45 or more or 50 consecutive amino acids.

  As used herein, the term “bivalent” means that an antigen-binding antibody polypeptide has two antigen-specific binding sites. The epitope recognized by the antigen binding site can be the same or different. An antibody polypeptide is “bispecific” when it binds two different epitopes (present on different or the same antigen) via each two antigen-specific binding sites. Is said.

  As used herein, the term “tetravalent” means that an antigen-binding polypeptide has four antigen-specific binding sites. The epitope recognized by the antigen binding site can be the same or different. A “bispecific” tetravalent antibody polypeptide has two binding sites for one epitope or antigen and two binding sites for different epitopes or antigens.

  As used herein, a “tetravalent bispecific antigen-binding polypeptide construct” is a native IgG in that it has two antigen-binding arms joined by heavy and light chain constant regions. It has a structure similar to However, unlike natural IgG, each arm has two antigen binding regions, an antigen binding region specific for the first antigen and an antigen binding region specific for the second antigen. In the tetravalent bispecific antigen binding polypeptide constructs described herein, each of the antigen binding regions is a single region antibody. That is, the antigen binding regions do not pair with each other to form a single binding site, eg, scFvs.

As used herein, the term “IgG format” is similar to native IgG in that the construct has two antigen-binding arms joined together by heavy and light chain constant regions. Means an artificial antigen-binding polypeptide having the above structure. As described herein, an antigen binding polypeptide in IgG format is a first antigen or epitope fused to an IgG heavy chain constant region (eg, C _H 1-C _H 2 -C _H 3). A single region that binds to a second antigen fused to two copies of a first fusion protein comprising a single region antibody polypeptide that binds to a light chain constant region (eg, C _λ or C _κ ) Consists of four polypeptide chains with two replicas of a second fusion protein containing an antibody polypeptide. In this format, when co-expressed in cells, the heavy chain constant regions are disulfide bonded to each other, and each of these heavy chain constant regions is also disulfide bonded to the light chain constant region. An antigen binding polypeptide in the IgG format is tetravalent, as that term is used herein. A single region antibody fused to a constant region binds a different antibody (eg, dAb1 fused to the heavy chain constant region binds one antigen and dAb2 fused to the light chain constant region binds the other antigen. Or bind different epitopes on the same antigen (eg, dAb1 fused to the heavy chain constant region binds one epitope on the antigen and dAb2 fused to the light chain constant region is the same) All four regions bind the same epitope on the same antigen (dAb1 and dAb2 bind the same epitope on the same antigen). Can be combined).

As described herein, “Fab format” refers to one single region antibody fused to a light chain constant region C _L (eg, C _λ or C _κ ), and the other single region antibody is , Refers to a bivalent antibody polypeptide construct fused to the C _H 1 constant region, wherein each C _H 1 and C _L constant region is disulfide bonded to each other. Single region antibodies can be selected to bind different antibodies (producing a bispecific Fab format), different epitopes on the same antigen (bispecific), or the same epitope on the same antigen. Is possible. Examples of Fab format bispecific antibody polypeptides include, for example, an anti-TNF-α single region antibody described herein, eg, fused to a _Cλ light chain, and two fusion proteins of each through the constant regions are disulfide bonded to each other, is fused to a human heavy chain C _{H 1} constant region, including the anti-VEGF single domain antibodies described herein. In this form of antibody polypeptide construct, the antigen binding regions pair with each other and do not form a single binding site, eg, scFvs, and each single region antibody binds its own antigen to construct the construct. Can be made bivalent.

“Rheumatoid arthritis” (RA) refers to a disease involving inflammation of the inner surface of joints and / or other internal organs. RA typically affects many different joints. It is typically chronic and can be a recurrent disease. RA is a disease that affects the entire body and is one of the most common forms of arthritis. RA is characterized by inflammation of the membrane lining the joint that causes pain, stiffness, warmth, redness and swelling. The inflamed intra-articular surface, ie the synovium, can commit and damage bone and cartilage. Inflammatory cells release enzymes that can digest bone and cartilage. Affected joints lose their shape and alignment, resulting in pain and reduced movement. Symptoms include joint inflammation, swelling, and difficulty in movement and pain. Other symptoms include decreased appetite, fever, loss of vitality, and anemia. Other features include an aneurysm (rheumatic aneurysm) under the skin of the part that is subjected to pressure (eg, the back of the elbow). Rheumatoid arthritis is clinically scored based on a number of clinically approved criteria such as those described in US Pat. No. 5,698,195, which is incorporated herein by reference. In short, a clinical response test can evaluate the following parameters:
1. Evaluation of the number of tender joints and pain / tenderness The following scoring method is used.
0 = no pain / tenderness 1 = slight pain. Patients say tenderness when asked.
2 = moderate pain. The patient says he has tenderness and is depressed.
3 = severe pain. The patient says that there is tenderness, and winks and withdraws.
2. Number of swollen joints tenderness and swelling are assessed individually for each joint.
3. Morning stiffness (minutes)
4). 4. Grip strength Visual analog pain standard (0-10cm)
6). Patients and blinded evaluators are asked to evaluate the clinical response to the drug. Clinical response is assessed using the following subjective scoring system.
5 = Excellent response (best response expected)
4 = good response (less than the best expected response)
3 = Reasonable response (although improved but can be improved further)
2 = No response (no effect)
1 = worsening (disease)

  The cause of rheumatoid arthritis is not yet known. However, RA is an autoimmune disease where the immune system attacks healthy joint tissue, causing inflammation and subsequent joint damage. Many people with RA have a certain genetic marker called HLA-DR4.

  As used herein, the phrase “TNF-α related disease” refers to the administration of an agent that neutralizes or antagonizes the function of TNF-α, alone or in combination with other treatments. Means a disease or disorder that is effective in treating (the term “treatment” is as defined herein).

  As used herein, the term “treat” or “treatment” refers to the prevention of the onset of a disease or condition of the disease, the suppression of progression of the disease or condition of the disease, or the transformation of the disease or condition of the disease. Means.

  As described herein, the phrase “preventing the onset of a disease” refers to an individual who is susceptible to a given disease, such as one or more symptoms or measurable parameters of rheumatoid arthritis. It means not happening.

  As described herein, the phrase “inhibition of disease progression” refers to a disease that has already appeared in the individual being treated compared to progression in the absence of such treatment. It means to stop or relax the increase in the severity of symptoms.

  As described herein, the phrase “disease transformation” means that one or more symptoms or measurable parameters of a disease are compared to the symptoms or parameters before administration of the drug after administration of the drug. Means improvement. An “improvement” of a symptom or measurable parameter is evidenced by a statistically significant, preferably at least 10% significant difference in the measurable parameter.

  Measurable parameters can include both directly measurable parameters as well as indirectly measurable parameters. Non-limiting examples of parameters that can be directly measured include joint size, joint mobility, arthritis and histopathological scores or indicators, and serum levels of indicators such as cytokines. Indirectly measurable parameters include, for example, patient awareness of movement discomfort or reduction, or clinically accepted criteria for assessing disease severity.

  As used herein, an “increase” in a parameter, such as an arthritis score or other measurable parameter, means a statistically significant increase in that parameter. Alternatively, “increase” means an increase of at least 10%. Similarly, a “decrease” in the parameter means a statistically significant decrease in the parameter, or a decrease of at least 10%.

  As used herein, the term “antagonize” means that an agent interferes with activity. Where the activity is, for example, TNF-α, VEGF or other biologically active molecule or cytokine, the term includes, as a non-limiting example, binding or interaction to the receptor (in vitro or in culture). (At least 10) of the activity of the molecule or cytokine, including cell surface or in vivo), intracellular signaling, cytotoxicity, mitogenesis, or other downstream effects or processes mediated by the molecule or cytokine (eg, gene activation) %) Suppression. Antagonizing includes interference with receptor binding by factors such as TNF, VEGF, etc., as well as interference with the activity of the factor when the factor binds to a cell surface receptor.

  As used herein, the term “greater than” means that one value is equal to another value or is statistically significant (p <0.1, preferably p <0.05, more It means that it is preferably greater than that value (p <0.01). For example, if the effect of one composition is compared to the effect of another composition in treating disease or antagonizing receptor binding, the comparison should be made equimolar.

  As used herein, “conjugated” means the attachment of a polymer component, such as PEG, to an amino acid residue of an antibody polypeptide, eg, a single region antibody as described herein. To do. The attachment of a PEG polymer to an amino acid residue of an antibody polypeptide, such as a single domain antibody, is referred to as “PEGylation” and includes N-hydroxyl succinimide (NHS) active ester, succinimidyl propionate (SPA), maleimide (MAL), It can be achieved using a number of PEG-binding components including but not limited to vinyl sulfonate (VS) or thiol. A PEG polymer or other polymer can be attached to the antibody polypeptide at a predetermined location, or can be randomly attached to the antibody molecule. However, it is preferred to attach the PEG polymer to the antibody polypeptide at a predetermined location. The PEG polymer can be attached to any residue in the antibody polypeptide, but the polymer is naturally occurring in the antibody polypeptide, or the antibody is e.g. by mutagenesis of the natural residue in the antibody polypeptide to cysteine or lysine It is bound to lysine or cysteine that has been recombined into a polypeptide. As used herein, “coupled” refers to two or more antibody single variable region monomers that form a dimer, trimer, tetramer, or other multimer. It can also mean a meeting. expressing a dAb monomer as a fusion protein, linking two or more monomers via a peptide linker between the monomers, or translating the monomer to a disulfide bond, or di, tri or DAb monomers by several methods known in the art including, but not limited to, chemically linking directly to each other through a linker to a multivalent linking component (eg, multi-arm PEG) or through a linker Can be combined to form a multimer.

  As used herein, the phrase “directly attached” to a polymer that is “directly attached” to an antibody polypeptide, eg, a single variable region polypeptide, is essentially part of the variable region. Means, for example, the situation where the polymer is attached to a residue not included in the constant region, hinge region or linker peptide. Conversely, as used herein, the phrase “directly bound” to an antibody polypeptide refers to an amino acid residue that is part of a naturally occurring variable region (eg, that can be bound to a hinge region). It means the attachment of a polymer molecule to an antibody single variable region to which no polymer is attached to the group. A polymer is “indirectly linked” when it is attached to an antibody polypeptide via a binding peptide. That is, the polymer is not conjugated to amino acid residues that are part of the antibody itself. Alternatively, the polymer is “indirectly attached” to the antibody polypeptide when attached to the C-terminal hinge of the polypeptide, or attached to any residue in the constant region that may be present as part of the antibody polypeptide. The

As used herein, the term “homology” or “similarity” means the degree to which two nucleotide or amino acid sequences are structurally similar to each other. As used herein, sequence “similarity” is a measure of the degree to which amino acid sequences share similar amino acid residues at corresponding positions in a sequence alignment. Amino acids are similar to each other when their side chains are similar. Specifically, “similarity” encompasses amino acids that are conservative substitutions for one another. A “conservative” substitution is any substitution with a positive score in the blosum62 substitution matrix (Hentikoff and Hentikooff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919). By the expression “sequence A has n% similarity to sequence B”, n% of the positions of the optimal global alignment between sequences A and B consist of identical amino acids or conservative substitutions. Optimal global alignment can be achieved using the following parameters in the Needleman-Wunsch alignment algorithm.
About polypeptides:
Permutation matrix: blosum62.
Gap scoring function: -A -B * LG (where A = 11 (gap penalty), B = 1 (gap length penalty), LG is the length of the gap).
About nucleotide sequences:
Permutation matrix: 10 for matching, 0 for mismatching.
Gap scoring function: -A -B * LG (where A = 50 (gap penalty), B = 3 (gap length penalty), LG is the gap length).

  Typical conservative substitutions are between Met, Val, Leu and lle; between Ser and Thr; between residues Asp, Glu and Asn; between residues Gln, Lys and Arg; or the aromatic residue Phe And Tyr.

  As used herein, the two sequences are either Needleman-Wunsch algorithm, or Tatusova & Madden, 1999, FEMS Microbiol Lett. 174: 247-250 are “homologous” or similar to each other when they have at least 85% identity to each other when arranged using the “BLAST2 sequence”. If the amino acid sequence is sequenced using the “BLAST2 sequence algorithm”, the Blosum62 matrix is the default matrix.

  “Identity” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences, as is known in the art. In the art, “identity” also means the degree of sequence relationship between polypeptides or polypeptide sequences as determined by the match between sequences of sequences, depending on the situation. Percent identity is calculated according to Computational Molecular Biology, Lesk, A .; M.M. Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. et al. W. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A .; M.M. , And Griffin, H .; G. Ed., Human Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G .; Academic Press, 1987; Sequence Analysis Primer, Gribskov, M .; and Devereux, J. et al. Ed., M Stockton Press, New York, 1991; Carillo, H .; and Lipman, D.M. SIAM J .; Applied Math. 48: 1073 (1988), and can be readily calculated by known methods, including but not limited to those described above. Preferred methods for determining identity are designed to give the highest match between the sequences tested. The method for determining identity is compiled with publicly available computer programs. Preferred computer program methods for determining percent identity between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA (Attschul). S. F., et al., J. Molec. Biol. 215: 403-410 (1990), but are not limited to the BLAST X program, NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBINLM NIH Bethesda, Md. 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)). A polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” with respect to a reference nucleotide in “column number A” means that the polynucleotide sequence is every 100 nucleotides of the reference nucleotide sequence in “SEQ ID NO: A”. Except that it can contain up to 5 point mutations, it means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, in other words at least 95% of the reference nucleotide sequence. To obtain a polynucleotide having a nucleotide sequence with identity, up to 5% of the nucleotides in the reference sequence can be removed or replaced with other nucleotides, or up to 5% of all nucleotides in the reference sequence A number of nucleotides can be inserted into the reference sequence. These reference sequence variations are those 5 or 3 terminal positions of the reference nucleotide sequence, or those interspersed individually between nucleotides in the reference sequence, or in one or more consecutive groups within the reference sequence. Similarly, a polypeptide having an amino acid sequence with at least 95% identity to the reference amino acid sequence of “SEQ ID NO: B” can be any place between the terminal positions. Means that the amino acid sequence of the polypeptide is identical to the reference sequence, except that every 100 amino acids of the reference amino acid of “SEQ ID NO: B” can contain up to 5 amino acid modifications. In other words, to obtain a polypeptide having at least 95% amino acid sequence identity to the reference amino acid sequence, Or removing up to 5% of the amino acid residues or other amino acids may be substituted, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence are amino or carboxy terminal positions of the reference amino acid sequence, or individually scattered between residues in the reference sequence, or those within one or more consecutive groups within the reference sequence. Can occur anywhere between the end positions.

  As described herein, the terms “low stringency”, “medium stringency”, “high stringency” or “very stringent conditions” refer to conditions for nucleic acid hybridization and washing. Show. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N., incorporated herein by reference in its entirety. Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to in this specification are as follows. (1) Low stringency hybridization that is soaked in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C. and then washed twice with 0.2 × SSC, 0.1% SDS at least at 50 ° C. Conditions (Washing temperature can be raised to 55 ° C under low stringency conditions). (2) Medium stringent hybridization conditions of immersing in 6 × SSC at about 45 ° C. and then washing once or multiple times with 0.2 × SSC, 0.1% SDS at about 60 ° C. (3) High stringency hybridization conditions of immersing in 6 × SSC at about 45 ° C. and then washing once or multiple times with 0.2 × SSC, 0.1% SDS at 65 ° C. And preferably (4) very high stringency hybridization conditions are as follows: after immersion in 0.5 M sodium phosphate, 7% SDS at 65 ° C., once in 65 ° C. with 0.2 × SSC, 1% SDS Alternatively, it is washed multiple times.

  As used herein, the phrase “in a concentration of” means that a given polypeptide is dissolved in a solution (preferably an aqueous solution) in the stated mass or molar amount per unit volume. Thus, a polypeptide present “at a concentration of x” or “at least at a concentration of x” excludes dried and crystallized preparations of the polypeptide.

  As used herein, the term “repertoire” refers to a collection of diverse variants, eg, polypeptide variants that differ in their primary sequence. A library used in the present invention will encompass a repertoire of polypeptides comprising at least 1000 members.

  As used herein, the term “library” means a mixture of heterologous polypeptides or nucleic acids. A library consists of members each having a single polypeptide or nucleic acid sequence. To this extent, a library is synonymous with a repertoire. Sequence differences between library members are responsible for the diversity present in the library. The library can take the form of a simple mixture of polypeptides or nucleic acids, or it can take the form of organisms or cells transformed with a library of nucleic acids, such as bacteria, viruses, animals and plant cells. it can. Each individual organism or cell preferably contains one or a limited number of library members. Advantageously, the nucleic acid is incorporated into an expression vector to allow expression of the nucleic acid encoded polypeptide. Thus, in a preferred embodiment, the library contains one or more replicas of an expression vector comprising a single member of the library in nucleic acid form that can be expressed to produce its corresponding polypeptide member. Each can take the form of a population of host organisms. Therefore, the population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants.

  As used herein, “polymer” means a macromolecule composed of repeating monomer units, optionally substituted linear or branched polyalkylene, polyalkenylene. Or it can mean a polyoxyalkylene polymer or a non-synthetic or natural polymer such as a branched or unbranched polysaccharide. As used herein, “polymer” specifically refers to optionally substituted or branched poly (ethylene glycol), poly (propylene glycol) or poly (vinyl alcohol) and derivatives thereof. means.

As used herein, “PEG” or “PEG polymer” means polyethylene glycol, more specifically N-hydroxylsuccinimide (NHS) active ester of PEG, such as succinimidyl propionate, It may mean a derivatized form of PEG including, but not limited to, benzotriazole active esters, maleimide, vinyl sulfone or PEG derivatized with a thiol group. Particular PEG _{formulations, PEG-O-CH 2 CH} 2 CH 2 -CO 2 -NHS, PEG-O-CH 2 -NHS, PEG-O-CH 2 CH 2 -CO 2 -NHS, PEG-S- _{CH 2 CH 2 -CO-NHS,} PEG-O 2 CNH-CH (R) -CO 2 -NHS, PEG-NHCO-CH 2 CH 2 -CO-NHS, and _{PEG-O-CH 2 -CO 2} -NHS Where R is (CH ₂ ) ₄ ) NHCO ₂ (mPEG). PEG polymers useful in the present invention may be linear molecules or branched molecules in which multiple PEG moieties are present in a single polymer. Some particularly preferred PEG structures useful in the present invention include, but are not limited to, the following structures:

  As used herein, “sulfhydryl selective reagents” are useful reagents for conjugating PEG polymers to thiol-containing amino acids. The thiol group on the amino acid residue cysteine is particularly useful for interaction with sulfhydryl selective reagents. Sulfhydryl selective reagents useful in the present invention include, but are not limited to, maleimide, vinyl sulfone and thiol. The use of sulfhydryl selective reagents for conjugation to cysteine residues is known in the art and can be modified as needed according to the present invention (eg Zalipsky, 1995, Bioconjug. Chem. 6: 150; Greenwald). 2000, Crit. Rev. Ther. Drug Carrier Syst. 17: 101; Herman et al., 1994, Macromol. Chem. Phys. 195: 203).

  As used herein, the term “antigen” means a molecule bound by an antibody or antibody binding region (eg, a variable region). Typically, an antigen is capable of eliciting an antibody response in vivo. Antigens can be peptides, polypeptides, proteins, nucleic acids, lipids, carbohydrates or other molecules. In general, immunoglobulin variable regions are selected for target specificity for a particular antigen.

As used herein, the term “epitope” means a unit of structure conventionally bound by an immunoglobulin V _H / V _L pair. An epitope represents a target for the specificity of an antibody in order to define a minimal binding site for the antibody. In the case of a single region antibody, an epitope represents a unit of structure that is individually bound by variable regions.

  As used herein, the term “neutralize” when used with reference to a single immunoglobulin variable region polypeptide described herein, the polypeptide is the target antigen. Means interfering with a measurable activity or function. A polypeptide has a measurable activity or function of a target antigen of at least 50%, preferably 95% or more including at least 60%, 70%, 80%, 90%, or 100% (ie, detection of target antigen). A “neutralizing” polypeptide is one that reduces the measurable activity or function of the target antigen (by the absence of a possible effect or function). This reduction in the measurable activity or function of the target antigen can be assessed by one skilled in the art using standard methods that measure one or more indicators of that activity or function. As an example, if the target is TNF-α, TNF-α-induced expression of ELAM-1 on HUVEC using standard L929 cell killing test or a measure of TNF-α-induced cell activation. Neutralizing activity can be assessed by measuring the ability of a single immunoglobulin variable region to suppress. Similar to “neutralization” as used herein, “inhibiting cytotoxicity” as used herein means that cytotoxicity is suppressed and cell death is reduced by at least 10% or more. Means a reduction in cell death as measured, for example, using the standard L929 cell killing test.

  As used herein, “measurable activity of a target antigen” includes cell signaling, enzyme activity, binding activity, ligand-dependent internalization, cell killing, cell activation, promoting cell survival, and Including but not limited to gene expression. One skilled in the art can perform a test to measure the activity against a given target antigen. Preferably, “activity” as used herein is determined by (1) ND50 in a cell-based test, (2) affinity for a target ligand, (3) ELISA binding, or (4) receptor binding test. It is prescribed. Methods for performing these tests are known to those skilled in the art and are described in further detail below.

  As used herein, “retain activity” means at least 10 levels of the activity of a non-PEG-conjugated antibody polypeptide whose activity is measured as described herein. %, Preferably at least 29%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, preferably 95%, 98% and 90% of the activity of non-PEG-conjugated antibody polypeptides of the same sequence It means a level of activity of a PEG-conjugated antibody polypeptide, eg, a single variable region, that is up to 100%. More specifically, the activity of a PEG-conjugated antibody polypeptide compared to a non-PEG-conjugated antibody polypeptide should be measured on the basis of antibody moles. That is, an equivalent molar number of each PEG-conjugated and non-PEG-conjugated antibody polypeptide should be used in each test. In determining whether a particular PEG-conjugated antibody polypeptide “retains activity”, it is preferable to compare the activity of the PEG-conjugated antibody polypeptide with the activity of the same antibody polypeptide in the absence of PEG.

As used herein, the terms “homodimer”, “homotrimer”, “homotetramer”, and “homomultimer” each refer to a given single immunoglobulin variable region. A molecule comprising two or more (eg, 4, 5, etc.) monomers of a polypeptide sequence. For example, a homodimer will contain two copies of the same _VH sequence. A “monomer” of a single immunoglobulin variable region polypeptide is a single V _H or V _L sequence that specifically binds an antigen. Monomers in homodimers, homotrimers, homotetramers or homomultimers can be expressed, for example, by expression as a fusion protein having a peptide linker between the monomers, or after translation. They can be linked by disulfide bonds, or chemically linked to each other directly or through a linker via a bond to a di, tri or multivalent linking moiety. In one embodiment, the monomers in a homodimer, trimer, tetramer or multimer can be linked by a multi-arm PEG polymer, dimer, trimer, tetramer or Each monomer of the multimer is linked to the PEG component of the multi-arm PEG as described above.

As used herein, the terms “heterodimer”, “heterotrimer”, “heterotetramer” and “heteromultimer” each refer to two or more different single immunizations. A molecule comprising two or more (eg, 4, 5, 6, 7 or 8 or more) monomers of a globulin variable region polypeptide sequence. For example, a heterodimer will contain two _VH sequences, such as _VH1 and _VH2 , or may contain a combination of _VH and _VL . Similar to a homodimer, trimer or tetramer, a monomer in a heterodimer, heterotrimer, heterotetramer or heteromultimer has a peptide linker between the monomers, for example They can be linked by expression as a fusion protein, or by chemically linking the translated monomers to each other directly or through a linker by disulfide bonds or bonds to di, tri or multivalent binding components. In one embodiment, the monomers in the heterodimer, trimer, tetramer or multimer can be linked by a multi-arm PEG polymer, dimer, trimer, tetramer or Each monomer of the multimer is linked to the PEG component of the multi-arm PEG as described above.

  As used herein, the term “half-life” refers to the mechanism by which serum concentration of a ligand (eg, an antibody polypeptide such as a single immunoglobulin variable region), eg, degradation of the ligand, and / or natural mechanism. Means the time taken to decrease 50% in vivo due to clearance or sequestration of the ligand by Antibody polypeptides are stabilized in vivo, and their half-life is increased by binding to molecules that resist degradation and / or clearance or sequestration, such as PEG. The half-life of an antibody polypeptide, such as dAb), is increased if its functional activity persists in vivo over a longer period than a similar dAb that does not bind to the PEG polymer. Typically, the half-life of a PEGylated dAb is increased by 10%, 20%, 30%, 40% or 50% or more compared to a non-PEGylated dAb. It is possible to increase the half-life in the range of 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times or more. Alternatively, or in addition, a half-life increase of 30, 40, 50, 60, 70, 80, 90, 100, 150 is possible. According to the present invention, the PEG-conjugated antibody single variable region can be 0.25 to 170 hours, preferably 1 to 100 hours, more preferably 30 to 100 hours, even more preferably 50 to 100 hours, 170, 180, Has a half-life of up to 190 and 200 hours.

  As used herein, “resisting to degradation” or “resisting to degradation” when used against PEG or other polymer-bound dAb monomer or multimer. PEG or other polymer-bound dAb monomer or multimer means that it degrades at a level of 10% or less when contacted with pepsin at pH 2 for 30 minutes, preferably no degradation at all. When specifically addressing PEG or other polymer-bound dAb multimers (eg, hetero or homodimers, trimers, tetramers, etc.), molecules that are resistant to degradation can span 30 minutes at pH 2. In the presence of pepsin, it degrades at a level of less than 5% and preferably does not degrade at all.

  As used herein, “hydrodynamic size” means the apparent size of a molecule (eg, a protein molecule) based on the diffusion of an aqueous solution of the molecule. The diffusion or movement of the protein solution can be processed to derive the apparent size of the protein given by the “Stokes radius” or “hydrodynamic radius” of the protein particles. The “hydrodynamic size” of a protein depends on both mass and shape (structure) so that two proteins with the same molecular mass have different hydrodynamic sizes based on the overall structure of the protein. The hydrodynamic size of a PEG-conjugated antibody polypeptide, eg, a single variable region (including the antibody variable region multimers described herein) is 24 kDa to 500 kDa, 30 to 500 kDa, 40 to 500 kDa, 50 to 500 kDa, It may be in the range of 100 to 500 kDa, 150 to 500 kDa, 200 to 500 kDa, 250 to 500 kDa, 300 to 500 kDa, 350 to 500 kDa, 400 to 500 kDa, and 450 to 500 kDa. Preferably, the hydrodynamic size of the PEGylated dAb of the present invention is 30 to 40 kDa, 70 to 80 kDa or 200 to 300 kDa. If an antibody variable region multimer is desired for imaging applications, the multimer should have a hydrodynamic size of 50 to 100 kDa. Alternatively, if an antibody single region multimer is desired for therapeutic use, the multimer should have a hydrodynamic size greater than 200 kDa.

Bispecific Antibody Polypeptides In our international patent application WO2004 / 003019, the specificity of one of the ligands acts to increase the half-life of the ligand by binding to it. A further improvement of bispecific ligands directed to proteins or polypeptides present in vivo in organisms that can be described has been described. WO2004 / 003019 includes a first immunoglobulin single variable region having binding specificity for a first antigen or epitope and a second complementary immunoglobulin single variable having binding activity for a second antibody or epitope. Wherein one or both of said antigens or epitopes act to increase the half-life of the ligand in vivo, said severe isomeric ligand comprising an anti-HSA VH region and If not composed of an anti-p galactosidase VK region, a bispecific ligand is described in which the first and second regions lack a region complementary to each other sharing the same specificity.

  Antigens or epitopes that increase the half-life of the ligand as described herein are advantageously present in proteins or polypeptides found in organisms in vivo.

  Examples include extracellular matrix proteins, blood proteins, and proteins that are present in various tissues of an organism. These proteins act to reduce the rate of ligand secretion from the blood, for example by acting as a bulking agent or by immobilizing the ligand at the desired site of action. Examples of antigens / epitopes that increase in vivo half-life are shown in Appendix 1 below.

  Increased half-life is beneficial for in vivo use of immunoglobulins, particularly antibodies, most particularly small sized antibody fragments. The fragments (Fvs, disulfide bond Fvs, Fabs, scFvs and dAbs) are rapidly secreted from the body. Thus, most parts of the body can be reached quickly, are generated quickly and are easily processed, but their in vivo use has been limited by their short duration in vivo. The present invention solves this problem by increasing the half-life of the ligand in vivo, resulting in a longer duration of the functional activity of the ligand in the body.

  Methods for pharmacokinetic analysis and measurement of ligand half-life will be familiar to those skilled in the art. For details, see Kenneth, A. et al., “Chemical Stability of Pharmaceuticals: A Handbook for Pharmaceuticals” and Peters et al., “Pharmacological Analysis: A Practical App. Reference is also made to “Pharmacocetics”, M Gibaldi & D Perron, publication: Marcel Dekker, 2nd edition (1982), describing pharmacokinetic parameters such as tα and tβ half-lives and area under the curve (AUC).

  Half-life (T1 / 2α and T1 / 2β) and AUC can be determined from a curve of ligand serum concentration versus time. For example, a curve can be modeled using the WinNonlin analysis package (available from Pharsight Corp., Mountain View, Calif.). In the first phase (alpha phase), the ligand is mainly distributed in the patient and part is removed. The second phase (β phase) is the terminal phase when the serum concentration decreases as the ligand is distributed and secreted from the patient. The tα half-life is the half-life of the first phase and the tβ half-life is the half-life of the second phase. Thus, advantageously, the present invention provides a ligand according to the present invention having a tα half-life in the range of 15 minutes or more, or a composition comprising a ligand. In one embodiment, the lower end of the range is 30 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition or alternatively, a ligand or composition according to the invention will have a tα half-life in the range of 12 hours or less. In one embodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours. Examples of suitable ranges are 1 to 6 hours, 2 to 5 hours or 3 to 4 hours. Advantageously, the present invention provides a ligand according to the present invention or a composition comprising a ligand having a tβ half-life in the range of 2.5 hours or more.

  In one embodiment, the lower end of the range is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition or alternatively, a ligand or composition according to the invention has a tβ half-life in the range of 21 days or less. In one embodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Advantageously, a ligand or composition according to the invention will have a tβ half-life in the range of 12 to 60 hours.

  In a further embodiment, the half-life ranges from 12 to 48 hours. In a further embodiment, the half-life ranges from 12 to 26 hours.

  In addition to or in lieu of the above criteria, the present invention provides a ligand according to the present invention having a AUC value (area under the curve) in the range of 1 mg min / ml or more, or a composition comprising a ligand. In one embodiment, the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 mg min / ml. In addition or alternatively, a ligand or composition according to the invention has an AUC in the range of up to 600 mg min / ml.

  In one embodiment, the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg min / ml. Advantageously, the ligand according to the invention has an AUC in the range selected from the group consisting of 15 to 150 mg min / ml, 15 to 100 mg min / ml, 15 to 75 mg min / ml and 15 to 50 mg min / ml become.

  In a first embodiment, the bispecific ligand is in the two complementary variable regions, ie, in their native environment, even if they individually bind to a cognate epitope within the scope of the present invention. It contains two variable regions that can function together as a cognate pair or group. For example, the complementary variable regions can be immunoglobulin heavy and light chain variable regions (VH and VL). The VH and VL regions are advantageously provided by scFv or Fab antibody fragments. For example, a hinge region is provided at the C-terminus of each V region, and cysteines are disulfide bonded in the hinge region, dAbs each having a cysteine are provided at the C-terminus of the region, and cysteines are disulfide bonded to each other, or Fab format By using V-CH & V-CL to produce a dimer, trimer, or even a peptide linker (eg, Gly4Ser linker described below) to produce a multimer. Variable regions can be linked together to form a valent ligand. The inventors have found that the use of complementary variable regions allows the surfaces of the two regions to be coated together and sequestered from the solvent. Also, complementary regions can stabilize each other. In addition, it is possible to create bispecific IgG antibodies without the disadvantages of hybrid hybrid cells used in the prior art or the need to modify heavy or light chains at the subunit interface by protein engineering It is.

  The bispecific ligand of the first aspect of the invention has at least one VH / VL pair. Thus, a bispecific IgG according to the present invention comprises two such pairs, one pair present on each arm of the Y-shaped molecule. Thus, unlike conventional bispecific antibodies or diabodies, where the ratio of chains used determines the success of the preparation and presents practical difficulties, the bispecific ligands of the present invention have a chain balance. There is no problem about. Chain imbalance in conventional bispecific antibodies allows VL chain 1 to bind to antigen or epitope 1 along with VH chain 2 and VH chain 2 binds to antigen or epitope 2 along with VH chain 2 It is possible and due to the association of two different VL chains and two different VH chains that the two proper pairs bind together in some way. Thus, bispecificity is only provided in a single molecule when VL chain 1 is paired with VH chain 1 and VL chain 2 is paired with VH chain 2. Such bispecific molecules can be made in two different ways. First, it can be made by the association of two existing VH / VL pairs that each bind to a different antigen or epitope (eg, in bispecific IgG). In this case, the VH / VL pairings must all bind in a 1: 1 ratio to create a population of molecules, all of which are bispecific. This does not occur (even if the complementary CH region has been enhanced by “knob-in-hole” engineering) and can bind to a bispecific molecule and one antigen or epitope. However, it produces a mixture of molecules that cannot bind to other antigens or epitopes. A second method of making bispecific antibodies is by the simultaneous association of two different VH chains and two different VL chains (eg, in a bispecific diabody). In this case, there is a tendency for VL chain 1 to pair with VH chain 1 and VL chain 2 to pair with VH chain 2 (which can be enhanced by “knob-in-hole” engineering of the VL and VH regions). The combination is not achieved with all molecules, and producing a mixed formulation results in inappropriate pairings that cannot bind to either antigen or epitope.

  Bispecific antibodies constructed according to the bispecific ligand approach according to the first aspect of the invention have binding to antigen or epitope 1 in the VH or VL region, respectively, and complementary binding to antigen or epitope 2 It overcomes all these problems because it exists in the target VL or VH region. Since the VH and VL regions are paired 1: 1, all VH / VL pairs are bispecific and thus all forms constructed using these VH / VL pairs (Fv, scFvs, Fabs, minibodies, IgGs, etc.) will have 100% bispecific activity.

  Within the scope of the present invention, first and second “epitope” are understood to be epitopes that are not identical and are not bound by a single monospecific ligand. In the first configuration of the invention, they are advantageously present on different antigens, one of which acts to increase the half-life of the ligand in vivo. Similarly, the first and second antigens are advantageously not identical.

  The bispecific ligands of the present invention do not include ligands as described in WO02 / 02773. Thus, the ligands of the invention do not include complementary VH / VL pairs that jointly bind any one or more antigens or epitopes. Instead, the ligand according to the first aspect of the invention comprises VH / VL complementary pairs in which the V regions have different specificities.

  Furthermore, the ligand according to the first aspect of the invention comprises VH / VL complementary pairs with different specificities for non-specifically related epitopes or antigens. A structurally related epitope or antigen is an epitope or antigen having sufficient structural similarity to be bound by a conventional VH / VL complementary pair that cooperates to bind the antigen or epitope, In the case of epitopes that are related, the epitopes are sufficiently similar in structure that they “fit” in the same binding pocket formed by VH / VL in the antigenic antigen binding site.

  In a second aspect, the present invention comprises a first immunoglobulin variable region having a first antigen or epitope binding specificity and a second immunoglobulin variable region having a second antigen or epitope binding specificity. A ligand comprising, wherein one or both of the first and second variable regions binds to an antigen that increases the half-life of the ligand in vivo, and the variable regions provide ligands that are not complementary to each other.

  In one embodiment, binding to one variable region modulates ligand binding to a second variable region.

  In the present embodiment, the variable region may be, for example, a pair of VH regions or a pair of VL regions. The binding of the antigen at the first binding site can be regulated such that the binding of the antigen at the second site is enhanced or suppressed. For example, binding at the first site at least partially inhibits antigen binding at the second site. In such embodiments, the ligand is in vivo of the subject organism through binding to a protein that increases the half-life of the ligand, for example, until it is bound to a second target antigen and dissociates from the protein that increases the half-life. Can be maintained in the body.

  Regulation of binding in the context is achieved as a result of structural proximity of the antigen binding sites relative to each other. Such structural proximity can be achieved by the nature of the structural component that binds two or more antigen binding sites, for example by providing a ligand with a relatively stiff structure that holds the antigen binding site in the vicinity. Advantageously, two or more antigen binding sites are physically linked to each other such that one site regulates antigen binding at another site by a process involving steric hindrance and / or structural changes within the immunoglobulin molecule. Close.

  The first and second antigen binding regions can be associated covalently or non-covalently. When these regions are covalently associated, the association is mediated by, for example, a disulfide bond or a polypeptide linker such as (Gly4Ser) n (n = 1 to 8, eg 2, 3, 4, 5 or 7). be able to.

  While integrating a ligand according to this aspect of the invention into a non-immunoglobulin multiligand structure to form a multivalent complex that binds the target molecule of the same antigen can provide superior binding activity, At least one variable region binds antigen and increases the half-life of the multimer. For example, a natural bacterial receptor such as SpA has been used as a framework for transplantation of CDRs to generate ligands that specifically bind to one or more epitopes. Details of this procedure are described in US Pat. No. 5, S31,012. Other suitable scaffolds include those based on fibronectin and affibodies. Details of suitable procedures are described in WO 98/58965. Other suitable frameworks include van den Beuken et al. Mol. Biol. (2001) 310, 591-601, including lipocalin and CTLA4, and the framework as described in WO0069907 (Medical Research Council), for example based on the ring structure of bacterial GroEL or other chaperone polypeptides. .

Protein scaffolds can be attached, for example, CDRs can be transplanted into CTLA4 scaffolds and used with immunoglobulin _VH or _VL regions to form ligands.

  Similarly, fibronectin, lipocalin and other frameworks can be combined.

  If the variable regions are selected from a V gene repertoire selected using, for example, the phage display techniques described herein, these variable regions may be identified as specific universal ligands as defined herein. Can include inclusive framework regions, as can be recognized by. The use of inclusive frameworks, universal ligands, etc. is described in WO 99/20749. In the present invention, reference to phage display includes phage and / or phagemid.

  When a V gene repertoire is used, the polypeptide sequence variation is preferably located within the structural loop of the variable region. The polypeptide sequence of any variable region can be altered by DNA shuffling or mutation to enhance the interaction of each variable region with its complementary pair.

  In a preferred embodiment of the invention, the “dual specific ligand” is a single chain Fv fragment. In an alternative embodiment of the invention, the “bispecific ligand” consists of the Fab region of an antibody. The term “Fab region” includes Fab-like regions where two VH or two VL regions are used.

  The variable region can be derived from an antibody directed against the target antigen or epitope. Alternatively, variable regions can be derived from a repertoire of single antibody regions, such as those expressed on the surface of filamentous bacteriophages. Selections can be made as described below and in the examples.

Preparation of dAbs Aspects of the invention include not only bispecific ligands, but also ligands that bind TNF-α alone, TNF-α and HSA, or other half-life-enhancing polypeptides of the bispecific form, and two It also relates to various constructs of ligands that bind TNF-α and VEGF in a bispecific format. It is also possible to prepare ligands that bind VEGF and HSA or other half-life extending polypeptides. The bispecific TNF-α / VEGF construct can further comprise a binder for HSA or other half-life extending nucleotides. In each of these embodiments, the individual ligand, ie the ligand that binds TNF-α, HSA or VEGF, may preferably be dAbs. The production of dAbs is described below and in the examples.

  In various embodiments, the dAbs disclosed herein can exist in monomeric, dimeric, trimeric, tetrameric, or even higher multimeric forms. Multimeric constructs can be homomultimers, ie homodimers, homotrimers, homotetramers, etc., in addition to heterodimeric forms such as bispecific constructs. Heterotrimers, heterotetramers, and higher order heteromultimers are also specifically contemplated. To further increase the serum half-life of the polypeptide construct, each of the various dAb structures can be further conjugated with additional components such as polyethylene glycol (PEG). PEGylation is known in the art and is described herein.

  Single immunoglobulin variable regions or dAbs are prepared in several ways. In a preferred embodiment, dAbs are human single immunoglobulin variable regions. For each of these techniques, well-known nucleic acid sequence preparation (eg, amplification, mutation, etc.) and processing methods can be applied.

One means of preparing dAbs is to amplify and express the _VH or _VL regions of the heavy and light chain genes for cloned antibodies known to bind the desired antigen. The boundaries of the V _H and V _L regions are set by Kabat et al. (1991, supra). Information about the boundaries of the _VH and _VL regions of the heavy and light chain genes amplifies the V region from cloned heavy and light chain coding sequences encoding antibodies known to bind a given antigen. Used to design PCR primers. The amplified V region is inserted into a suitable expression vector such as pHEN-1 (Hoogenboom et al., 1991, Nucleic Acids Res. 19: 4133-4137) and expressed as a single body or as a fusion with other polypeptide sequences. Is done. The expressed _VH or _VL region is then screened for high affinity that binds to the desired antigen separate from the rest of the heavy or light chain polypeptide. In all aspects of the invention, screening for binding is performed as known in the art or as described below.

The repertoire of _VH or _VL regions is screened, for example, by phage display, panning against the desired antigen. Methods for constructing bacteriophage display libraries and lambda phage expression libraries are well known in the art and are described in McCafferty et al., 1990, Nature 348: 552; Kang et al., 1991, Proc. Natl. Acad. Sci. U. S. A. 88: 4363; Clackson et al., 1991, Nature 352: 624; Lowman et al., 1991, Biochemistry 30: 10832; Burton et al., 1991, Proc. Natl. Acad. Sci U. S. A. 88: 10134; Hoogenboom et al., 1991, Nucleic Acids Res. 19: 4133; Chang et al., 1991, J. MoI. Immunol. 147: 3610; Breitling et al., 1991, Gene, 104: 147; Marks et al., 1991, J. MoI. Mol. Biol. 222: 581; Barbas et al., 1992, Proc. Natl. Acad. Sci. U. S. A. 89: 4457; Hawkins and Winter (1992) J. MoI. Immunol. 22: 867; Marks et al. (1992) J. MoI. Biol. Chem. 267: 16007; and Lerner et al. (1992) Science 258: 1313. scFv phage libraries are described in, for example, Huston et al., 1988, Proc. Natl. Acad. Sci U. S. A. 85: 5879-5883; Chaudhary et al., 1990, Proc. Natl. Acad. Sci U. S. A. 87: 1066-1070; McCafferty et al., 1990, supra; Clackson et al., 1991, supra; Marks et al., 1991, supra; Chiswell et al., 1992, Trends Biotech. 10:80; and Marks et al., 1992, supra. Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Improved versions of phage display techniques are also known, as described, for example, in WO 96/06213 and WO 92/01047 (Medical Research Council et al.) And WO 97/08320 (Morphosys, supra).

The repertoire of _VH or _VL regions can be a natural or synthetic repertoire of immunoglobulin sequences. A natural repertoire is, for example, prepared from immunoglobulin-expressing cells harvested from one or more individuals. The repertoire can be prepared from “protozoa”, eg, human fetal or neonatal immunoglobulin-expressing cells, or rearranged, ie, prepared from, eg, adult human B cells. Natural repertoires are described, for example, in Marks et al., 1991, J. MoI. Mol. Biol. 222: 581 and Vaughan et al., 1996, Nature Biotech. 14: 309. If desired, clones identified from the natural repertoire or any repertoire that bind the target antigen are mutated and further screened to generate and select variants with improved binding properties.

  A synthetic repertoire of single immunoglobulin variable regions is prepared by artificially introducing diversity into the cloned V region. Synthetic repertoires are described, for example, in Hoogenboom & Winter, 1992, J. Am. Mol. Biol. 227: 381; Barbas et al., 1992, Proc. Natl. Acad. Sci. U. S. A. 89: 4457; Nissim et al., 1994, EMBO J. et al. 13: 692; Griffiths et al., 1994, EMBO J. et al. 13: 3245; DeKriuf et al., 1995, J. MoI. Mol. Biol. 248: 97; and WO99 / 20749.

The antigen binding region of a conventional antibody includes two separate regions, a heavy chain variable region (V _H ) and a light chain variable region (V _L , which can be V _κ or V _λ ). The antigen-binding site of the antibody consists of 6 polypeptide loops, 3 polypeptide loops from the _VH region (H1, H2 and H3) and 3 polypeptide loops from the _VL region (L1, L2 and L3). Formed by a polypeptide loop. The boundaries of these loops are described, for example, in Kabat et al. (1991, supra). A diverse primary repertoire of V genes encoding _VH and _VL regions is generated in vivo by combinatorial rearrangement of gene segments. The _VH gene is produced by recombination of three gene segments _VH , D and _JH . In humans, depending on the haplotype, approximately 51 functional V _H segments (Cook and Tomlinson (1995) Immunol Today 16: 237), 25 functional D segments (Corbett et al. (1997) J. Mol. Biol. 268: 69) and six functional _JH segments (Ravetch et al. (1981) Cell 27: 583). The V _H segment encodes the region of the polypeptide chain that forms the first and second antigen binding loops of the V _H region (H1 and H2), whereas the V _H , D and J _H segments bind. To form a third antigen-binding loop of the V _H region (H3).

V _L gene is produced by the recombination of gene segments only two _{V L} and _{J L.} The humans, there are approximately 40 functional V _kappa segments (Schable and Zachau (1993) Biol.Chem.Hoppe-Seyler 374 : 1001), 31 functional V _kappa segments (Williams et al., (1996) J. Mol. Biol. 264: 220; Kawasaki et al. (1997) Genome Res. 7: 250) and five functional J _kappa segments (Hieter et al. (1982) J. Biol. Chem. 257: 1516) and 4 There are two functional J _λ segments (Vasenik and Leder (1990) J. Exp. Med. 172: 609). The _VL segment encodes the region of the polypeptide chain that forms the first and second antigen binding loops of the _VL region (L1 and L2), whereas the _VL and _JL segments bind , Form a third antigen-binding loop of the _VL region (L3). Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all antigens with at least moderate affinity. High affinity antibodies are generated in vivo by “affinity maturation” of reconstituted genes in which point mutations are generated and selected by the immune system based on improved binding.

  Analysis of antibody structure and sequence shows that five of the six antigen binding loops (H1, H2, L1, L2, L3) have a limited number of main chain structures or standard structures. (Chothia and Lesk (1987) J. Mol. Biol. 196: 901; Chothia et al. (1989), Nature 342: 877). The backbone structure is determined by (i) the length of the antigen binding loop, and (ii) specific residues or types of residues at certain major positions in the antigen binding loop and antibody framework. Analysis of loop length and major residues made it possible to predict the backbone structure of H1, H2, L1, L2 and L3 encoded by the majority of human antibody sequences (Chothia et al. (1992) J. MoI. Mol. Biol.227: 799; Tomlinson et al. (1995) EMBO J.14: 4628; Williams et al. (1996) J. Mol.Biol.264: 220). The H3 region is much more diverse in terms of sequence, length and structure (through the use of D segments), but the length and presence of certain residues at major positions in the loop and antibody framework, or residues Form a limited number of backbone structures for short loop lengths depending on the type of (Martin et al. (1996) J. Mol. Biol. 263: 800; Shirai et al. (1996) FEBS Letters 399: 1 ).

According to one embodiment of the present invention, it is possible to add diversity in the synthetic repertoire to any site in the CDRs of various antigen binding loops, but this approach does not properly overlay and binds antigen. The ratio of the V region contributing to the decrease in the ratio of molecules having the potential to increase is increased. Understanding the residues that contribute to the backbone structure of the antigen binding loop allows for the identification of specific residues that diversify in the synthetic repertoire of _VH or _VL regions. That is, diversity is optimally introduced into residues that are not essential for maintaining the backbone structure. As an example, for loop L2 diversification, the conventional approach diversifies all residues in the corresponding CDR (CDR2) defined by Kabat et al. (1991, supra), ie, 7 residues. become. However, for L2, positions 50 and 53 are known to be different in natural antibodies and observed to contact antigen. A preferred approach would diversify only those two residues in this loop. This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities.

In one aspect, the synthetic variable region repertoire is prepared in the background of _VH or _Vκ based on artificially diversified germline _VH or _Vκ . For example, the _VH region repertoire is based on the cloned germline _VH gene segment V3-23 / DP47 (Tomlinson et al., 1992, J. Mol. Biol. 227: 7768) and JH4b (see FIGS. 1 and 2). V _kappa region repertoire, for example, germline V _kappa gene segments O2 / O12 / DPK9 (Cox et al., 1994, Eur.J.Immunol.24: 827) and J _kappa based on 1 (see FIG. 3). Diversity is introduced into these or other gene segments by, for example, PCR mutagenesis. Diversity can be introduced randomly, eg, by error-prone PCR (Hawkins et al., 1992, J. Mol. Biol. 226: 889) or chemical mutagenesis. However, as noted above, the introduction of diversity is preferably directed to specific residues. Codon NNK (IUPAC nomenclature (N = G, A, T or C, and K = G or T) using mutagenic primers, encoding all amino acids and TAG stop codons for the desired residue. ) Is preferably targeted for introduction. NNN codon (generates additional stop codons TGA and TAA), DVT codon (A / G / T) (A / G / C) T), DVC codon ((A / G / T) (A / G / C) C) and DVY codons ((A / G / T) (A / G / C) (C / T) are also used to achieve a similar purpose.) Encodes 22% serum and 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and cysteine, most similar to the distribution of amino acid residues to the antigen binding site of the repertoire. The PCR mutagenesis is well known in the art, but is useful for primer construction useful in the methods of the present invention. And to examine the PCR mutagenesis in the chapter of the following "PCR mutagenesis".

In one embodiment, as shown in FIG. 1, the diversity corresponding to the diversity in CDRs 1, 2 and 3 is represented by sites H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, NNK codons are used for H95, H97 and H98, and are introduced into the human germline _VH gene segment V3-23 / DP47 (Tomlinson et al., 1992, J. Mol. Biol. 227: 7768) and JH4b.

In other embodiments, as shown in FIG. 2, the diversity corresponding to the diversity in CDRs 1, 2 and 3 can be expressed, for example, at sites H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56. , H58, H95, H97, H98, H99, H100, H100a and H100b are introduced into human germline _VH gene segments V3-23 / DP47 and JH4b using NNK codons.

In other embodiments, as shown in FIG. 3, the diversity corresponding to the diversity in CDRs 1, 2 and 3 can be expressed, for example, at sites L30, L31, L32, L34, L50, L53, L91, L92, L93, L94. and L96 using the NNK codon, introduced into the human germline V _kappa gene segments O2 / O12 / DPK9 and J _kappa 1.

  The diversified repertoire is cloned into a phage display vector as is known in the art and as described, for example, in WO 99/20749. In general, nucleic acid molecules and vector constructs required to practice the present invention are available in the art and are described in Sambrook et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor, USA. Constructed and operated as described in standard laboratory manuals such as

  The nucleic acid manipulation in the present invention is typically performed with a recombinant vector. As used herein, a “vector” is an individual element used to introduce heterologous DNA into cells for its expression and / or replication. Methods for selecting or constructing and then using the vectors are well known to those skilled in the art. Many vectors are suitably available including bacterial plasmids, bacteriophages, artificial chromosomes and episomal vectors. The vector can be used for simple cloning and mutagenesis. Alternatively, a gene expression vector is employed as a typical vector carrying the repertoire (pre-repertoire) member of the present invention. The vectors used in accordance with the present invention are selected to correspond to a polypeptide coding sequence of the desired size, typically 0.25 kilobases (kb) to 40 kb in length. A suitable host cell is transformed with the vector after the in vitro cloning operation. Each vector generally includes various functional components including a cloning (or “polylinker”) site, an origin of replication, and at least one selectable marker gene. If the given vector is an expression vector, it is an enhancer element, promoter, transcription terminator located near the cloning site, respectively, so that it is functionally linked to a gene encoding a polypeptide repertoire member according to the present invention. And a signal sequence.

  Both cloning and expression vectors generally contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (eg SV40, adenovirus) are useful for cloning vectors in mammalian cells. is there. In general, an origin of replication is not required for a mammalian expression vector unless it is used for mammalian cells capable of replicating high levels of DNA, such as COS cells.

  Advantageously, the cloning or expression vector also contains a selection gene, also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in selective media. Thus, host cells that are not transformed with the vector containing the selection gene will not survive in the medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, supplement nutrient deficiencies, or provide important nutrients that cannot be obtained in growth media. To do.

  Since the replication of the vector according to the invention is most conveniently carried out in E. coli, an E. coli selectable marker is used, such as the β-lactamase gene that confers resistance to the antibiotic ampicillin. These can be obtained from E. coli plasmids such as pBR322 or pUC plasmids such as pUC18 or pUC19.

  Expression vectors usually contain a promoter that is recognized by the host organism and is operably linked to the coding sequence of interest. The promoter can be inducible or constitutive. The term “operably linked” means a proximal sequence in which the described components are in a relationship that allows them to function as intended. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

  Suitable promoters for use with prokaryotic host organisms include, for example, hybrid promoters such as β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and tac promoters. Promoters used in bacterial systems will generally also contain a Shine-Dalgarno sequence operably linked to the coding sequence.

  In the libraries or repertoires described herein, preferred vectors are expression vectors that allow expression of nucleotide sequences corresponding to polypeptide library members. Thus, selection is performed by individual propagation and expression of single clones expressing polypeptide library members, or by use of any selection display system. As mentioned above, the preferred selection display system uses bacteriophage display. Thus, phage or phagemid can be used. A preferred vector is a phagemid vector having an E. coli origin of replication (for double stranded replication) and a phage origin of replication (for production of single stranded DNA). Manipulation and expression of the vector is well known in the art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the vector is an expression consisting of a selectable marker gene that confers selectivity for β-lactamase, or a plasmid, N-to-C-terminal (leading the expressed polypeptide to the periplasmic space) pelB leader sequence. A lac promoter upstream of the cassette, multiple cloning sites (for cloning nucleotide versions of library members), optionally one or more peptide tags (for detection), optionally one or more TAGs Contains stop codon and phage protein pIII. Using various suppressor and non-suppressor strains of E. coli and adding a helper phage such as glucose, isopropylthio β-D-galactoside (IPTG), or VCS M13, the vector replicates as an unexpressed plasmid, It will be possible to generate only polypeptide library members or phages some of which contain at least one copy of polypeptide-pIII on their surface.

  Examples of preferred vectors include the pHEN1 phagemid vector (Hoogenboom et al., 1991, Nucl. Acids Res. 19), which is repressed in the presence of glucose and generates pIII fusion protein under the control of the LacZ promoter induced by IPTG. 4133-4137; for example, a sequence such as SEQ ID NO: 7 in WO03 / 031611 is available). When grown on a suppressor strain of E. coli, such as TG1, a gene III fusion protein is produced and contained in phage, whereas when grown on a non-suppressor strain, such as HB2151, the bacterial periplasm of soluble fusion protein and Allows secretion into the medium. Since expression of gene III prevents later infection by helper phage, bacteria containing the phagemid vector are grown in the presence of glucose prior to infection with VCSM13 helper phage for phage release.

  Conventional linking techniques are employed in the construction of the vector according to the present invention. The isolated vector or DNA fragment is cleaved, adjusted and recombined in the form desired to produce the required vector. If desired, sequence analysis to confirm that the correct sequence is present in the constructed vector is performed using standard methods. Suitable methods for constructing expression vectors, preparing transcripts in vitro, introducing DNA into host cells, and performing analyzes to assess expression and function are known to those skilled in the art. Detect the presence of a gene sequence in a sample and amplify and / or express its Southern or Northern analysis, Western blot, DNA, RNA or protein dot blot, in situ hybridization, immunocytochemistry, or nucleic acid or Quantification is by conventional methods such as sequence analysis of protein molecules. Those skilled in the art will readily recognize how these methods can be modified as desired.

PCR mutagenesis The primer is complementary to a portion of the target molecule present in the pool of nucleic acid molecules used to prepare a collection of nucleic acid repertoire members encoding polypeptide repertoire members. Primers are most often prepared by chemical or enzymatic synthesis methods. Mutagenic oligonucleotide primers are generally 15 to 100 nucleotides in length, ideally 20 to 40 nucleotides, although oligonucleotides of different lengths are also used.

  Typically, two nucleic acid sequences are substantially complementary (at least about 65% complementarity over a range of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 85% or 90% complementarity). Kanehisa, 1984, Nucleic Acids Res. 12: 203. As a result, it is expected that some mismatch at the priming site is tolerated. The discrepancy can be as small as mono, di or trinucleotide. Alternatively, it can comprise a nucleotide loop where the mismatch is defined herein as a region encompassing 4 or more consecutive nucleotides.

  In general, five factors affect the efficiency and selectivity of primer hybridization to a second nucleic acid molecule. These factors require that (i) primer length, (ii) nucleotide sequence and / or composition, (iii) hybridization temperature, (iv) buffer chemistry and (v) primer be hybridized. The potential for steric hindrance in the region, an important factor when non-random priming sequences are designed.

There is a positive correlation between the length of the primer and the efficiency and accuracy of annealing the primer to the target sequence. Longer sequences have a higher melting temperature (T _M ) than shorter sequences and are less likely to be repeated within a given target sequence, thereby minimizing promiscuous hybridization. Since single molecule rather than bimolecular hybridization movements are generally preferred in solution, primer sequences that have a high GC content or include palindromic sequences, such as their intended target sites, are self-directed. There is a tendency to hybridize. At the same time, designing a primer containing a sufficient number of GC nucleotide pairs to bind tightly to the target sequence is found in each such pair when the A and T bases are paired. This is important because it is linked by three hydrogen bonds rather than two. Hybridization temperature varies inversely with primer annealing efficiency, such as the concentration of organic solvents, such as formamide, that can be included in the hybridization mixture, but increasing salt concentration promotes binding. Under stringent hybridization conditions, longer probes hybridize more efficiently than shorter probes, which are sufficient under more permissive conditions. Stringent hybridization conditions for the primer typically include a salt concentration of less than about 1M, more commonly less than about 500 mM, preferably less than about 200 mM. Hybridization temperatures range from 0 ° C to above 22 ° C, above about 30 ° C, and (most often) above about 37 ° C. Longer fragments may require higher hybridization temperatures for specific hybridizations. A combination of parameters is more important than an absolute measurement of a single parameter because several factors affect the stringency of hybridization.

  Primers are designed with these elements in mind. Although the relative advantages of many sequences can be estimated by one skilled in the art, computer programs have been designed to assist in the evaluation of some of these parameters and optimization of primer sequences. Examples of such programs include the DNAStar ™ software package ("PrimerSelect" from DNAStar, Inc. (Madison, Wis.)) And OLIGO 4.0 (National Biosciences, Inc.) Suitable oligonucleotides are designed. And suitable methods, such as the phosphoramidite method described in Beaucage and Carruthers, 1981, Tetrahedron Lett. Chem.Soc.103: 3185, or a commercially available automated nucleotide synthesizer, or for example V Prepared by other chemical methods using LSIPS ™ technology.

  PCR is performed using template DNA (at least 1 fg, more practically 1-1000 ng) and at least 25 pmol of oligonucleotide primers. Because each sequence is represented by a small portion of the primer pool molecule and is limited in volume in later amplification cycles, it is advantageous to use a larger amount of primer if the primer pool is very heterogeneous. sell. A typical reaction mixture is 2 μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10 × PCR buffer 1 (Perkin-Elmer), 0.4 μ of 1.25 μM dNTP, 0.15 μl (or 2.5 Unit) Taq DNA polymerase (Perkin Elmer), and a total volume of 25 μl of deionized water. The mineral oil is overlaid and PCR is performed using a programmed thermal cycler.

  The length and temperature of each step of the PCR cycle, and the number of cycles are adjusted according to the actual stringency requirements. Annealing temperature and timing are both determined by the efficiency of annealing the primer to the template and the degree of mismatch allowed. Obviously, if the nucleic acid molecule is amplified and mutagenized simultaneously, inconsistencies are required in at least the first round of synthesis. In an attempt to amplify a population of molecules using a mixed pool of mutagenic primers, the loss of potential mutation products under strict (high temperature) annealing conditions due simply to the low melting temperature is reduced to the target site of the primer. Weigh against indiscriminate annealing to other sequences. The ability to optimize the stringency of primer annealing conditions is well within the knowledge of those skilled in the art. An annealing temperature between 30 ° C. and 72 ° C. is used. Initial denaturation of the template molecule usually occurs over 4 minutes at a temperature between 92 ° C and 99 ° C, followed by denaturation (94-99 ° C, 15 seconds to 1 minute), annealing (temperature determined as described above). 1 to 2 minutes) and extension (72 ° C., 1 to 5 minutes depending on the length of the amplified product) followed by 20 to 40 cycles. The final extension is generally performed at 72 ° C. for 4 minutes, followed by an indefinite (0-24 hour) step at 4 ° C.

Screening for dAbs for antigen binding Following expression of the repertoire of dAbs on the surface of the phage, select by contacting the phage repertoire with the immobilized target antigen, washing to remove unbound phage, and growing the bound phage. The entire process is called “panning”. Alternatively, phage are pre-selected for expression of properly superimposed member variants by panning against a fixed universal ligand (eg, protein A or protein L) that is bound only by the overlapping members. This has the advantage of increasing the proportion of members likely to bind the target antigen by reducing the proportion of non-functional members. Preselection with universal ligands is taught in WO 99/20749. Screening of phage antibody libraries is described, for example, by Harrison et al., 1996, Meth. Enzymol. 267: 83-109.

  Screening is generally performed using generated antigens immobilized on an individual support, such as a plastic tube or well, or a chromatography matrix, such as Sepharose ™ (Pharmacia). Screening or selection can also be performed on complex antigens such as the surface of cells (Marks et al., 1993, BioTechnology 11: 1145; de Kruif et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3938). An alternative method involves selection by binding biotinylated antigen in solution and then capturing on streptavidin-coated beads.

  In a preferred embodiment, panning is performed by immobilizing the antigen (generic or specific) to a well in a tube or plate, such as a Nunc MAXISORP ™ immunotube 8-well strip. Wells are coated with 150 μl of antigen (100 μg / ml in PBS) and incubated overnight. The wells are then washed 3 times with PBS and blocked with 400 μl PBS-2% skim milk (2% MPBS) for 2 hours at 37 ° C. The mixture is incubated for 90 minutes at room temperature to remove the liquid containing unbound phage. The wells are rinsed 10 times with PBS-0.1% Tween 20 and then 10 times with PBS to remove the detergent. Bound phage is eluted by adding 200 μl of freshly prepared 100 mM triethylamine, mixing well, and incubating for 10 minutes at room temperature. The eluted phage is transferred to a tube containing 100 μl of 1M Tris HCl (pH 7.4) and stirred to neutralize the triethylamine. Exponentially growing E. coli host cells (eg, TG1) are infected with, eg, 150 ml of diluted phage by incubation at 37 ° C. for 30 minutes. Infected cells are spun down, resuspended in fresh media, and seeded in upper agarose. The phage plaques are eluted or plunged into a new culture of host cells and propagated for analysis or further selection rounds. If necessary, one or more rounds of plaque generation is performed to ensure a population of naïve selection phage. Other screening techniques are described in Harrison et al., 1996, supra.

  Allows for secretion of bacterial non-suppressor strains, such as soluble gene III fusion proteins, following identification of phage expressing a single immunoglobulin variable region that binds the desired target when a phagemid vector such as pHEN1 is used By infecting HB2151, the variable region fusion protein is readily produced in soluble form. Alternatively, V region sequences can be subcloned into an appropriate expression vector to produce soluble proteins according to methods known in the art.

Purification and enrichment of dAbs dAb polypeptides secreted into the periplasmic space or bacterial media are recovered and purified according to known methods (Harrison et al., 1996, supra). Skerra & Puckthun (1988, Science 240: 1038) and Breitling et al. (1991, Gene 104: 147) describe the recovery of antibody polypeptides from the periplasm, and Better et al. (1988, Science 240: 1041). Describes the recovery from the culture supernatant. Purification can also be achieved by binding to a universal ligand such as protein A or protein L. Alternatively, the variable region can be expressed with a peptide tag that facilitates purification by affinity chromatography, such as Myc, HA or 6 × -His tag.

  The polypeptide is concentrated by several methods well known in the art, including, for example, ultrafiltration, diafiltration and tangential flow filtration. The ultrafiltration process uses semipermeable membranes and pressure to separate molecular species based on size and shape. The pressure is applied by gas pressure or centrifugation. Commercial limits by Millipore (Bedford, Mass .; examples include Centricon ™ and Microcon ™ concentrators) and Vivascience (Hanover, Germany; examples include Vivaspin ™ concentrators) Outer filtration products are widely available. By selecting a molecular weight cut-off smaller than the target polypeptide (a difference of as much as 10 kD can be used without problems, but usually 1/3 to 1/6 the molecular weight of the target polypeptide), the solvent and smaller solute are reduced. The polypeptide is retained when passing through the membrane. Thus, a molecular weight cutoff of about 5 kD is useful for enrichment of the dAb polypeptides described herein.

  Diafiltration using an ultrafiltration membrane for the “washing” process is used when it is desirable to remove or replace salts or buffers in the polypeptide preparation. The polypeptide is concentrated by passing a solvent and small solutes through the membrane, and the remaining salt or buffer is limited while diluting the retained polypeptide with fresh buffer or salt solution or water as desired. Removed by continuing filtration. In continuous diafiltration, fresh buffer is added at the same rate as the filtrate passes through the membrane. Diafiltration volume is the volume of polypeptide solution before the start of diafiltration, and with continuous diafiltration, wash 6 diafiltration volumes with fresh buffer. Makes it possible to remove more than 99.5% of fully permeable solutes. Alternatively, the process is performed in a discontinuous manner that repeatedly dilutes the sample and then returns to its original volume to remove or replace salts or buffers and ultimately concentrate the polypeptide. Is possible. Devices for diafiltration and detailed procedures for their use are available, for example, from Pall Life Sciences (Ann Arbor, Michigan) and Sartorius AG / Vivascience (Hanover, Germany).

  Tangential flow filtration (TFF), also known as “cross flow filtration”, also uses ultrafiltration membranes. A fluid containing the target polypeptide is aspirated tangentially along the surface of the membrane. Pressure causes some of the fluid to pass through the membrane while the target polypeptide is retained above the filter. However, unlike standard ultrafiltration, retained molecules do not accumulate on the membrane surface and are carried away by tangential flow. The solution that does not pass through the filter (containing the target polypeptide) can be repeatedly circulated through the membrane to achieve the desired concentration. Equipment for TFF and detailed approaches for its use are described, for example, in Millopore (eg ProFlux M12 ™ Benchtop TFF system and Pellicon ™ system) and Pall Life Sciences (eg Minim ™ tangential flow). Available from the Filtration System).

  Protein concentration is measured by several methods well known in the art. These methods include, for example, amino acid analysis, absorption at 280 nm, “Bradford” and “Lorry” methods, and SDS-PAGE. The most accurate method involves total hydrolysis followed by amino acid analysis by HPLC, and the concentration is then determined by comparison with the known sequence of the dAb polypeptide. This method is the most accurate, but expensive and time consuming. Protein measurements by measuring UV absorption at 280 nm are faster and much cheaper, but are relatively accurate and are preferred as a compromise for amino acid analysis. The protein concentration reported in the examples described herein was measured using absorbance at 280 nm.

“Bradford” and “Raleigh” protein tests (Bradford, 1976, Anal. Biochem. 72: 248-254; Lowry et al., 1951, J. Biol. Chem. 193: 265-275) most frequently Compare with a standard curve based on bovine serum albumin (BSA). These methods are less accurate and tend to underestimate the concentration of a single immunoglobulin variable region. However, it is possible to improve their accuracy by using _VH or _Vκ single region polypeptides as standards.

  Additional protein testing methods are described in US Pat. No. 4,839,295 (incorporated herein by reference), and Pierce as “BCA Protein Assay” (eg Pierce Catalog No. 23227). Bicinchoninic acid test sold by Biotechnology (Rockford, Ill.).

  The SDS-PAGE method uses known standard concentrations, eg, gel electrophoresis and Coomassie blue staining, which are compared to known amounts of a single immunoglobulin variable region polypeptide. It can be quantified by the naked eye or by concentration measurement.

  In a third aspect, the invention relates to a first immunoglobulin single variable region having a first binding specificity and a second single immunoglobulin single variable having a second (different) binding specificity. A method comprising: Selecting a first variable region by its ability to bind to a first epitope; (b) selecting a second variable region by its ability to bind to a second epitope; and (c) variable Combining the regions; and (d) selecting a ligand by its ability to bind to the first epitope and the second epitope.

  The ligand can bind to the first and second epitopes at the same time, or if there is competition for epitope binding between the binding regions, the binding of one region is the other for its cognate epitope. Region coupling can be eliminated. Thus, in one embodiment, step (d) above requires simultaneous binding to the first and second (and possibly further) epitopes, and in another embodiment, binding to the first and second epitopes. Are not done at the same time.

  The epitope is preferably present on a separate antigen.

  The ligand advantageously comprises a VH / VL combination of immunoglobulin variable regions or a combination of VH / VH or VL / VL, as described above. The ligand may also be a camel VHH region if the VHH region specific for an antigen that increases the half-life of the ligand in vivo does not bind chicken egg white lysozyme (HEL), porcine pancreatic α-amylase or NmC-A. Can be included. Conrath et al. (2001) JBC 276: 7346-7350 and WO 99/23221 have not described increasing the half-life of a ligand in vivo using specificity for an antigen that increases half-life. Pig, BSA-conjugated RR6 adenosine 5 dye or S. cerevisiae. Mutates HG982 cells.

  In one embodiment, the first variable region is selected for binding to the first epitope in the absence of a complementary variable region (ie, selected as a dAb as described above). In a further embodiment, the first variable region is different from the second variable region and in the presence of a third variable region that is complementary to the first region, the first epitope / antigen Selected for binding to. Similarly, the second region can be selected in the absence or presence of a complementary variable region.

  In addition to a protein that increases half-life, the antigen or epitope targeted by the ligand of the invention may be any antigen or epitope, but advantageously the antigen or epitope targeted for therapeutic benefit. It is an epitope. The present invention provides ligands comprising open structures, closed structures and sequestered dAb monomeric ligands specific for any such target, in particular the targets further specified herein. The target may be a polypeptide, protein or nucleic acid, which may be natural or synthetic, or part thereof. In this regard, the ligands of the invention can bind an epitope or antigen and act as an antagonist or agonist (eg, an EPO receptor agonist). One skilled in the art will appreciate that the choices are widely varied.

  They may be, for example, human or animal proteins, cytokines, cytokine receptors, enzymes, cofactors for enzymes, or DNA binding proteins. Suitable cytokines and growth factors that can be targeted by the mono- or bispecific binding polypeptides described herein include ApoE, Apo-SAA, BDNF, BLyS, cardiotrophin-1, EGF, EGF receptor, ENA-78, eotaxin, eotaxin-2, exodas-2, EpoR, acidic FGF, basic FGF, fibroblast growth factor-10, FLT3 ligand, fractalkine (CX3C), GDNF, G- CSF, GM-CSF, GF-01, insulin, IFN-y, IGF-I, IGF-II, IL-, IL-1p, 20IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11, IL-12, I -13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin B IP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphotactin, Murrelian inhibitor, single cell colony inhibitor, single cell attracting protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP- 4, MIG, MIP1α, MIP1β, MIP3α, MIP3β, MIP-4, bone marrow precursor inhibitor-1 (MPIF-1), NAP-2, neurturin, nerve growth factor, β-NGF, NT-3, NT-4 Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO / MGSA, GRO-β, GRO-8, HCC1, 1-309, HER1, HER2, HER3, HER4, TACE recognition site, TNF BP-I and TNF BP-II, and combinations described in the appendices herein, different combinations, or annex 2 present individually Any target disclosed in Appendix 3 may be mentioned, but is not limited thereto.

  As noted, preferred ligands include TNF-α and VEGF alone, together and / or with anti-HSA binding activity.

  Cytokine receptors include receptors for the aforementioned cytokines. It will be understood that this list is not exhaustive.

  In one embodiment of the invention, the variable region is derived from the respective antibody directed against the antigen or epitope. In a preferred embodiment, the variable region is derived from a repertoire of single variable antibody regions.

  In a further aspect, the present invention provides one or more nucleic acid molecules encoding at least one bispecific ligand as defined herein.

  Bispecific ligands can be encoded by a single nucleic acid molecule. Alternatively, the nuclear region can be encoded with individual nucleic acid molecules. If the ligand is encoded by a single nucleic acid molecule, the regions may be expressed as a fusion protein in the form of an scFv molecule or expressed separately and then bound to each other using, for example, a chemical binder. You may let them. Ligands expressed from individual nucleic acids will be bound to each other by suitable means.

  The nucleic acid can further encode a signal sequence that, when expressed, exports the polypeptide from the host cell and fuses to the surface component of the filamentous bacteriophage particle (or other component of the selection display system) upon expression. be able to.

  In a further aspect, the present invention provides a vector comprising a nucleic acid encoding a dual specific ligand according to the present invention.

  In a further aspect, the present invention provides a host cell transfected with a vector encoding a dual specific ligand according to the present invention.

  Expression from the vector can be configured to generate variable regions for selection, for example, on the surface of bacteriophage particles. This makes it possible to select the displayed variable region, thereby enabling selection of a “bispecific ligand” using the method of the present invention.

  The present invention further provides a kit comprising at least one bispecific ligand according to the present invention.

The bispecific ligand according to the present invention preferably comprises a combination of heavy and light chain regions. For example, a bispecific ligand can include a VH region and a VL region that can be bound to each other in the form of scFv. In addition, the ligand can include one or more CH or CL regions. For example, the ligand, CH1 region, CH2 or CH3 region, and / or _{C L} region, Cmu, Shimyu2, may include Cμ3 or Cμ4 domain, or combinations thereof. A hinge region can also be included. The combination of regions may be similar to a natural antibody such as IgG or IgM, or a fragment thereof such as an Fv, scFv, Fab or F (ab ′) 2 molecule. VH, other structures such as a single arm of an IgG molecule comprising VL, CH1 and _{C L} regions are also contemplated.

  In a preferred embodiment of the invention, the variable region is selected from a single region V gene repertoire. In general, a single antibody region repertoire is displayed on the surface of filamentous bacteriophages. In preferred embodiments, each single antibody region is selected by binding to an antigen of the phage repertoire.

  In a preferred embodiment of the invention, each single variable region can be selected for binding to its target antigen or epitope in the absence of a complementary variable region. In an alternative embodiment, a single variable region can be selected for binding to its target antigen or epitope in the presence of a complementary variable region. Thus, the first single variable region can be selected for the third complementary variable region, and the second variable region can be selected in the presence of the fourth complementary variable region. The complementary third or fourth variable region may be a natural cognate variable region having the same specificity as the single region being tested, or a non-cognate complementary region such as a “dummy” variable region. .

  Preferably, the bispecific ligands of the present invention comprise only two variable regions, but several such ligands can be incorporated together in the same protein, for example two such ligands such as IgG or IgM. It can be incorporated into multimeric immunoglobulins. Alternatively, in other embodiments, multiple bispecific ligands are combined to form a multimer. For example, two different bispecific ligands are combined to create a tetraspecific molecule.

  It should be noted that the light and heavy variable regions of the bispecific ligand produced according to the method of the present invention may be present on the same polypeptide chain or may be present on different polypeptide chains. The merchant will understand. If the variable regions are present on different polypeptide chains, they can be linked to a linker, generally a flexible linker (such as a polypeptide chain), a chemical linking group, or any other method known in the art. Can be coupled to each other.

  In a further aspect, the present invention provides a composition comprising a bispecific ligand obtained by the method of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

  Furthermore, the present invention provides a method for the treatment and / or prevention of diseases using a “dual specific ligand” or composition according to the present invention.

  In a second configuration, the present invention provides a multispecific ligand comprising at least two non-complementary variable regions. For example, a pair of VH regions or a pair of VL regions can be included. Advantageously, these regions are non-camel derived regions. They are preferably human regions or comprise a human framework region (FW) and one or more heterologous CDRs. CDR and framework regions are regions of immunoglobulin variable regions that are considered in the Kabat database of protein sequences for immunology.

  Preferred human framework regions are those encoded by germline gene segments DP47 and DPK9. Advantageously, FW1, FW2 and FW3 of the VH or VL region have the sequence of FW1, FW2 or FW3 from DP47 or DPK9. The human framework can optionally include mutations, for example, up to about 5 amino acid changes or up to about 10 amino acid changes in the human framework used for the ligands of the invention.

  The variable regions in the multispecific ligand according to the second configuration of the invention can be arranged in an open or closed structure. That is, the variable regions are arranged so that their cognate ligands can be bound independently and simultaneously, or only one of the variable regions can bind the cognate ligand at a time. be able to.

  The inventors have found that under certain structural conditions, a non-complementary variable region (eg, two light chain variable regions or two heavy chain variable regions) does not function together as a cognate pair. Nevertheless, it was recognized that the binding of the first epitope to the first variable region could exist to suppress the binding of the second epitope to the second variable region.

  Advantageously, the ligand comprises two or more variable regions. That is, it includes at least four variable regions. Advantageously, the four variable regions comprise a human-derived framework.

  In a preferred embodiment, the human framework is identical to the framework of the human germline sequence.

  We believe that the antibodies have particular application in ligand binding studies for therapy and other applications.

  Thus, in a first aspect of the second configuration, the present invention provides a method for producing a multispecific ligand comprising: a) its ability to bind a first epitope binding region to a first epitope. Selecting, b) selecting the second epitope binding region by its ability to bind to the second epitope, c) combining the epitope binding regions, d) a closed structure multispecific ligand Selecting by one second epitope and its ability to bind to said second epitope.

  In a further aspect of the second configuration, the present invention provides a first epitope binding region having a first epitope binding specificity and a non-complementary second epitope binding region having a second epitope binding specificity. Wherein the first and second binding specificities are for preparing a closed structure multispecific ligand that competes for epitope binding such that the closed structure multispecific ligand cannot bind both epitopes simultaneously. A) selecting a first epitope binding region by its ability to bind to a first epitope; and b) selecting a second epitope binding region by its ability to bind to a second epitope. C) combining the epitope-binding regions such that these regions are in a closed structure; and d) a closed structure multispecific ligand. , Said first second epitope and bind to the second epitope, the method comprising the steps of: selecting by the first and second its ability to not simultaneously bind to the epitope.

  The present invention further includes a first epitope binding region having a first epitope binding specificity and a non-complementary second epitope binding region having a second epitope binding specificity, A binding specificity of 2 provides a closed structure multispecific ligand that competes for epitope binding so that the closed structure multispecific ligand cannot bind both epitopes simultaneously.

  An alternative embodiment of the above aspect of the second configuration of the invention optionally includes a further step (bl) comprising selecting a third or further epitope binding region. Thus, the produced multispecific ligands contain more than one epitope binding specificity, whether open or closed. In a preferred embodiment of the second configuration of the invention, when the multispecific ligand comprises two or more epitope binding regions, at least two of the regions are closed structures and compete for binding. Other regions may compete for binding or may be free to associate independently with their cognate epitope.

  According to the present invention, the term “multispecific ligand” means a ligand having two or more epitope binding specificities as defined herein.

  As defined herein, the term “closed structure” (multispecific ligand) refers to an epitope of a ligand such that epitope binding by one epitope binding region competes with epitope binding by another epitope binding region. It means that the binding regions bind or associate with each other, possibly by a protein framework. That is, cognate epitopes can be bound individually by each epitope binding region, but not simultaneously. Using the methods described herein, it is possible to achieve a closed structure of the ligand.

  “Open structure” means that the epitope binding regions of a ligand bind or associate with each other, possibly by a protein framework, so that epitope binding by one epitope binding region does not compete with epitope binding by other epitope binding regions Means that.

  As referred to herein, the term “competing” means that binding of a first epitope to its cognate epitope binding region is inhibited when the second epitope is bound to its cognate binding region. Means. For example, binding can be sterically inhibited, for example, by physical blocking of the binding region, or by changing the structure or environment of the binding region such that its affinity or binding activity for the epitope is reduced.

  In a further aspect of the second configuration of the invention, the epitopes can be displaced from each other upon binding. For example, a first epitope on an antigen that, when bound to its cognate first binding region, displaces the epitope bound to the second binding region, causing steric hindrance of the second binding region or structural change thereof. Can exist.

  Advantageously, the binding is reduced to 25% or more, advantageously 40%, 50%, 60%, 70%, 80% or 90% or more, preferably close to 100% so that the binding is completely inhibited. The Epitope binding can be measured by conventional antigen binding tests such as ELISA, fluorescence-based techniques including FRET, or techniques such as surface plasmon resonance that measure the mass of the molecule.

  According to the method of the present invention, advantageously, each epitope binding region has a different epitope binding specificity.

  Within the scope of the present invention, first and second “epitope” are understood to be epitopes that are not identical and are not bound by a single monospecific ligand. They may be present on different or the same antigen, but may be separated by a distance that does not form a single object that can be bound by a single monospecific VH / VL binding pair of conventional antibodies. Experimentally, if both individual variable regions of a single chain antibody form (region antibody or dAbs) compete individually with monospecific VH / VL ligands for the two epitopes, those two epitopes Are not far enough to be considered individual epitopes according to the present invention.

  The closed structure monospecific ligands of the present invention do not include the ligands described in WO02 / 02773. Thus, the ligands of the invention do not include complementary VH / VL pairs that jointly bind any one or more antigens or epitopes. Instead, the ligand according to the invention preferably comprises a non-complementary VH or VL pair. Advantageously, each VH or VL in each VH or VL pair has a different epitope binding specificity, such that the epitope binding site competes with the epitope binding at one site for the epitope binding at the other site. Be placed.

  According to the present invention, advantageously, each epitope binding region comprises an immunoglobulin variable region. More advantageously, each immunoglobulin variable region is a variable light chain region (VL) or a variable heavy chain region VH. In the second configuration of the invention, the immunoglobulin region when present on the ligand according to the invention is non-complementary, i.e. does not associate to form a VH / VL antigen binding site. Thus, multispecific ligands contemplated in the second configuration of the invention comprise immunoglobulin regions of the same subtype that are variable light chain regions (VL) or variable heavy chain regions (VH). Furthermore, when the ligand according to the invention is a closed structure, the immunoglobulin region may be of the camel VHH type.

  In an alternative embodiment, the ligand according to the invention does not comprise a camel VHH region. More particularly, the ligands of the invention do not contain one or more amino acid residues specific for the camel VHH region as compared to the human VH region.

  Advantageously, the single variable region is derived from antibodies selected for binding activity against different antigens or epitopes. For example, the variable region can be isolated at least in part by human immunization. Alternative methods are known in the art, including isolation from human antibody libraries and synthesis of artificial antibody genes.

  The variable region advantageously binds a superantigen such as protein A or protein L. Binding to a superantigen is a property of an appropriately overlaid antibody variable region that allows the region to be isolated from, for example, a recombinant or mutated region. The epitope binding region according to the present invention comprises a protein framework and an epitope interaction region (advantageously present on the surface of the protein). The epitope binding region may be based on a protein framework or skeleton other than the immunoglobulin region. For example, natural bacterial receptors such as SpA have been used as frameworks for CDR grafting to generate ligands that specifically bind to one or more epitopes. Details of this procedure are described in US Pat. No. 5,831,012. Other suitable frameworks include those based on fibronectin and affibodies. Details of suitable procedures are described in WO 98/58965. Other suitable frameworks include van den Beuken et al. Mol. Biol. (2001) 310, 591-601, the lipocalin and CTLA4, and a skeleton as described in W00069907 (Medical Research Council), for example based on the ring structure of bacterial GroEL or other chaperone polypeptides. Can be mentioned. Protein scaffolds can be combined. For example, CDRs can be transplanted into a CTLA4 framework and used in combination with immunoglobulin VH or VL regions to form multivalent ligands. Similarly, fibronectin, lipocalin and other frameworks can be combined.

  One skilled in the art will appreciate that the epitope-binding regions of closed structural multispecific ligands produced according to the methods of the present invention can be present on the same polypeptide chain or on different polypeptide chains. If the variable regions are present on different polypeptide chains, they may be introduced via linkers, preferably flexible linkers (such as polypeptide chains), chemical linking groups, or any other method known in the art. Can be combined.

  The first and second epitope binding regions can be associated covalently or non-covalently. When these regions are covalently associated, the association can mediate, for example, a disulfide bond.

  In the second aspect of the present invention, the first and second epitopes are preferably different. They may be polypeptides, proteins or nucleic acids, which may be natural or synthetic, or parts thereof. In this regard, the ligands of the invention can bind epitopes or antigens and can act as antagonists or agonists (eg, receptor agonists). The epitope binding regions of a ligand in one embodiment have the same epitope specificity, eg, when multiple epitope copies are present on the same antigen, they can bind simultaneously. In other embodiments, these epitopes are provided on different antigens so that the ligand can bind the epitope and crosslink the antigen. One skilled in the art will appreciate that the choice of epitopes and antigens is wide and varied. They may be, for example, human or animal proteins, cytokines, cytokine receptors, enzymes, cofactors for enzymes, or DNA binding proteins.

  Suitable cytokines and growth factors that can be targeted by the mono- or bispecific binding polypeptides described herein include ApoE, Apo-SAA, BDNF, BLyS, cardiotrophin-1, EGF, EGF receptor, ENA-78, eotaxin, eotaxin-2, exodas-2, EpoR, acidic FGF, basic FGF, fibroblast growth factor-10, FLT3 ligand, fractalkine (CX3C), GDNF, G- CSF, GM-CSF, GF-01, insulin, IFN-y, IGF-I, IGF-II, IL-, IL-1p, 20IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11, IL-12, I -13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin B IP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphotactin, Murrelian inhibitor, single cell colony inhibitor, single cell attracting protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP- 4, MIG, MIP1α, MIP1β, MIP3α, MIP3β, MIP-4, bone marrow precursor inhibitor-1 (MPIF-1), NAP-2, neurturin, nerve growth factor, β-NGF, NT-3, NT-4 Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO / MGSA, GRO-β, GRO-8, HCC1, 1-309, HER1, HER2, HER3, HER4, TACE recognition site, TNF BP-I and TNF BP-II, and combinations described in the appendices herein, different combinations, or annex 2 present individually Any target disclosed in Appendix 3 may be mentioned, but is not limited thereto.

  Cytokine receptors include receptors for the aforementioned cytokines, such as IL-1 R1, IL-GR, IL-10R, IL-18R, as well as receptors for the cytokines described in Annex 2 or 3, and Annex 2 And the receptor disclosed in 3.

  It will be understood that this list is not exhaustive. If the multispecific ligand binds to two epitopes (on the same or different antigens), the antigen can be selected from this list.

  Advantageously, bispecific ligands targeting cytokines and other molecules that synergistically cooperate in the therapeutic context of the organism's body can be used. Accordingly, the present invention is a method for synergizing the activity of two or more cytokines, comprising administering a dual specific ligand capable of binding to said two or more cytokines. Provide a method. In this aspect of the invention, the bispecific ligand is any bispecific ligand, including a ligand composed of complementary and / or non-complementary regions, an open structure ligand, and a closed structure ligand. May be. For example, this aspect of the present specification relates to a combination of a VH region and a VL region, only a VH region, and only a VL region.

  Synergism from a therapeutic point of view can be achieved in several ways. For example, if both targets are ligand targets, the target combination may be therapeutically active, while targeting only one target is not therapeutically effective. In other embodiments, only one target can provide a low or minimal therapeutic effect, but when combined with a second target, the combination results in a synergistic increase in therapeutic effect.

  Preferably, the cytokine bound by the bispecific ligand of this aspect of the invention is selected from the list shown in Appendix 2.

  Furthermore, bispecific ligands can be used in the oncology field where one specificity targets CD89 expressed by cytokine cells and the other is tumor specific. Examples of tumor antigens that can be targeted are shown in Appendix 3.

  In one embodiment of the second configuration of the invention, the variable region is derived from an antibody directed against the first and / or second antigen or epitope. In a preferred embodiment, the variable region is derived from a repertoire of single variable antibody regions. In one example, the repertoire is a repertoire that is not made in an animal or synthetic repertoire. In other examples, the single variable region is not isolated (at least in part) by animal immunization. It is therefore possible to isolate a single region from a neural library.

  In another aspect, the second configuration of the invention comprises a first epitope binding region having a first epitope binding specificity and a non-complementary second epitope binding region having a second epitope binding specificity. And a multispecific ligand comprising: The first and second binding specificities may be the same or different.

  In a further aspect, the present invention comprises a first epitope binding region having a first epitope binding specificity and a non-complementary second epitope binding region having a second epitope binding specificity, And the second binding specificity provides a closed structure multispecific ligand that can compete for epitope binding such that the closed structure multispecific ligand cannot bind both epitopes simultaneously.

  In a further aspect, the present invention provides an open structure ligand comprising non-complementary binding regions that are specific for different epitopes on the same target. The ligand binds to the target with high binding activity.

  Similarly, the present invention includes non-complementary binding regions specific for the same epitope, and includes IL-5, PDGF-AA, PDGF-BB, TGFβ, TGFβ2, TGFβ3 and TNFα, such as human TNFα receptor 1 And a multivalent ligand directed to a target comprising multiple copies of said epitope, such as human TNFα.

  In a similar embodiment, binding to individual epitopes has no therapeutic significance, but the ligands according to the invention have low affinity so that the increased binding activity resulting from binding to two epitopes provides a therapeutic advantage. It can be configured to bind individual epitopes. In certain instances, epitopes that are present individually in normal cell types and are clustered only in abnormal or diseased cells such as tumor cells can be targeted. In this context, the bispecific ligand according to the present invention effectively targets only abnormal or tumor disease cells. Multiple copies of the same epitope, or a ligand specific for an adjacent epitope on the same target (known as chelated dAbs) is a trimer containing three or more non-complementary binding regions Alternatively, it may be a multimeric (tetramer or higher) ligand. For example, a ligand comprising 3 or 4 VH or VL regions can be constructed.

  In addition, ligands are provided that bind to multi-subunit targets, where each binding region is specific for the target subunit. The ligand may be a dimer, trimer or multimer. Preferably, the multispecific ligand according to the above aspect of the invention is obtainable by the method of the first aspect of the invention.

  According to the above aspect of the second configuration of the present invention, advantageously, the first epitope binding region and the second epitope binding region are non-complementary immunoglobulin variable regions as defined herein. . It is a VH-VH or VL-VL variable region.

  In particular, chelated dAbs constitute a library of dimers, trimers or multimeric dAbs using preferred embodiments of the invention, i.e., vectors containing construct dAbs upstream or downstream of the linker sequence, It can be prepared by the use of anchor dAbs that insert a third or additional dAbs on the other side of the linker. For example, the anchor or derived dAb may be TAR1-5 (VK), TAR1-27 (V), TAR2h-5 (VH) or TAR2h-6 (VK).

In an alternative approach, the use of linkers can be avoided, for example, by using non-covalent or natural affinity between binding regions such as VH and VL. Thus, the present invention is a method for preparing a chelating ligand comprising:
(A) providing a vector comprising a nucleic acid sequence encoding a single binding region specific for a first epitope on a target;
(B) providing a vector encoding a repertoire comprising a second binding region specific for a second epitope on the target, which may be the same as or different from the first epitope, wherein the second epitope The epitope is adjacent to the first epitope;
(C) expressing the first and second binding regions;
(D) isolating a combination of first and second binding regions that bind to form a target binding dimer.

  The first and second epitopes are adjacent so that the multimeric ligand can bind to both epitopes simultaneously. This gives the ligand the advantage of improved binding activity. If the epitopes are identical, the increased binding activity is due to the presence of multiple copies of the epitope on the target that allow at least two replicates to bind simultaneously to obtain an enhanced binding activity effect. can get.

  The binding regions can be associated in several ways, as well as through the use of linkers.

  For example, the binding region can include cysteine residues, avidin and streptavidin groups, or other means for synthesis after non-covalent bonding. Those combinations that bind efficiently to the target will be isolated. Alternatively, the linker comprises a first binding region and a second expressed as a single polypeptide from a single vector, including, for example, a repertoire of the first binding region, the linker, and the second binding region as described above. May exist between the bonding regions.

  In preferred embodiments, the first and second binding regions spontaneously associate when bound to an antigen. For example, when the VH and VK regions bind to adjacent epitopes, they naturally associate in a ternary interaction to form a stable dimer. The associated protein can be isolated in a target binding test. The advantage of this procedure is that only binding regions that bind to neighboring epitopes in the correct structure associate and are isolated as a result of improved binding activity to the target.

  In an alternative embodiment of the above aspect of the second configuration of the invention, the at least one epitope binding region comprises a non-immunoglobulin “protein scaffold” or “protein scaffold” as defined herein. Suitable non-immunoglobulin protein scaffolds include, but are not limited to, any of the aforementioned scaffolds selected from the group consisting of SpA, fibronectin, GroEL and other chaperones, lipocalin, CCTLA4 and affibodies.

  According to the above aspect of the second configuration of the present invention, the epitope binding region is advantageously bound to a “protein backbone”.

  Advantageously, the protein scaffold according to the invention is an immunoglobulin scaffold. According to the present invention, the term “immunoglobulin scaffold” means a protein that, as defined in the present invention, contains at least one immunoglobulin overlay and acts as a nucleus for one or more epitope binding regions. To do.

  A preferred “immunoglobulin scaffold” as defined herein comprises at least (i) an immunoglobulin molecule comprising the CL (kappa or lambda subclass) of an antibody or (ii) the CH1 region of an antibody heavy chain; And an immunoglobulin molecule comprising the CH2 region; an immunoglobulin molecule comprising the CH1, CH2 and CH3 regions of the antibody heavy chain; or an assembly (ii) used in conjunction with the antibody CL (kappa or lambda subclass) One of the skeletons. A hinge region can also be included. Such combinations of regions may be similar to natural antibodies such as IgG or IgM, or fragments thereof such as Fv, scFv, Fab or F (ab ') 2 molecules.

  Those skilled in the art will recognize that this list is not intended to be exhaustive.

  Backbone binding to the epitope binding regions defined herein can be achieved at the polypeptide level, ie after expression of the nucleic acid encoding the backbone and / or epitope binding region. Alternatively, the binding step can be performed at the nucleic acid level. Methods for conjugating a protein backbone according to the present invention to one or more epitope binding regions are well known to those of skill in the art and involve the use of protein chemistry and / or molecular biology techniques as described herein. Including.

  Advantageously, the closed structure multispecific ligand comprises a first region capable of binding a target molecule and a second region capable of binding a molecule or group that extends the half-life of the ligand. be able to. For example, the molecule or group may be a giant agent such as HSA or a cell matrix protein. As used herein, the phrase “a molecule or group that increases the half-life of a ligand” refers to the molecule or group when bound to a bispecific ligand as described herein. Means a molecule or chemical group that increases the in vivo half-life of the bispecific ligand when administered to an animal compared to an unbound ligand. Examples of molecules or groups that extend the half-life of the ligand are described below. In preferred embodiments, a closed structure multispecific ligand may be capable of binding a target molecule only to a molecule or group displacement that increases half-life. Thus, for example, closed structure multispecific ligands maintain circulation in the bloodstream of the subject by macromolecules such as HSA. When encountering a target molecule, competition between the binding regions of closed structural multispecific ligands results in HSA displacement and target binding.

Ligands according to any aspect of the invention, as well as dAb monomers useful for constituting the ligands, are advantageously between 300 nM and 5 pM (ie 3 × 10 ⁻⁷ to 5 × 10 ⁻¹² M), preferably 50 nM to 20 pM, or 5 nM to 200 pM, or 1 nM to 100 pM, 1 × 10 ⁻⁷ M or less, 1 × 10 ⁻⁸ M or less, 1 × 10 ⁻⁹ M or less, 1 × 10 ⁻¹⁰ M or less, or 1 × 10 5 × 10 ⁻¹ to 1 × 10 ⁻⁷ S ⁻¹ , preferably 1 × 10 ⁻² to 1 × 10 ⁻⁶ S ⁻¹ measured by a K _{d of} ⁻¹¹ M or less and / or surface plasmon resonance. Or 5 × 10 ⁻³ to 1 × 10 ⁻⁵ S ⁻¹ , or 5 × 10 ⁻¹ S ⁻¹ or less, or 1 × 10 ⁻² S ⁻¹ or less, or 1 × 10 ⁻³ S ⁻¹ or less, or 1 × ¹⁰ -4 ^{S -1} or less, or 1 × 10 ^- They can dissociate from their cognate 20 targets with a Koff rate constant of ⁵ S ⁻¹ or less, or 1 × 10 ⁻⁶ S ⁻¹ or less. K _d rate constant _is correlated defined as _K off _/ K _on.

In particular, the present invention provides an anti-TNF-α dAb monomer (or a bispecific ligand containing the dAb), homodimer, heterodimer or homotrimer in which each dAb binds to TNF-α. Provide a ligand. The ligand binds to TNF-α with a K _d of 300 nM to 5 pM (ie 3 × 10 ⁻⁷ to 5 × 10 ⁻¹² M), preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM, most preferably 1 nM to 100 pM. To do. In other words, K _d is 1 × 10 ⁻⁷ M or less, preferably 1 × 10 ⁻⁸ M or less, more preferably 1 × 10 ⁻⁹ M or less, advantageously 1 × 10 ⁻¹⁰ M or less, Most preferably 1 × 10 ⁻¹¹ M or less and / or 5 × 10 ⁻¹ to 1 × 10 ⁻⁷ S ⁻¹ , preferably 1 × 10 ⁻² to 1 × 10 ⁻⁶ measured by surface plasmon resonance. S ⁻¹ , more preferably 5 × 10 ⁻³ to 1 × 10 ⁻⁵ S ⁻¹ , for example 5 × 10 ⁻¹ S ⁻¹ or less, preferably 1 × 10 ⁻² S ⁻¹ or less, more preferably 1 × A K _{off of} 10 ⁻³ S ⁻¹ or less, preferably 1 × 10 ⁻⁴ S ⁻¹ or less, more preferably 1 × 10 ⁻⁵ S ⁻¹ or less, most preferably 1 × 10 ⁻⁶ S ⁻¹ or less. It is a rate constant.

  Preferably, the ligand is 500 nM to 50 pM, preferably 100 nM to 50 pM, advantageously 10 nM to 100 pM, more preferably 1 nM to 100 pM, such as 50 nM or less, preferably 5 nM or less, advantageously 500 pM, in a standard L929 test. Hereinafter, TNF-α is neutralized with an ND50 of 200 pM or less, most preferably 100 pM or less.

  Preferably, the ligand is 500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 510 nM to 100 pM, advantageously 1 nM to 100 pM, such as 50 nM or less, preferably 5 nM or less, more preferably 500 pM or less, advantageously 200 pM or less. Most preferably, binding of TNF-α to TNF-α receptor I (p55 receptor) is inhibited with an IC50 of 100 pM or less. Preferably, TNF-α is human TNF-α.

Also, the present invention provides 300 nM to 5 pM (ie, 3 × 10 ⁻⁷ to 5 × 10 ⁻¹² M), preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM, most preferably 1 nM to 100 pM, such as 1 × 10 ⁻ A K _{d of} ⁷ M or less, preferably 1 × 10 ⁻⁸ M or less, more preferably 1 × 10 ⁻⁹ M or less, advantageously 1 × 10 ⁻¹⁰ M or less, most preferably 1 × 10 ⁻¹¹ M or less. And / or 5 × 10 ⁻¹ to 1 × 10 ⁻⁷ S ⁻¹ , preferably 1 × 10 ⁻² to 1 × 10 ⁻⁶ S ⁻¹ , more preferably 5 × 10 ^{− 3} to 1 × 10 ⁻⁵ S ⁻¹ , for example 5 × 10 ⁻¹ S ⁻¹ or less, preferably 1 × 10 ⁻² S ⁻¹ or less, more preferably 1 × 10 ⁻³ S ⁻¹ or less, advantageously 1 x ^10-4 S- ^{1 or} higher Below, more advantageously, an anti-TNF receptor I dAb monomer that binds to TNF receptor I with a K _off rate constant of 1 × 10 ⁻⁵ S ⁻¹ or less, most preferably 1 × 10 ⁻⁶ S ⁻¹ or less. Or a dual specific ligand comprising the dAb.

  Preferably, the dAb monomer or ligand is 500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM, in standard tests (eg, the L929 or HeLa tests described herein), Advantageously, TNF-α is neutralized with an ND50 of 1 nM to 100 pM, such as 50 nM or less, preferably 5 nM or less, more preferably 500 pM or less, advantageously 200 pM or less, most preferably 100 pM or less.

  Preferably, the dAb monomer or ligand is 500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM, advantageously 1 nM to 100 pM, such as 50 nM or less, preferably 5 nM or less, more preferably 500 pM or less, Advantageously, binding of TNF-α to TNF-α5 receptor I (p55 receptor) is inhibited with an IC50 of 200 pM or less, most preferably 100 pM or less. Preferably, the TNF receptor I target is human TNF-α.

The present invention also provides a dAb monomer that binds to serum albumin (SA) with a K _d of 1 nM to 500 μM (eg, 1 × 10 ⁻⁹ to 5 × 10 ⁻⁴ ), preferably 100 nM to 10 uM (or the dAb). Bispecific ligands). Preferably, for bispecific ligands comprising a first anti-SA dAb and a second dAb to another target, the affinity of the second dAb for that target (eg, Kd and / or surface plasmon) By resonance, eg, Koff measured using BIAcore, the affinity of the first dAb to SA is 1 to 100,000 times (preferably 100 to 100,000 times, more preferably 1000 to 100,000 times, or 10,000 times to 100,000 times). Double). For example, a first dAb binds SA with an affinity of about 10 μM and a second dAb binds its target with an affinity of 100 pM. Preferably, the serum albumin is human serum albumin (HSA).

In one embodiment, the first Adb (or dAb monomer) binds SA (eg, HSA) with a K _d of about 50, preferably 70, more preferably 100, 150 or 200 nM.

  The present invention provides dimers, trimers and polymers of the dAb monomer according to the above-described embodiments of the present invention.

  Ligands according to the invention comprising dAb monomers, dimers and trimers can be bound to antibody Fc regions comprising one or both of the CH2 and CH3 regions and optionally the hinge region. For example, the polypeptide can be prepared using a vector that encodes a ligand that binds to the Fc region as a single nucleotide sequence.

  In a further aspect of the second configuration of the invention, the invention provides one or more nucleic acid molecules that encode at least a multispecific ligand as defined herein. In one embodiment, the ligand is a closed structure ligand. In other embodiments, the ligand is an open structure ligand. Multispecific ligands can be encoded on a single nucleic acid molecule. Alternatively, each epitope binding region can be encoded by a separate nucleic acid molecule. If the ligand is encoded by a single nucleic acid molecule, those regions may be expressed as a fusion polypeptide or may be expressed separately and subsequently bound to each other using, for example, a chemical binding agent. Good. Ligands expressed from individual nucleic acids will be bound to each other by appropriate means.

  The nucleic acid can encode a single sequence for expression and export of the polypeptide from the host cell at the same time as it is expressed and at the same time is fused with the surface component of the filamentous bacteriophage particle (or other component of the selection display system). be able to. Leader sequences that can be used for bacterial expression and / or phage or phagemid display include pelB, stII, ompA, phoA, bla and pelA.

  In a further aspect of the second configuration of the present invention, the present invention provides a vector comprising a nucleic acid according to the present invention.

  In a further aspect, the present invention provides a host cell transfected with a vector according to the present invention.

  Expression from the vector can be configured to generate epitope binding regions for selection, eg, on the surface of bacteriophage particles. This allows the selection of the display area and the selection of “multispecific ligands” using the method of the invention.

  In a preferred embodiment of the second configuration of the invention, the epitope binding region is an immunoglobulin variable region and is selected from a single region V gene repertoire. In general, a single antibody region repertoire is displayed on the surface of filamentous bacteriophages. In preferred embodiments, each single antibody region is selected by binding a phage repertoire to an antigen.

  The invention further provides a kit comprising at least one multispecific ligand according to the invention, which may be an open or closed structure ligand. The kit according to the present invention may be, for example, a diagnostic kit, a treatment kit, a chemical or biological species detection kit, or the like.

  In a further aspect of the second configuration of the invention, the invention provides a homogeneous immunity test using a ligand according to the invention.

  In a further aspect of the second configuration of the invention, the invention comprises a closed structure multispecific ligand obtainable by the method of the invention and a pharmaceutically acceptable carrier, diluent or excipient. I will provide a. Furthermore, the present invention provides a method for treating a disease using a closed structure multispecific ligand or composition according to the present invention. In a preferred embodiment of the invention, the disease is cancer or an inflammatory disease such as rheumatoid arthritis, asthma or Crohn's disease.

  In a further aspect of the second configuration of the present invention, the present invention provides a diagnostic method comprising diagnosis of a disease using a closed structure multispecific ligand or composition according to the present invention.

  Thus, in general, the binding of an analyte to a closed structure multispecific ligand can be utilized to displace the agent and generate a signal related to the displacement. For example, binding of an analyte (second antigen) causes the enzyme (first antigen) to bind to the antibody, particularly when the enzyme is retained by the antibody through its active site, providing the basis for an immunoassay. It is possible.

Thus, in the last aspect of the second configuration, the present invention is a method for detecting the presence of a target molecule comprising:
(A) providing a closed structure multispecific ligand bound to an antigen, wherein the ligand is specific for a target molecule and an agent, and the agent bound by the ligand is Generating a detectable signal related to the displacement;
(B) contacting the closed structure multispecific ligand with a target molecule;
(C) detecting a signal generated as a result of the displacement of the agent.

  According to the above aspect of the second configuration of the present invention, advantageously the agent is an enzyme that becomes inactive when bound by a closed structure multispecific ligand. Alternatively, the agent may be any selected from the group consisting of a substrate for an enzyme and a fluorescent, luminescent or chromogenic molecule that becomes inactive or disappears when bound by a ligand.

  Sequences similar or homologous (eg, having at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the amino acid level sequence identity is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99. % Or more. At the nucleic acid level, sequence identity is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Or more. Alternatively, substantial identity exists when a nucleic acid segment hybridizes to the complement of a strand under selective hybridization conditions (eg, very stringent hybridization conditions). The nucleic acid may be present in whole cells, may be present in cell lysates, or may be present in a partially purified form or a substantially naive form.

  Calculations of “homology” or “sequence identity” or “similarity” between two sequences (the terms are used interchangeably herein) are performed as follows. Align sequences for purposes of optimal comparison (eg, introduce gaps in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and heterologous sequences for comparison purposes) Can be ignored).

  In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60% of the length of the reference sequence, Even more preferred is at least 70%, 80%, 90%, 100%. The amino acid residues or nucleotide residues at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, Amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”). The percent identity of two amino acids is the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. It is a function.

  Advantageously, the BLAST algorithm (version 2.0) with parameters set to default values is employed for alignment of the sequences. The BLAST algorithm is based on the National Center for Biotechnology Information (".ncbi") of the National Institutes of Health ("nib") of the US Government (".gov") in the "/ Blast!" Directory of the "blast_help.html" file. It is described in detail on the wide web site ("www"). The search parameters are defined as follows and are preferably set to defined default parameters.

  BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm employed by the blastp, blastn, blastx, tblastn and tblastx programs. These programs are described in Karlin and Altschul, 1990, 20 Proc. Natl. Acad. Sci. USA 87 (6): 2264-8 (representing their significance using the statistical methods referenced in “blast_help.html” above. The BLAST program specifies homology to query sequences, for example. Created for sequence identity searches, these programs are generally not useful for motif style searches, see Altschul et al. (1994) for a discussion of basic issues in sequence database similarity searches. .

  The five BLAST programs available on the National Center for Biotechnology Information website perform the following tasks: “Blastp” compares an amino acid query sequence against a protein sequence database. “Blastn” compares a nucleotide query sequence against a nucleotide sequence database. “Blastx” compares a six-frame conceptual translation product of nucleotide query sequences (both strands) against a protein sequence database. “Tblastn” compares a protein query sequence against a dynamically translated nucleotide sequence in all six reading frames (both strands). “Tblastx” compares the 6-frame translation of the nucleotide query sequence against the 6-frame translation of the nucleotide sequence database.

  BLAST uses the following search parameters:

  Histogram (HISTOGRAM): Displays a histogram of scores for each search. The default is yes (see parameter H in the BLAST Manual).

  Description (DESCRIPTIONS): Limits the number of short descriptions that make a reported sequence correspond to a specified number. The default limit is 100 descriptions (see parameter V on the manual page). See also EXPECT and CUTOFF.

  ALIGNMENT: Limit database sequences to the specified number for which high score segment pairs (HSPs) are reported. The default limit is 50. If more database sequences meet the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only correspondences that are attributed to maximum statistical significance are reported. (See parameter B in the BLAST Manual).

  EXPECT: Statistical significance threshold for reporting correspondence to database sequences. The default value is 10, according to Karlin and Altschul (1990), where 10 correspondences are expected to be found simply by chance. If the statistical significance attributed to the correspondence is above the EXPECT threshold, the correspondence is not reported. Lower EXPECT thresholds are more stringent and have fewer chance responses to be reported. Fractional values are also allowed (see parameter E in the BLAST Manual).

  Cut-off (CUTOFF): Cut-off score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for database sequences only if the statistical significance attributed to them is at least as high as that attributed to an isolated HSP with a score equal to the cut-off (CUTOFF) value. Higher cutoff (CUTOFF) values are more rigorous and have fewer chance responses reported (see parameter S in the BLAST Manual). Typically, the significance threshold can be managed more intuitively using EXPECT.

  MATRIX: A surrogate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992, Proc. Natl. 30 Acad. Sci. USA 89 (22): 10915-9). Valid alternative choices include PAM40, PAM120, PAM: 250, and IDENTITY. There is no substitute scoring matrix available for BLASTN. If a matrix (MATRIX) directory in the BLASTN array is specified, an error response is returned.

  STRAND: restricts the TBLASTN search to the top or bottom strand of the database sequence. Alternatively, the BLASTN, BLASTX, or TBLASTX search is limited to the reading frame at the top or bottom strand of the query sequence.

  Filter (FILTER): Woton & Federhen (1993) Computers and Chemistry 17: 149-163 query sequence segment with low compositional complexity as measured by SEG program, or Claverie & States, 1993, Computers and Chemistry 19: Mask segments composed of short-period intra-repetitions as measured by the 201 XNU program or, for BLASTN, by the DUST program of the Tatusov and Lipman (see NCBI World Wide Web site). Filtering eliminates reports that are statistically significant but not biologically significant from the blast output (eg common acidic, basic or proline rich regions) and can be used for specific mapping to database sequences It is possible to leave a more biologically important region of the query sequence. The low complexity sequence found by the filter program uses the letter “N” in the nucleotide sequence (eg “N” repeated 13 times) and the letter “X” in the protein sequence (eg “X” repeated 9 times). Replaced.

  Filtering applies only to query sequences (or their translation products), not to database sequences. The default filtering is DUST for BLASTN and SEG for other programs. When applied to arrays in SWISS-PROT, filtering should not always be expected to be effective, as it is not uncommon to have nothing masked by SEG, XNU, or both. The sequence may also be masked entirely, indicating that any reported statistical significance for the unfiltered query sequence is questionable.

  NCBI-gi: Causes the NCBI gi identifier to be shown in the output in addition to the acquisition and / or location name.

  Most preferably, the sequence comparison is performed using a simple BLAST search algorithm provided on the NCBI World Wide Web site in the “/ BLAST” directory.

Preparation of Immunoglobulin-Based Multispecific Ligands Bispecific ligands according to the present invention can be used to produce scFv, “phage” antibodies, and structures that are open or closed according to the desired configuration of the present invention. Other engineered antibody molecules can be prepared according to already established techniques used in the field of antibody engineering to prepare. Techniques for preparing antibodies, particularly bispecific antibodies, are described, for example, in Winter & Milstein, (1991) Nature 349: 293-299; ) Crit. Rev. Immunol. 12: 125-168; Holliger, P .; & Winter, G.G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter et al. (1995) J. MoI. Hematother. 4, 463-470; A. & Hawkins, R .; E. (1995) Trends Biotechn. 13, 294-300; Hoogenboom, H .; R. (1997) Nature Biotechnol. 15, 125-126, Fearon, D.M. (1997) Nature Biotechnol. 15, 618-619; & Pack, P.A. (1997) Immunotechnology 3,83-105; Carter, P. et al. & Merchant, A.M. M.M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Hollinger, P .; & Winter, G .; (1997) Cancer Immunol. Immunother. 45, 128-130, and references cited therein.

  The present invention contemplates the selection of variable regions for two different antigens or epitopes, and subsequent variable region combinations. Libraries and selection procedures known in the art are employed as techniques employed for variable region selection. A natural library that uses rearranged V genes harvested from human B cells (Marks et al., (1991) J. Mol. Biol., 222: 581; Vaughan et al., (1996) Nature Biotech., 14: 309) is Are well known to those skilled in the art. Synthetic libraries (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381; Barbas et al., (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al., (1994) EMBO J. et al. , 13: 692; Griffiths et al. (1994) EMBO], 13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97) are usually used by cloning immunoglobulin V genes using PCR. Prepared. Errors in the PCR process can lead to a high degree of randomization. VH and / or VL libraries can be selected individually (in this case, single region binding is selected directly) or together for the target antigen or epitope.

  A preferred method for producing a bispecific ligand according to the present invention selects a variable region repertoire for binding to a first antigen or epitope and a variable region repertoire for binding to a second antigen or epitope. Including using a selection system. The selected first and second variable regions are then combined and a bispecific ligand is selected for binding to both the first and second antigens or epitopes. The closed structural ligand is selected for binding both the first and second antigens or epitopes individually rather than simultaneously.

A. Library Vector Systems Various selection systems suitable for use in the present invention are known in the art. Examples of such systems are described below.

Any of the bacteriophage lambda expression systems can be directly screened as bacteriophage plaques or colonies of lysogens as previously described (Muse et al. (1989) 20 Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad.Sci.U.S.A., 87; Mullinax et al., (1990) Proc.Natl.Acad.Sci.U.S.A. Sci.U.S.A., 88: 2432), used in the present invention. Although the expression system can be used to screen up to 10 ⁶ different members of the library, they are in fact not suitable for screening a larger number of members (over 10 ⁶ members). .

  Of particular use in the construction of a library is a selection display system that allows a nucleic acid to be bound to a polypeptide it expresses. As used herein, a selection display system is a system that allows selection of individual members of a library by a suitable display means by binding generic and / or target ligands.

  Selection protocols for isolating desired members of large libraries, represented by phage display technology, are known in the art. A variety of peptide sequences are displayed on the surface of filamentous bacteriophages (Scott and Smith (1990) Science, 249: 386). The system uses antibody fragments for in vitro selection and amplification of specific antibody fragments that bind target antigens ( And nucleotide sequences encoding them) proved useful (McCafferty et al., WO 92/01047). Nucleotide sequences encoding the VH and VL regions are linked to gene fragments that encode leader signals that direct them to the periplasmic space of E. coli. As a result, the resulting antibody fragments are displayed on the surface of bacteriophage, typically as a fusion to a bacteriophage coat protein (eg, pIII or pVIII).

  Alternatively, the antibody fragment is externally displayed on a lambda phage capsid (phage body). The advantage of phage-based display systems is that because they are biological systems, selected library members can be amplified by simply growing phage containing the selected library members in bacterial cells. In addition, since the nucleotide sequence encoding the polypeptide library member is contained in a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation are relatively simple.

  Methods for constructing bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al. (1990) Nature, 348, incorporated herein by reference). Kang et al., (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et al., (1991) Nature, 352: 624; Lowman et al., (1991) Biochemistry, 30: 10832. Burton et al., 20 (1991) Proc. Natl. Acad. Sci USA, 88: 10134; Hoogenboom et al., (1991) Nucleic Acids Res., 19: 4133; 1) J. Immunol., 147: 3610; Breitling et al., (1991) Gene, 104: 147; Marks et al., (1991) supra; Barbas et al., (1992) supra; Hawkins and Winter (1992) J. Immunol. Marks et al., 1992, J. Biol. Chem., 267: 16007; Lerner et al., (1992) Science, 258: 1313).

  One particularly advantageous approach is to use scFv phage libraries (Huston et al., 1988, Proc. Natl. Acad. Sci USA, 85: 5879-5883; Chaudhary et al., (1990). Proc. Natl. Acad. Sci USA, 87: 1066-1070; McCafferty et al., (1990) supra; Clackson et al., (1991) Nature, 352: 624; Marks et al., (1991) J. Mol. Biol., 222: 581; Chiswell et al., 30 (1992) Trends Biotech., 10:80; Marks et al. (1992) J. Biol. Chem., 267). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. For example, improved versions of the phage display techniques described in WO 96/06213 and WO 92/01047 (Medical Research Council) and WO 97/08320 (Morphosys), which are incorporated herein by reference, are also known.

  Another system for generating a library of polypeptides involves the use of a cell-free enzyme mechanism for in vitro synthesis of library members. In one method, RNA is selected by alternating selection for target ligands and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). Similar techniques can be used to identify DNA sequences that bind a given human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635). WO92 / 05258 and WO92 / 14843). Similarly, polypeptides can be synthesized using in vitro translation as a method for generating large libraries. In general, these methods involving stabilized polysome complexes are further described in WO88 / 08453, WO90 / 05785, WO90 / 07003, WO91 / 02076, WO91 / 05058 and WO92 / 02536. An alternative non-phage based display system disclosed in WO95 / 22625 and WO95 / 11922 (Affymax) uses polysomes to display polypeptides for selection.

  Yet another category of techniques involves the selection of a repertoire in an artificial compartment that allows a gene to bind to its gene product. For example, selection systems that can select nucleic acids encoding the desired gene product in microcapsules formed with water-in-oil emulsions are described in WO99 / 02671, WO00 / 40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16 ( 7), 652-6. Genetic elements encoding gene products having the desired activity are partitioned into microcapsules and then transcribed and / or translated to produce each gene product (RNA or protein) within the microcapsules. Subsequently, genetic elements that produce gene products having the desired activity are classified. This technique selects the gene product of interest by detecting the desired activity by various means.

B. Library Configuration A library for selection purposes may be constructed using techniques known in the art, for example as described above, or may be purchased from commercial sources. Libraries useful for the present invention are described, for example, in WO 99/20749. Once a vector system has been selected and one or more nucleic acid sequences encoding the polypeptide of interest have been cloned into a library vector, it is subject to diversity within the cloned molecule by undergoing mutagenesis prior to expression. Can be generated. Alternatively, the encoded protein can be expressed and selected as described above before mutagenesis and further selection is performed. Mutagenesis of nucleic acid sequences encoding structurally optimized polypeptides is performed by standard molecular methods. Of particular use is the polymerase chain reaction or PCR (Mullis and Faloona (1987) Methods E'nzymol., 155: 335, incorporated herein by reference). PCR that amplifies a target sequence of interest using multiple cycles of DNA replication catalyzed by a thermostable DNA-dependent DNA polymerase is well known in the art. For the construction of various antibody libraries, see Winter et al. (1994) Ann. Rev. Immunology 12, 433-55, and references cited therein.

  PCR is performed using template DNA (at least 1 fg, more practically 1-1000 ng) and at least 25 pmol of oligonucleotide primers. Because each sequence is represented by a small portion of the primer pool molecule and is limited in volume in later amplification cycles, it is advantageous to use a larger amount of primer if the primer pool is very heterogeneous. sell. A typical reaction mixture is 2 μl DNA, 25 pmol oligonucleotide primer, 2.5 μl 10 × PCR buffer 1 (Perkin-Elmer (Foster City, Calif.)), 0.4 μl 1.25 μM dNTP,. 15 μl (or 2.5 units) Taq DNA polymerase (Perkin Elmer (Foster City, Calif.)) And an amount of deionized water to a total volume of 25 μl. The mineral oil is overlaid and PCR is performed using a programmed thermal cycler. The length and temperature of each step of the PCR cycle, and the number of cycles are adjusted according to the actual stringency requirements. Annealing temperature and timing are both determined by the efficiency of annealing the primer to the template and the degree of mismatch allowed. Obviously, if the nucleic acid molecule is amplified and mutagenized simultaneously, inconsistencies are required in at least the first round of synthesis. The ability to optimize the stringency of primer annealing conditions is well within the knowledge of those skilled in the art. An annealing temperature between 30 ° C. and 72 ° C. is used. Initial denaturation of the template molecule usually occurs over 4 minutes at a temperature between 92 ° C and 99 ° C, followed by denaturation (94-99 ° C, 15 seconds to 1 minute), annealing (temperature determined as described above). 1 to 2 minutes) and extension (72 ° C., 1 to 5 minutes depending on the length of the amplification product) followed by 20 to 40 cycles. The final extension is generally performed at 72 ° C. for 4 minutes, followed by an indefinite (0-24 hour) step at 4 ° C.

C. Single Variable Region Combinations Once regions useful in the present invention are selected, they can be combined by a variety of methods known in the art, including covalent and non-covalent methods.

  A preferred method involves the use of the described polypeptide linker in combination with, for example, a scFv molecule (Bird et al. (1988) Science 242: 423-426). Bird et al., Science 242, 423-426; Hudson et al., Journal Immunol. Methods 231 (1999) 177-189; Hudson et al., Proc Nat Acad Sci USA 85, 5879 5883 discusses suitable linkers. The linker is preferably flexible and allows two single regions to interact. An example of one linker is a (Gly4 ser) n linker (n = 1-8, eg 2, 3, 4, 5 or 7). Linkers used in diabodies that are less flexible may be employed (Holliger et al. (1993) PNAS (USA) 90: 6444-6448).

  In one embodiment, the employed linker is not an immunoglobulin hinge region.

Various regions can be combined using methods other than linkers. For example, the use of disulfide bridges provided through natural or engineered cysteine residues is utilized to stabilize V _H -V _H , V _L -V _L or V _H -V _L dimers (Reiter et al., ( 1994) Protein Eng. 7: 697-704), or remodeling of the interface between variable regions can be used to improve the “compatibility” and stability of the overall action (Ridgeway et al., (1996) Protein Eng. 7: 617-621; Zhu et al. (1997) Protein Science 6: 781-788).

  Other techniques for conjugating or stabilizing immunoglobulin variable regions, particularly antibody VH regions, can be employed as appropriate.

  According to the present invention, the bispecific ligand can be in a “closed” structure in the dissolved state. A “closed” structure is a structure in which two regions (eg, VH and VL) exist in an associated form, such as the form of an associated VL pair that forms the antibody binding site. For example, the scFv may be a closed structure depending on the arrangement of the linker used to join the VH and VL regions. If this is flexible enough to allow the regions to meet or if they are held securely in the meeting position, they will likely employ a closed structure.

  Similarly, VH region pairs and VL region pairs can exist in a closed structure. In general, this becomes a function of a closed structure of the region by a rigid linker or the like in the ligand molecule. A closed ligand cannot bind both a molecule that increases the half-life of the ligand and a second target molecule. Thus, the ligand will typically bind only the second target molecule upon dissociation from the molecule that increases the half-life of the ligand.

  Furthermore, VH / VH, VL / VL or VH / VL dimer configurations without linkers provide competition between regions.

  The ligand according to the present invention may further be an open structure. In this structure, the ligand can simultaneously bind both a molecule that increases the half-life of the ligand and a second target molecule. Typically, the variable regions in an open structure are retained so far that they do not interact to form an antibody binding region and do not compete for binding to their respective epitopes (in the case of VH VL pairs). ). In the case of VH / VH or VI / VL dimers, these regions are not joined by a rigid linker. Originally, the region pair does not compete for antigen binding and does not form an antibody binding site.

  Fab fragments and whole antibodies exist primarily in a closed structure, whereas open and closed bispecific ligands presumably exist in various equilibrium states under different atmospheres. Binding of the ligand to the target probably shifts the equilibrium balance to an open structure. Thus, a particular ligand according to the present invention can exist in two structures in solution, one of which (open form) can independently bind two antigens or epitopes, while the other structure (Closed form) can bind only one antigen or epitope. Thus, antigens or epitopes compete for binding to the ligand in this structure.

  Thus, while the open form of the bispecific ligand can exist in equilibrium with the closed form in the dissolved state, it is assumed that equilibrium is advantageous for the closed form. Furthermore, the open form can be blocked by target binding to become a closed structure. Preferably, therefore, certain bispecific ligands of the present invention exist in equilibrium between the two structures (open structure and closed structure).

Bispecific ligands according to the invention can be modified to favor open and closed structures. For example, stabilizing the V _H -V _L interaction on disulfide bonds stabilizes the closed structure. Furthermore, the linker used to join the regions comprising the VH region and _VL region pairs can be configured to favor the open form. For example, linkers can sterically inhibit region association by incorporating large amino acid residues in the opposite direction, or by designing suitable rigid constructs that physically separate the regions.

D. Characterization of the bispecific ligand The binding of the bispecific ligand to its specific antigen or epitope can be tested by methods well known to those skilled in the art and including ELISA. In a preferred embodiment of the invention, binding is tested using a monoclonal phage ELISA.

  Phage ELISA can be performed according to any suitable procedure. The Eri-temporal protocol is described below.

  The population of phage generated in each selection can be screened for binding by ELISA to selected antigens or epitopes to identify “polyclonal” phage antibodies. Phages from single infected bacterial colonies from these populations can then be screened by ELISA to identify “monoclonal” phage antibodies. It is also desirable to screen soluble antibody fragments for binding to an antigen or epitope, which can also be performed by ELISA, eg, using reagents for the C or N-terminal tag (eg, Winter et al., ( 1994) Ann. Rev. Immunology 12, 433-55 and references cited therein).

  The diversity of selected phage monoclonal antibodies was examined by gel 5 electrophoresis of PCR products (Marks et al., 1991, supra; Nissim et al., 1994, supra), exploration (Tomlinson et al., 1992). Nol. Biol. 227, 776) or vector DNA sequences.

E. Structure of “dual specific ligand” As noted above, an antibody herein comprises at least one heavy and light chain variable region, at least two heavy chain variable regions, or at least two light chain variable regions. Defined as an antibody (eg, IgG, IgM, IgA, IgA, IgE) or fragment (Fab, Fv, disulfide bond Fv, scFv, diabody) (the term antibody also includes dAbs). Antibodies can be derived at least in part from any species that naturally produces antibodies, or can be made by recombinant DNA technology (isolated from serum, B cells, hybridomas, transfectomas, yeasts or bacteria). )

In a preferred embodiment of the invention, the bispecific ligand comprises at least one 20 single heavy chain variable region of an antibody and one single light chain variable region of an antibody, or two single heavy or light chain variable regions. Including. For example, the ligand can comprise a VH / VL pair, a pair of VH regions, or a pair of _VL regions.

  The first and second variable regions of the ligand may be present on the same polypeptide chain. Alternatively, they may be present on individual polypeptide chains. When present on the same polypeptide chain, they can be joined by a linker, preferably a peptide sequence, as described above.

  The first and second variable regions can associate covalently or non-covalently. If they are covalently associated, the covalent bond may be a disulfide bond.

  If the variable regions are selected from a V gene repertoire selected using, for example, the phage display techniques described herein, these variable regions are specific universal ligands described herein. The generic framework area is included so that it can be recognized by The use of generic frameworks and generic ligands is described in WO 99/20749.

  When a V gene repertoire is used, the polypeptide sequence variation is preferably located within the structural loop of the variable region. The polypeptide sequence of any variable region can be altered by DNA shuffling or mutation to enhance the interaction of each variable region with its complementary pair. DNA shuffling is known in the art and taught in Stemmer, 1994, Nature 370: 389-391 and US Pat. No. 6,297,053, both of which are incorporated herein by reference. Yes.

  In a preferred embodiment of the invention, the “dual specific ligand” is a single chain Fv fragment. In an alternative embodiment of the invention, the “bispecific ligand” consists of the Fab format.

  In a further aspect, the present invention provides a nucleic acid encoding at least one “dual-specific ligand” as defined herein.

  One skilled in the art will appreciate that, depending on aspects of the invention, both antigen and epitope can bind simultaneously to the same antibody molecule. Alternatively, they can compete for the same antibody molecule. For example, if both epitopes are bound simultaneously, both variable regions of the bispecific ligand can bind their target epitopes independently. If the regions compete, one variable region can bind its target, but not at the same time as another variable region binds its cognate target. Alternatively, the first variable region can bind its target, but not at the same time as the second variable region binds its cognate target.

  The variable region can be derived from an antibody directed against the target antigen or epitope. Alternatively, it can be derived from a repertoire of single antibody regions, such as those expressed on the surface of filamentous bacteriophages. The following can be selected.

  In general, the nucleic acid molecules and vector constructs required to practice the present invention are described in standard laboratory manuals such as Sambrook et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor, USA. Can be constructed and operated as is.

  The manipulation of nucleic acids useful in the present invention is typically performed with recombinant vectors.

  Accordingly, in a further aspect, the present invention provides a vector comprising a nucleic acid encoding at least one “dual-specific ligand” as defined herein.

  As used herein, vector means an individual element used to introduce heterologous DNA into cells for its expression and / or replication. Methods of selecting or constructing the vector and then using it are well known to those skilled in the art. Many vectors are publicly available, including bacterial plasmids, bacteriophages, artificial chromosomes and episomal vectors. The vector can be used for simple cloning and gene induction. Alternatively, a gene expression vector is employed. The vectors used in accordance with the present invention can be selected to correspond to a polypeptide coding sequence of the desired size, typically from 0.25 kilobases (kb) to 40 kb or more. Suitable host cells are transformed with the vector after an in vitro cloning process. Each vector generally contains various functional components including a cloning (or “polylinker”) site, an origin of replication and at least one selectable marker gene. When the predetermined vector is an expression vector, the vector is an enhancer element, promoter, transcription termination and signal sequence located in the vicinity of the cloning site, respectively, so as to be functionally linked to the gene encoding the ligand according to the present invention. Have one of the following.

  In general, both cloning and expression vectors contain a nucleic acid sequence that enables the vector to be expressed in one or more selected host cells. Typically, in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.

  The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (eg SV40, adenovirus) are useful for cloning vectors in mammalian cells. is there. In general, an origin of replication is not required for these vectors unless the mammalian expression vector is used in mammalian cells capable of replicating high levels of DNA, such as COS cells.

  Advantageously, the cloning or expression vector may contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in selective media. Thus, host cells that are not transformed with the vector containing the selection gene will not survive in the medium. Typical selection genes are antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, which provide resistance to supplemental nutritional deficiencies, important nutrients that cannot compensate for nutritional deficiencies or cannot be obtained in growth media. Encodes a protein that supplies Since replication of the vector encoding the ligand according to the present invention is most conveniently performed in E. coli, an E. coli selectable marker is used, such as the β-lactamase gene that confers resistance to the antibiotic ampicillin. These can be obtained from E. coli plasmids such as pBR322 or pUC plasmids such as pUC18 or pUC19.

  Expression vectors usually contain a promoter that is recognized by the host adult and is operably linked to the coding sequence of interest. The promoter can be inducible or structural. The term “operably linked” means a proximal sequence in which the described components are in a relationship that allows them to function as intended. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

  Suitable promoters for use with prokaryotic host organisms include, for example, hybrid promoters such as p-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and tac promoters. Promoters used in bacterial systems also generally include a Shine-Dalgarno sequence that is operably linked to the coding sequence. Preferred vectors are expression vectors that allow for expression of nucleotide sequences corresponding to polypeptide library members. Thus, selection by the first and / or second antigen or epitope can be performed by individual growth and expression of a single clone expressing a polypeptide library member, or by use of any selection display system. As mentioned above, the preferred selection display system is bacteriophage display. Thus, phage or phagemid vectors such as pIT1 or pIT2 can be used. Leader sequences useful in the present invention include pelB, stII, ompA, phoA, bla and pelA. An example is a phagemid vector having a replicating E. coli origin (for double-stranded replication) and a replicating phage origin (for production of single-stranded DNA). The processing and expression of the vectors are well known in the art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the vector consists of the β-lactamase gene, the lac promoter upstream of the expression cassette consisting of the pelB leader sequence (which directs the expressed polypeptide to the periplasmic space) (N to C terminus), (library member A multiple cloning site (to clone the nucleotide version of), optionally one or more peptide tags (for detection), optionally one or more TAG stop codons, and the phage protein pIII. Therefore, using various suppressor and non-suppressor strains of E. coli and adding helper phages such as glucose, isopropylthio-β-D-galactoside (IPTG), or VCS M13, the vector replicates as a plasmid without expression. It will be possible to generate only large quantities of polypeptide library members, or phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.

  Conventional binding techniques are employed in the construction of the vector encoding the ligand according to the present invention. The isolated vector or DNA fragment is cleaved, adjusted and recombined in the form desired to produce the required vector. If desired, analysis to confirm that the correct sequence is present in the constructed vector can be performed by known methods. Suitable methods for constructing expression vectors, preparing transcripts in vitro, introducing DNA into host cells, and performing analyzes to assess expression and function are known to those skilled in the art. Detect the presence of a gene sequence in a sample and amplify and / or express its Southern or Northern analysis, Western blot, DNA, RNA or protein dot blot, in situ hybridization, immunocytochemistry, or nucleic acid or Quantification is by conventional methods such as sequence analysis of protein molecules. Those skilled in the art will readily recognize how these methods can be modified as desired.

Closed Structure Multispecific Ligand Structure According to an aspect of the second configuration of the invention, two or more non-complementary epitope binding regions are combined to form a closed structure as defined herein. Advantageously, instead of or in addition to the linkers described herein, those regions are further linked to a backbone that can facilitate the formation and / or maintenance of the closed structure of the epitope binding site. Can be made.

(I) Skeleton The skeleton may be based on immunoglobulin molecules or non-immunoglobulins in the above-mentioned origins. A preferred “immunoglobulin backbone” as defined herein comprises at least (i) an immunoglobulin molecule comprising the CL (kappa or lambda subclass) region of an antibody or (ii) the CH1 region of an antibody heavy chain; From any of the immunoglobulin molecules comprising the CH1 and CH2 regions; immunoglobulin molecules comprising the CH1, CH2 and CH3 regions of the antibody heavy chain; Including any of the selected skeletons. A hinge region can also be included. Such a combination of regions may be similar to, for example, a natural antibody such as IgG or IgM, or a fragment thereof such as an Fv, scFv, Fab or F (ab ′) 2 molecule. Those skilled in the art will recognize that this list is not intended to be exhaustive.

(II) Protein framework Each epitope binding region comprises a protein framework and one or more CDRs involved in the specific interaction of that region with one or more epitopes. Advantageously, the epitope binding region according to the invention comprises three CDRs. Preferred protein scaffolds include immunoglobulin domain based scaffolds, fibronectin based scaffolds, affibody based scaffolds, CTLA4 based scaffolds, chaperone based scaffolds such as GroEL, lipocalin based scaffolds, and bacterial Fc receptors SpA and SpD. Any of the skeletons selected from the group consisting of skeletons based on: One skilled in the art will appreciate that this list is not intended to be exhaustive.

F: The selection of the backbone backbone structure used to construct the bispecific ligands The immunoglobulin superfamily all share a similar overlap for their polypeptide chains. For example, antibodies are highly diversified in terms of their temporal sequences, but by comparison of sequences and crystal structures, unexpectedly, five of the six antigen-binding loops of antibodies (H1, H2 , L1, L2, L3) have been shown to adopt a limited number of backbone structures, or standard structures (Chothia and Lesk, (1987), J. Mol. Biol., 196: 901; Chothia et al. (1989), Nature, 342: 877). Thus, analysis of loop length and major residues allowed prediction of the backbone structures of H1, H2, L1, L2 and L3 found in the majority of human antibodies (Chothia et al. (1992), J. Biol. Mol. Biol., 227: 799; Tomlinson et al. (1995), EMBO J., 14:20 4628; Williams et al. (1996), J. Mol. Biol., 264: 220). The H3 region is much more diverse in terms of sequence, length and structure (due to the use of the D segment), but the length and presence of particular residues or residues at key positions in the loop and antibody framework. It also forms a limited number of backbone structures for type-dependent short loop lengths (Martin et al. (1996), J; Mol. Biol. 263: 800; Shirai et al. (1996), FEDS Letters, 399. : 1).

The dual specific ligands of the invention are advantageously assembled from a library of regions such as a library of VH regions and / or a library of _VL regions. Furthermore, the bispecific ligands of the invention can themselves be provided in the form of a library. In one aspect of the invention, a library of bispecific ligands and / or regions is designed in which specific loop lengths and major residues are selected so that the backbone structure of the members is known. Advantageously, these are the true structures of naturally occurring immunoglobulin superfamily molecules that minimize the probability of being non-functional, as described above. Germline V gene segments serve as one suitable basic framework for constructing antibody or T cell receptor libraries. Other sequences are also used. Changes can occur infrequently so that a small number of functional members can have an altered backbone structure that does not affect their function.

  Standard structure theory can also be used to evaluate the number of different backbone structures encoded by a ligand, to predict backbone structures based on the ligand sequence, and to select residues for diversification that does not affect the standard structure . In the human VK region, the L1 loop can adopt one of four standard structures, the L2 loop has a single standard structure, and 90% of the human VK region has 4 relative to the L3 loop. Alternatively, it is known to adopt a standard structure of 5 (Tomlinson et al. (1995), supra). Thus, in a single VK region, different standard structures can be combined to create a series of different backbone structures. Suppose that the Vα region encodes a different set of standard structures for the L1, L2 and L3 loops, and that the VK and Vα regions can be paired with any VH region that can code several standard structures for the H1 and H2 loops. The number of standard structure combinations observed for these five loops is quite large. This implies that the generation of diversity in the backbone structure can be essential for the generation of a wide range of binding specificities. However, by constructing an antibody library based on a single known backbone structure, contrary to prediction, it generates enough diversity to target virtually all antigens. It was found that diversity in the main chain structure is not required. Even more surprising is that the single backbone structure need not be a common structure. A single natural structure can be used as the basis for the entire library. Thus, in a preferred embodiment, the bispecific ligand of the present invention has a single known backbone structure.

  The single backbone structure chosen is preferably generalized among the immunoglobulin superfamily type molecules in question. The structure is generalized when it is recognized that a significant number of natural molecules adopt it. Thus, in a preferred embodiment of the present invention, the natural occurrence of different backbone structures for each binding loop of an immunoglobulin region is individually considered and then the natural variable regions having the desired combination of backbone structures for the different loops are selected. . If no match is found, select the closest match. The desired combination of backbone structures for the different loops is preferably made by selecting germline gene segments that encode the desired backbone structure. More preferably, the selected germline gene segments are naturally expressed at a high frequency, and most preferably they are expressed in all natural germline gene segments.

  In designing a bispecific ligand or library thereof, the incidence of different backbone structures for each of the six antigen binding loops can be examined individually. For H1, H2, L1, L2 and L3, a predetermined structure adopted for 20% to 100% of the antigen binding loop of the natural molecule is selected.

  Typically, the observed incidence is 35% or higher (ie 35% to 100%), ideally 50% or higher, or even 65% or higher. Since the majority of H3 loops do not have a standard structure, it is preferable to select a backbone structure common to those loops exhibiting the standard structure. Thus, for each of those loops, the structure most frequently observed in the natural repertoire is selected. In human antibodies, the most conventional standard structure (CS) for each loop is H1-CS1 (79% of the expressed repertoire), H2-CS3 (46%), VK L1-CS2 (39%), L2- CS1 (100%), VK L3-CS1 (36%) (calculation assumes αk: λ ratio is 70:30. Hood et al. (1967) Cold Spring Harbor Symp. Quant. Biol, 48: 133). For the H3 loop having a standard structure, the salt bridge is a CDR3 length of 7 residues from residue 94 to residue 101 (Kabat et al. (1991), Sequence of Immunological Interests, USA (Ministry of Health and Welfare) seems to be the most common. Protein data that can be used as a basis for antibody modeling, with at least 16 human antibody sequences present in the EMBL data library containing the necessary H3 length and major residues that form the structure. Present in the bank (2cgr and 1tet). The most frequently expressed germline gene segments of this standard structure combination are VH segment 2-23 (DP-47), JH segment JH4b, VK segment 02/012 (DPK9) and JK segment JK1. VH segments DP45 and DP38 are also suitable. It is therefore possible to use these segments together as a basis for forming a library with the desired single backbone structure.

  Alternatively, for each of the binding loops, instead of individually selecting a single main chain structure based on the natural occurrence of different main chain structures, a combination of main chain structures as a basis for selecting a single main chain structure The natural occurrence rate is used. In the case of antibodies, for example, it is possible to determine the natural occurrence rate of standard structure combinations for any two, three, four, five or all six antibodies in the antibody binding loop. Here, the selected structure is preferably common to natural antibodies, and most preferably observed most frequently in the natural repertoire. Thus, in human antibodies, for example, when considering the natural combination of five antigen binding loops H1, H2, L1, L2 and L3, the most frequent combination of standard structures is sought, and then a single main chain As the basis for selecting the structure, it is combined with the most popular structure for the H3 loop.

ii. Diversification of standard sequences Selecting several selected backbone structures, or preferably a single known backbone structure, to generate a repertoire with structural and / or functional diversity, It is possible to construct a bispecific ligand according to the present invention or a library used in the present invention by changing the binding site of the molecule. This means that the variants are generated with sufficient diversity in their structure and / or their function to provide a range of activities.

  The desired diversity is typically generated by changing selected molecules at one or more positions. The position to be changed can be selected at random or is preferably selected. Then, in the meantime, the normal amino acid is replaced with any natural or synthetic amino acid or analog thereof, and a large number of variants are generated, or the normal amino acid is replaced with one of the amino acids or The change can be achieved by substituting with multiple defined subsets to generate a more limited number of variants.

Various methods for introducing such diversity have been reported. Error-prone PCR (Hawkins et al., (1992), J. Mol. Biol., 226: 889), chemical mutagenesis (Deng et al., (1994), J. Biol. Chem., 269: 9533) or bacterial mutagenesis A gene strain (Low et al. (1996) J. Mol;. Biol., 260: 359) can be used to introduce random mutations into the gene encoding the molecule. Methods for mutating selected positions are also well known in the art and include using maladaptive or degenerate oligonucleotides with or without PCR. For example, several synthetic antibody libraries have been created by directing mutations to the antigen binding loop. A series of new binding specificities were created by randomizing the H3 region of human tetanus toxoid binding Fab (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457). Random or semi-random H3 and L3 regions were added to germline V gene segments to generate large libraries with unmutated framework regions (Hoogenboom & Winter, (1992), J: Mol. Biol. 227: 381; Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994), EMBO J., 13: 692; Griffiths et al. (1994), EMBO J. 13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97). The diversification has been extended to include some or all of the other antigen binding loops (Crameri et al., (1996), Nature Med., 52: 100; Riechmann et al., (1995), Bio / Technology, 13 : 475; Morphosys, WO 97/08320, supra). Because loop randomization has the potential to create more than about 10 ¹⁵ structures for H3 only and create a large number of variants for the other 5 loops as well, using current transformation techniques Or, using a cell-free system, it is not feasible to generate a library that represents all possible combinations. For example, one of the largest libraries constructed to date has produced only 6 × 10 ¹⁰ different antibodies, only a small percentage of the potential diversity for a library of this design. Yes (Griffiths et al. (1994), supra).

  In preferred embodiments, only those residues that are directly involved in creating or remodeling the desired function of the molecule are diversified. For many molecules, the function is to bind the target, thus targeting diversity while avoiding inclusive filling of the molecule or residue changes that are important for maintaining the selected backbone structure It is necessary to concentrate on the binding site.

Standard sequence diversification when applied to antibody regions In the case of antibody bispecific ligands, binding to the target is most often the antigen binding site. Thus, in a highly preferred embodiment, the present invention provides a library of antibody bispecific ligands or libraries for their assembly in which only residues at the antigenic sites are changed. These residues are extremely diverse in the human antibody repertoire and are known to make contacts in high resolution antibody / antigen complexes. For example, at L2, positions 50 and 53 are diverse in the natural antibody and are observed to contact the antibody. In contrast, the conventional approach diversifies all residues in the corresponding complementary determining region (CDR1) as defined by Kabat et al. (1991, supra) The group is compared to two residues diversified in the library for use according to the present invention. This represents a significant improvement in terms of the functional diversity required to generate a range of antigen binding specificities.

  In essence, antibody diversity is the somatic recombination of germline V, D and J gene segments (so-called germline and junctional diversity) to create a naive primary repertoire and the resulting rearranged V gene. Is the result of two processes with somatic hypermutation. Analysis of human antibody sequences shows that diversity in the primary repertoire is concentrated in the middle of the antigen binding site, whereas somatic hypermutation occurs in the region surrounding the antigen binding site that is highly conserved in the primary repertoire. Diversity is widespread (see Tomlinson et al. (1996) J. Mol. Biol. 256: 813). It is probably developed in an complementary manner as an efficient way to examine sequence space and is clearly specific to antibodies, but can be easily applied to other polypeptide repertoires. is there. The residues that change are a subset of the residues that form the binding site for the target. Different subsets of residues (including overlapping ones) at the target binding site are diversified at different stages upon selection, as desired.

  In the case of an antibody repertoire, an initial “naive” repertoire is created when some but not all of the residues in the antigen binding site are diversified. As used herein in this context, the term “naive” means an antibody molecule that does not have a given target. These molecules are encoded by the immunoglobulin genes of individuals who have not undergone immune diversification, as in the case of fetal and neonatal individuals whose immune system has not yet undergone extensive antigenic stimulation. . This repertoire is then selected against a series of antigens or epitopes. Then, if necessary, further diversity can be introduced outside the region diversified in the initial repertoire. This mature repertoire can be selected for remodeled function, specificity or affinity.

  The present invention provides two different naive repertoires of binding regions for the construction of bispecific ligands, or naive libraries of bispecific ligands in which some or all of the residues in the antigen binding site vary. . A “primary” library is diverse in germline V gene segments (germline diversity) or diversified during the recombination process (junctional diversity). Similar to the limited natural primary repertoire. Those diversified residues include H50, H52, H52a, H53, H55, H56, HS8, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. , But not limited to them. In “somatic” libraries, diversity is limited to residues that are diversified during recombination (junctional diversity) or highly somatically mutated. Those residues that are diversified include, but are not limited to, H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96. All residues listed as suitable for diversification in these libraries are known to contact at one or more antibody-antigen complexes. In both libraries, not all of the residues in the antigen binding site will change, so if you want to do so, changing the remaining residues introduces more diversity during selection . One skilled in the art will appreciate that any subset of any of these residues (or additional residues including the antigen binding site) can be used for the initial and / or later diversification of the antigen binding site.

  In the construction of the library used herein, diversification of the selected position typically allows several possible amino acids (all 20 amino acids or subsets thereof) to be introduced at that position, This is accomplished at the nucleic acid level by altering the coding sequence that specifies the sequence of the polypeptide. Using IUPAC nomenclature, the most universal codons are the NNK codon that encodes all amino acids, as well as the TAG stop codon. NNK codons are preferably used to introduce the required diversity. Other codons are also used that achieve the same purpose, including NNN codons that result in the generation of additional stop codons TGA and TAA.

The characteristic of side chain diversity at the antigen binding site of human antibodies is a clear bias that favors certain amino acid residues. VH, when the total amino acid composition of the most diverse positions of 10 in each of the V _k and Vλ regions 76 percent of side-chain diversity is only seven different residues, i.e. serine (24%), tyrosine ( 14%), asparagine (11%), glycine (9%), alanine (7%), aspartate (6%) and threonine (6%). This bias towards hydrophilic and small residues that can provide backbone flexibility probably reflects the development of a surface that is likely to bind a wide range of antigens or epitopes, and antibody promiscuity in the primary repertoire Help explain what is needed.

  Since it is preferable to resemble this amino acid distribution, the amino acid distribution at the altered position is preferably similar to that found in the antigen binding site of the antibody. Such bias in amino acid substitutions that allows for the selection of specific polypeptides (not just antibody polypeptides) against a range of target antigens is easily applied to any polypeptide repertoire. There are various methods for biasing the amino acid distribution at the position to be changed (including using trinucleotide mutagenesis (see WO 97/08320)), among which the preferred method for ease of synthesis is the conventional method. Is to use degenerate codons. Calculate the most typical codon by comparing the amino acid profile encoded by all combinations of degenerate codons (with equal proportions of single, double, triple and quadruple degeneracy at each position) with the use of natural amino acids Is possible. Codons (AGT) (AGC) T, (AGT) (AGC) C and (AGT) (AGC) (CT), respectively, using IUPAC nomenclature, ie DVT, DVC and DVY are the codons closest to the desired amino acid profile. is there. They encode 22% serine, 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and cysteine. Preferably, therefore, the library is constructed with DVT, DVC or 30 DVY codons at each of the diversified positions.

G: Antigen capable of increasing ligand half-life The bispecific ligand according to the invention binds in one configuration to one or more molecules capable of increasing the half-life of the ligand in vivo. Is possible. Typically, the molecule is a polypeptide that occurs naturally in vivo and resists its removal by endogenous mechanisms that degrade and remove unwanted substances from the organism. For example, a molecule that increases the half-life of an organism can be selected from the following molecules:

  Proteins from the extracellular matrix such as collagen, laminin, integrins and fibronectin. Collagen is the main protein of the extracellular matrix. About 15 types of collagen molecules are currently known, such as type I collagen (accounting for 90% of body collagen) found in bones, skin, tendons, ligaments, corneas and internal organs, or cartilage, invertebral disc, notochord, And the type II collagen found in the vitreous of the eye.

  Plasma proteins such as fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobulin, protein, ubiquitin, uterine globulin and β-2 microglobulin, and plasminogen, lysozyme, cystatin C, Proteins found in blood, including enzymes and inhibitors such as α-1-antitrypsin and pancreatic kypsin inhibitors. Plasminogen is an inactive precursor of the trypsin-like serine protease plasmin. It is usually seen circulating in the bloodstream. When plasminogen is activated and converted to plasmin, it opens a strong enzyme domain that dissolves fibrinogen fibers that engulf blood cells into the clot. This is called fibrinolysis.

  Immune system proteins such as IgE, IgG and IgM.

  Transport proteins such as retinol binding protein and α-1 microglobulin.

  Defensins such as β-defensin 1, neutrophil defensins 1, 2 and 3.

  Proteins found in blood brain barriers or nerve tissues such as melanocortin receptor, myelin and ascorbate transporter.

  Transferrin receptor specific ligand neuropharmaceutical fusion protein (see US5977307), brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-like growth factor 1 (IGF1) receptor, insulin-like growth factor 2 (IGF2) Receptors and insulin receptors.

  Proteins localized to the kidney such as polycystin, type IV collagen, organic anion transporter K1 and Hayman antigen.

  For example, alcohol dehydrogenase, G250 and other proteins localized in the liver.

Blood coagulation factor X
α-1 Antitrypsin HNF1α
Proteins localized in the lung, such as secretory components (binding IgA).

  For example, a protein localized in the heart such as HSP27. This is associated with dilated cardiomyopathy.

  For example, a protein localized in the skin such as keratin.

  A bone-specific protein such as bone morphogenic protein (BMP), which is a subset of the transforming growth factor β superfamily that exhibits osteogenic activity.

  Examples include BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-1)) and -8 (OP-2).

  Tumor specific proteins including human trophoblast antigens, herceptin receptors, estrogen receptors, and cathepsins such as cathepsin B (found in liver and spleen).

  LAG-3 (lymphocyte activating gene); bone protegerin ligand (OPGL) (see Nature 402, 304-309, 1999); OX40 (activated T cells and human T cell leukemia virus type I (HTLV-I)) A member of the TNF receptor family expressed on the only costimulatory T cell molecule known to be specifically upregulated in the producer cell—see J. Immunol. 2000 Jul 1; 16561): 263-70. Metalloproteases including CG6512 Drosophila, human parapreggin, human FtsH, human AFG3L2 and mouse ftsH (associated with arthritis / cancer); and acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF- 2) Vascular endothelial growth factor / vascular permeability factor VEGF / VPF), transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), Only expressed on activated T cells, including angiogenic growth factors including plasma-derived endothelial growth factor (PD-ECGF), placental growth factor (PIGF), midkine platelet-derived growth factor-BB (PDGF) and fractalkine Disease-specific proteins such as antigens.

Stress protein (heat shock protein).
HSP is usually found in cells. If they are found extracellularly, it is an indicator that the cells have died and have released their contents. This non-programmed cell death (necrosis) occurs only as a result of trauma, disease or injury, and thus in vivo, when extracellular HSP elicits a response from the immune system that resists infection and disease. Bispecific binding to extracellular HSP can be localized to the disease site.

Proteins involved in Fc transport Bramber receptor (also known as FcRB)
This Fc receptor has two functions, both of which are potentially useful for delivery. Their function is to (1) transport IgG from mother to offspring through the placenta, and (2) extend its serum half-life of IgG by protecting it from degradation. The receptor is thought to regenerate IgG from endosomes. See Holliger et al., Nat Biotechnol, 1997, Jul; 15 (7): 632-6.

  Ligands according to the present invention can be specifically designed for the above targets without requiring or increasing the increase in half-life in vivo. For example, ligands according to the present invention can be bispecific ligands, or increase half-life by being tissue specific, but bind tissue-specific therapeutic-related targets regardless of increased half-life. It may be specific for a target selected from the aforementioned targets that allow tissue specific targeting of the dAb monomer. Furthermore, if the ligand or dAb monomer is directed to the kidney or liver, it can transfer the ligand or dAb monomer to an alternative clearance pathway in vivo (eg, ligand to liver Can be transferred from clearance to kidney clearance).

Other approaches to increase in vivo half-life In addition to designing bispecific ligands corresponding to target proteins that increase the serum half-life of antibody polypeptide constructs, chemical components that increase serum half-life Binding to can further stabilize the antibody polypeptides described herein. In order to improve the pharmacokinetics of antibody molecules, the present invention provides single region variable region polypeptides attached to polymers that increase stability and increase half-life. Binding of polymer molecules (eg polyethylene glycol; PEG) to proteins has been well established and has been shown to modulate the pharmacokinetic properties of modified proteins. For example, PEG modification of proteins has been shown to alter in vivo circulation half-life, antigenicity, solubility, and resistance to protein degradation (Abuchowski et al., J. Biol. Chem. 1977, 252: 3578; Nucci et al., Adv. Drug Delivery Reviews, 1991, 6: 133; Francis et al. Stabilization ", 1991, pp 235-263 (P, NY) enum whereabouts)).

  Both site-specific and random PEGylation of protein molecules are known in the art (see, for example, Zalipsky and Lee, “Poly (ethyleneglycol) Chemistry: Biotechnical and Biomedical Applications”, 1992, pp 347-370). Plenum, NY; Goodson and Katre, 1990, Bio / Technology, 8: 343; Hershfield et al., 1991, PNAS, 88: 7185). More specifically, random PEGylation of antibody molecules at lysine residues and thiolated derivatives has been described (Ling and Mattiasson, 1983, Immunol. Methods, 59: 327; Wilkinson et al., 1987, Immunol. Letters, 15:17; Kitamura et al., 1991, Cancer Res., 51: 4310; Delgado et al., 1996, Br. J. Cancer, 73: 175; Pedley et al., 1994, Br. J. Cancer, 70: 1126).

  The method of PEGylation is described below. Examples of antibody polypeptides, and in particular PEGylation of dAbs, are co-pending applications PCT / GB2004 filed June 30, 2004, designated in the United States, each of which is incorporated herein by reference in its entirety. / 002829, and US Provisional Application No. 60 / 535,076, filed Jan. 8, 2004.

又はさらに５０ｐＭ程度の親和性（解離定数、Ｋ_ｄ＝Ｋ_ｏｆｆ／Ｋ_ｏｎ）を有する。 Affinity / Activity Measurements Isolated single domain antibody (eg, dAb) polypeptides described herein have at least 300 nM or less, preferably at least 300 nM to 50 pM, 200 nM to 50 pM, more preferably at least

Alternatively, it has an affinity (dissociation constant, K _d = K _off / K _on ) of about 50 pM.

The antigen binding affinity of variable region polypeptides can be conveniently measured by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, NY). In this method, antigen is bound to a BIAcore chip at a known concentration and a variable region polypeptide is introduced. Specific binding between the variable region polypeptide and the immobilized antigen results in an increase in protein concentration relative to the chip substrate and a change in the SPR signal. Changes in the SPR signal are recorded as resonance units (RU) and are displayed versus time along the Y axis of the sensorgram. The reference signal is collected with only the solvent that passes through the chip (eg, PBS). The net difference between the reference signal and the signal after completion of the variable region polypeptide injection represents the binding value for a given sample. BIAcore kinetic evaluation software (eg, version 2.1) is used to determine the off rate (K _off ), on rate (K _on ), and dissociation rate (K _d ) constants.

  High affinity depends on the complementarity of the surface of the antigen and the CDR of the antibody or antibody fragment. Complementarity is measured by the type and strength of possible molecular interactions between the target moiety and the CDR, such as potential ionic interactions, van der Waals attraction, hydrogen bonding, or other interactions that can occur. CDR3 tends to contribute more to antigen-binding interactions than CDR1 and 2, probably because of its generally larger size, and provides more opportunities for favorable surface interactions (eg, Padlan et al., 1994). , Mol. Immunol. 31: 169-217; Chothia & Lesk, 1987, J. Mol. Biol. 196: 904-917; Chothia et al., 1985, J. Mol. Biol. 186: 651-663). High affinity indicates a single immunoglobulin variable region / antigen pair with a high degree of complementarity and is directly related to the structure of the variable region and target.

  Using molecular modeling software that allows docking of antigen and polypeptide structure, it is possible to highlight structures that confer high affinity of a single immunoglobulin variable region polypeptide for a given antigen. Generally, a computer model of the structure of a single immunoglobulin variable region of known affinity is docked with a computer model of a polypeptide of known structure or other target antigen to measure the interaction surface It is possible. Given the structure of the interaction surface for the known interaction, predict the positive or negative effect of conservative or less conservative substitutions in variable region sequences on the strength of the interaction Can allow for rational design of improved binding molecules.

Multimeric forms of antibody single variable regions In one aspect, the antibody polypeptide constructs (eg, dAbs) described herein can be, for example, hetero- or homo-dimers, hetero- or homo-trimers, hetero- or homo-tetras. It is multimerized as a mer or higher dimensional hetero or homo multimer (eg, from hetero or homo pentamer to octamer). Multimerization can increase the strength of antigen binding through a binding activity effect in which the strength of binding is related to the sum of the binding affinities of multiple binding sites.

Hetero or homomultimers are prepared, for example, through the expression of a single region antibody fused through a peptide linker that results in a dAb-linker-dAb configuration, or a higher order multiple structure of that configuration. Multimers can be linked to additional components such as polypeptide sequences that increase serum half-life, or other effector components such as toxins or target components such as PEG. Hetero or homomultimers can be generated using any linker peptide sequence, eg, linker sequences used in the art to generate scFv. One generally useful linker is peptide sequence (Gly ₄ Ser) _n where n is from 1 to about 10 (eg n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) Including repetition of (SEQ ID NO: 7). For example, the linker can be (Gly ₄ Ser) ₃ (SEQ ID NO: 8), (Gly ₄ Ser) ₅ (SEQ ID NO: 9), (Gly ₄ Ser) ₇ (SEQ ID NO: 10), or another diverse (Gly ₄ Ser Ser) (SEQ ID NO: 7) sequence.

An alternative to the expression of multimers as monomers linked by peptide sequences is to link monomeric immunoglobulin variable regions through, for example, disulfide bonds or other post-translational chemical bonds. For example, a free cysteine is made, for example, at the C-terminus of a monomeric polypeptide, allowing for disulfide bonds between monomers. In this embodiment, or in other embodiments requiring a free cysteine, the cysteine is a dAb sequence (in C-terminal cysteine, the sequence in the primer is introduced into the downstream PCR primer, so in fact reverse complement, ie ACA or GCA The PCR primer is introduced by including a two-cysteine codon (TGT, TGC) immediately adjacent to the codon and immediately preceding one or more stop codons. If desired, a linker peptide sequence such as (Gly ₄ Ser) _n (SEQ ID NO: 7) is placed between the dAb sequence and the free cysteine. Expression of a monomer having a free cysteine residue results in an approximately 1: 1 mixture of monomeric and dimeric forms. The dimer is separated from the monomer using gel chromatography, for example ion exchange chromatography with salt gradient elution.

  Alternatively, using the free cysteines made, the monomers can be trimerized maleimide molecules (eg tris [2-maleimidoethyl] amine, TMEA) or dimaleimide PEG molecules (eg available from Nektar (Shearwater)). To a multivalent chemical linker such as

In one embodiment, homodimers or heterodimers of the invention include V _H or V _L region is a double bond to an immunoglobulin C _{H 1} region or C _K region respectively at the C-terminus amino acids. Accordingly, hetero-or homodimer, the antigen-binding region may be a Fab-like molecule comprising V _H and / or V _L regions associated covalently attached to the C _H1 and C _K domain respectively at the C-terminus . In addition or alternatively, dAb multimers of the invention can be modeled with camelid species that express the majority of fully functional, highly specific antibodies without light chain sequences. Camel heavy chain antibodies are found as single heavy chain homodimers that dimerize through their constant regions. The variable regions of these camel heavy chain antibodies, referred to as V _H H regions, retain the ability to bind antigens with high specificity when isolated as fragments of the V _H chain (Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahrodi et al., 1997, FEBS Lett. 414: 521-526). Thus, antibody single variable region multimers of the present invention having camel species heavy chain antibody V _H H structures can be constructed using methods known in the art and described above.

PEGylation of antibody polypeptides The present invention relates to PEGylated antibody polypeptides that increase half-life and decrease resistance to degradation without a decrease in activity (eg, binding affinity) compared to non-PEGylated antibody polypeptides (Eg dAb) monomers and multimers are provided.

が挙げられるが、それらに限定されない。
反応基（例えばＭＡＬ、ＮＨＳ、ＳＰＡ、ＶＳ又はチオール）はＰＥＧポリマーに直接結合されていてもよいし、リンカー分子を介してＰＥＧに結合されていてもよい。 Using methods known in the art, the antibody polypeptide molecules described herein can be made into polymer molecules (preferably PEG, preferably) to achieve increased half-life and improved degradation resistance properties. ). The polymer components that can be used in the present invention are synthetic or natural components, branched or unbranched, such as linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymers, or homo- or heteropolysaccharides. Polysaccharides can be included, but are not limited to them. Preferred examples of synthetic polymers that can be used in the present invention include linear or branched poly (ethylene glycol) (PEG), poly (propylene glycol), or poly (vinyl alcohol) and derivatives or substituted forms thereof. It is done. Particularly preferred substituted polymers for conjugation to the antibody polypeptides described herein include substituted PEGs including methoxy (polyethylene glycol). Natural polymer components that can be used in addition to or in place of PEG include lactose, amylose, dextran or glycogen, and derivatives thereof as would be recognized by one skilled in the art. Derivative forms of the polymer molecules include, for example, derivatives in which additional components or reactive groups are present that allow interaction with amino acid residues of the antibody polypeptides described herein. Such derivatives include N-hydroxyl succinimide (NHS) active esters, succinimidyl propionate polymers, maleimides, vinyl sulfones and sulfhydryl selective reagents such as thiols. Particularly preferred derivatized _{polymers, PEG-O-CH 2 CH} 2 CH 2 -CO 2 -NHS, PEG-O-CH 2 -NHS, PEG-O-CH 2 CH 2 -CO 2 -NHS, PEG-S _{-CH 2 CH 2 -CO-NHS,} PEG-O 2 CNH-CH (R) -CO 2 -NHS, PEG-NHCO-CH 2 CH 2 -CO-NHS and _{PEG-O-CH 2 -CO 2} -NHS (Wherein R is (CH ₂ ) ₄ ) NHCO ₂ (mPEG)), including but not limited to PEG polymers. PEG polymers can be linear molecules or can be branched molecules where multiple PEG moieties are present in a single polymer. Some particularly preferred PEG derivatives useful in the present invention include:

But are not limited to these.
The reactive group (for example, MAL, NHS, SPA, VS or thiol) may be directly bonded to the PEG polymer, or may be bonded to PEG via a linker molecule.

  The size of the polymers useful in the present invention can range from 500 Da to 60 kDa, for example 1000 Da to 60 kDa, 10 kDa to 60 kDa, 20 kDa to 60 kDa, 30 kDa to 60 kDa, 40 kDa to 60 kDa, and 50 kDa to 60 kDa. The polymer used in the present invention, particularly PEG, may be a linear polymer or may have a branched structure. Depending on the combination of molecular weight and structure, the polymer molecule will produce a molecule having an average hydrodynamic size of 24 to 500 kDa when bound to an antibody construct (eg, dAb) monomer or multimer. As used herein, the hydrodynamic size of a polymer molecule refers to the apparent size of a molecule (eg, a protein molecule) based on the diffusion of the molecule in an aqueous solution. The diffusion or movement of the protein in solution can be processed to derive an apparent size of the protein whose size is given by the Stokes radius or hydrodynamic radius of the protein particle. The “hydrodynamic size” of a protein depends on both mass and shape (structure) so that two proteins with the same molecular weight can have different hydrodynamic sizes based on the overall structure of the protein. Hydrodynamic sizes of PEG-conjugated antibody single variable regions (including single region antibody multimers described herein) are 24 kDa to 500 kDa, 30 to 500 kDa, 40 to 500 kDa, 50 to 500 kDa, 100 to 500 kDa. 150 to 500 kDa, 200 to 500 kDa, 250 to 500 kDa, 300 to 500 kDa, 350 to 500 kDa, 400 to 500 kDa, 450 to 500 kDa. Preferably, the hydrodynamic size of the PEGylated dAb is 30 to 40 kDa, 70 to 80 kDa, or 200 to 300 kDa. Thus, the size of polymer molecules attached to antibody polypeptides such as dAbs or dAb multimers can be varied depending on the desired application. For example, if the PEGylated dAb is intended to exit the blood and enter the surrounding tissue, it may be desirable to keep the size of the binding polymer small to facilitate overflow from the bloodstream. Alternatively, higher molecular weight polymers (eg, 30-60 kDa polymers) can be used if it is desirable for the PEGylated dAb to remain in the blood for a longer period of time.

  Methods that are well known in the art can be used to attach polymer (PEG) molecules useful in the present invention to antibody polypeptide constructs. The first step in the conjugation of PEG or other polymer components to the antibody polypeptide monomer or multimer of the present invention is the replacement of the hydroxyl end groups of the PEG polymer with spherical electron-containing groups. In particular, PEG polymers are attached to cysteine or lysine residues present in antibody polypeptide monomers or multimers. Cysteine and lysine residues are natural or can be reshaped into antibody polypeptide molecules. For example, a cysteine residue can be recombined at the C-terminus of a dAb polypeptide, or a residue at a particular solvent accessible position in a dAb or other antibody polypeptide can be replaced with cysteine or lysine. In preferred embodiments, the PEG moiety is linked to a cysteine residue present in the hinge region at the C-terminus of the dAb monomer or multimer described herein.

In one embodiment, the PEG polymer is attached to one or more cysteine or lysine residues present in the framework region (FW) and one or more heterologous CDRs of the dAb. The CDR and framework regions are regions of the immunoglobulin variable region defined in the Kabat database of protein sequences related to immunology (Kabat et al., (1991), Sequences of Proteins of Immunological Institute, US Department of Health and Human Services). In a preferred embodiment, PEG polymers are attached to the cystine or lysine residue in the _{V H} framework segment DP47, or _{V k} framework segment DPK9. The cysteine and / or lysine residues of DP47 that can be attached to PEG include the cysteine at position 22 or 96 and the lysine at position 43, 65, 76 or 98 of SEQ ID NO: 1 (Figure 21). The cysteine and / or lysine residues of DPK9 that can be attached to the PEG according to the present invention are the cysteine residues at position 23 or 88 and lysine at positions 39, 42, 45, 103 or 107 of SEQ ID NO: 2 (FIG. 22). Contains residues. In addition, specific cysteine or lysine residues can be attached to the PEG in the _VH standard framework region DP38 or DP45.

Ｖ_ｋダミーの一次アミノ酸配列（配列番号６）

加えて、Ｖ_ｋダミーｄＡｂ（上記参照）の溶解結晶構造を用いて、残基Ｖａｌ−１５、Ｐｒｏ−４０、Ｇｌｙ−４１、Ｓｅｒ−５６、Ｇｌｙ−５７、Ｓｅｒ−６０、Ｐｒｏ−８０、Ｇｌｙ−７１、Ｇｌｎ−１００、Ｌｙｓ−１０７及びＡｒｇ−１０８が溶媒接触可能なものとして識別され、ＰＥＧポリマーの結合のためのシステイン又はリジン残基への変異の魅力的な候補となった。一実施形態において、ＰＥＧポリマーは、多溶媒接触可能システイン又はリジン残基、或いはシステイン又はリジン残基に変異された溶媒接触可能残基に結合される。或いは、特定の抗体ポリペプチド構築物が１つの溶媒接触可能システイン又はリジン（或いはシステイン又はリジンに修飾された残基）を有するにすぎない場合、或いは特定の溶媒接触可能残基が、ＰＥＧ化のためのいくつかの当該残基から選択される場合には、１つの溶媒接触可能残基のみがＰＥＧに結合される。 In addition, certain solvent accessible sites in dAb molecules that are not natural cysteine or lysine residues can be mutated to cysteine or lysine for attachment of PEG polymers. Solvent accessible residues in any given dAb monomer or multimer can be determined using methods known in the art such as analysis of the crystal structure of a given dAb. For example, using the dissolved crystal structure of V _H dAb HEL4 (binding chicken lysozyme (see below)) residues Gln-12, Pro-41, Asp-62, Glu-89, Gln-112, Leu-115 , Thr-117, Ser-119 and Ser-120 have been identified as solvent accessible and have become attractive candidates for mutation to cysteine or lysine residues for attachment of PEG polymers.
HEL4 primary amino acid sequence (SEQ ID NO: 5)

Primary amino acid sequence of V _k dummy (SEQ ID NO: 6)

In addition, using the dissolved crystal structure of the V _k dummy dAb (see above), residues Val-15, Pro-40, Gly-41, Ser-56, Gly-57, Ser-60, Pro-80, Gly -71, Gln-100, Lys-107 and Arg-108 were identified as solvent accessible and became attractive candidates for mutations to cysteine or lysine residues for attachment of PEG polymers. In one embodiment, the PEG polymer is attached to a multi-solvent accessible cysteine or lysine residue, or a solvent accessible residue mutated to a cysteine or lysine residue. Alternatively, if a particular antibody polypeptide construct has only one solvent accessible cysteine or lysine (or a residue modified to cysteine or lysine), or a particular solvent accessible residue is When selected from several such residues, only one solvent accessible residue is attached to the PEG.

  Several coupling schemes useful for the present invention are provided by Nektar (San Carlos, Calif.). For example, if attachment to lysine residues of PEG or other polymers is desired, an active ester of a PEG polymer derivatized with N-hydroxysuccinimide such as succinimidyl propionate can be used. If conjugation to cysteine residues is intended, PEG polymers derivatized with sulfhydryl selective reagents such as maleimide, vinyl sulfone or thiol can be used. Examples of specific embodiments of PEG derivatives that can be used in accordance with the present invention to produce PEGylated dAbs can be found in the Nektar catalog (available at nektar.com on the World Wide Web). In addition, several derivatized forms of PEG can be used according to the present invention to facilitate attachment of PEG polymers to the dAb monomers or multimers of the present invention. PEG derivatives useful in the present invention include PEG-succinimidyl succinate, urethane-linked PEG, PEG phenyl carbonate, PEG succinimidyl carbonate, PEG-carboxymethyl azide, dimethylmaleic anhydride PEG, PEG dithiocarbonate derivatives, PEG-tresylate (2,2,2-trifluoroethane sulfonate), mPEGimide ester, and Zalipsky and Lee, (1992) ("Use of functionalized poly (ethylene glycol) s for modification of peptides", "poly (h) Biotechnical and Biomedical Applications , J.Milton Harris eds, Plenum Press, although other materials can be mentioned as described in NY), but are not limited to.

  In each of the above embodiments, the PEG polymer can be coupled to any applicable residue present in the antibody polypeptide construct peptide, or preferably one or more residues of the construct are cysteine or lysine. The residue can be modified or mutated and then used as the point of attachment for the PEG polymer. Preferably, the residues so modified are solvent accessible residues, ie residues that are accessible to the aqueous environment and to the derivatized PEG polymer when the antibody polypeptide construct is natively superimposed. It is. When any of these residues are mutated to cysteine residues according to the present invention, they are available for PEG conjugation using linear or branched MAL derivatized PEG (MAL-PEG).

  In one embodiment, an antibody construct is provided comprising an antibody single variable region and a PEG polymer, wherein the ratio of PEG polymer to antibody single variable region is a molar ratio of at least 0.25: 1. In a further embodiment, the molar ratio of PEG polymer to antibody single variable region is 0.33: 1 or greater. In a further embodiment, the molar ratio of PEG polymer to antibody single variable region is 0.5: 1 or greater.

H: Use of the ligands described herein 1) Use of multispecific ligands according to the second configuration of the present invention Multispecific ligands according to the second configuration of the present invention for in vivo therapeutic and prophylactic applications, in vitro and It can be employed for in vivo diagnostic applications, in vitro tests and reagent applications. For example, antibody molecules can be used in antibody-based testing techniques, such as μELISA technology, according to methods known to those skilled in the art.

  As mentioned above, multispecific ligands according to the present invention are used in diagnostic, prophylactic and therapeutic procedures. Multispecific antibodies according to the present invention are used diagnostically for Western analysis and in situ protein detection by standard immunohistochemical procedures. When used in these applications, the ligand can be tagged according to techniques known in the art. In addition, the antibody polypeptide can be used preliminarily in affinity chromatography procedures when synthesized on a chromatographic support such as a resin. All such techniques are well known to those skilled in the art.

  The diagnostic use of a closed structure multispecific ligand according to the present invention is the ability of a closed structure multispecific ligand to bind two targets competitively (closed structure), or two targets so that the two targets cannot bind simultaneously. Homologous tests for analysis utilizing the ability to simultaneously bind (open structure).

  A true homologous immunity test format has been eagerly sought by manufacturers of diagnostic tests and research test systems used in drug invention and development. Major diagnostic markets include human testing in hospitals, clinics and clinics, commercial related laboratories, blood banks, households, non-human diagnostics (eg food testing, water testing, environmental testing, bioprotection and veterinary testing ), And finally research (drug development, basic research and academic research).

  Currently, all these markets utilize immunoassay systems built around chemiluminescence, ELISA, fluorescence, or rarely radioimmunoassay techniques. Each of these test formats requires a separation step (step of separating the binding reagent from unbound reagent). In some cases, several separation steps are required. As these additional steps are added, reagents and automated operations are added, taking time and affecting the ultimate outcome of the test. In human diagnosis, the separation process can be automated, which hides the problem but does not eliminate it. The robot system, additional reagents, additional incubation time, etc. add significant cost and complexity. In drug development, such as high-throughput screening, where virtually millions of samples are tested at once with very low levels of test molecules, the ability to screen can be lost by adding additional separation steps. However, avoiding separation causes excessive noise in reading. Therefore, there is a need for a true homologous format that provides the range of sensitivity available from current test formats. Advantageously, the test has a fully quantitative reading with high sensitivity and large dynamic range. Sensitivity is an important requirement that reduces the amount of sample required. Both of these characteristics are characteristics of the homologous system. This is very important in terms of control testing and in drug development where the sample is expensive. The heterologous systems currently available in the art require large amounts of samples and expensive reagents.

  Applications for homologous tests include cancer tests where the largest test is a test for prostate specific antigen used to screen men for prostate cancer. Other uses include fertilization tests that provide a series of tests on women who attempt to include β-hogs for pregnancy. Testing for infectious diseases including hepatitis, HIV, rubella, and other viruses and microorganisms and sexually transmitted diseases. Tests, particularly those for HIV, A, B, C, non-A, non-B hepatitis, are used for blood banks. Therapeutic drug monitoring trials include monitoring prescription drug levels in patients, eg, digoxin for arrhythmias, phenobarbital levels in psychosis, theophylline for asthma to avoid effects and toxicity. Diagnostic tests are further useful for drug addiction tests, such as tests for cocaine and marijuana. Metabolic tests are used to measure thyroid function, anemia and other physiological diseases and functions.

  Homologous immunoassay formats are also useful for the production of standard clinical chemistry tests. Inclusion of immunological and chemical tests on the same instrument is extremely advantageous for diagnostic tests. Suitable chemical tests include tests for glucose, cholesterol, potassium and the like.

  A further major application for homologous immunity tests is drug invention and development. High-throughput screening involves testing combinatorial chemical libraries versus ultra-large targets. A signal is detected and then the positive group is grouped into smaller groups and ultimately tested in cells and then animals. Homologous tests can be used for all these types of tests. Immunoassays are often used in drug development, especially in animal and clinical trials. Homologous testing significantly accelerates and simplifies these procedures. Other uses include food and beverage testing (testing meat and other foods for E. coli and Salmonella, etc.), water testing including testing in aquatic plants against all types of contaminants including E. coli, and veterinary testing including.

  In a broad embodiment, the present invention relates to a binding test comprising a detectable agent that is bound to a closed structure multispecific ligand according to the present invention and whose detectable property is altered by binding of an analyte to said closed structure multispecific ligand. I will provide a. The test can be configured in a number of different ways, each utilizing the above properties of a closed structure multispecific ligand.

  The test relies on direct or indirect displacement of the drug by the analyte that results in a change in the drug's detection characteristics. For example, if the agent is an enzyme that is capable of catalyzing a reaction with a detectable endpoint, the enzyme is bound to a ligand and its active site is blocked, etc. Can be inactivated. An analyte that is also bound to a closed-structure multispecific ligand displaces the enzyme and activates it through liberalization of the active site. The enzyme then reacts with the substrate to trigger a detectable event. In an alternative embodiment, the ligand can bind an enzyme outside the active site to affect the structure of the enzyme and alter its activity. For example, the structure of the active site can be limited by ligand binding, or the binding of cofactors required for activity can be avoided.

  The physical implementation of the test can take any form known in the art. For example, a closed structure multispecific ligand / enzyme complex can be provided on the test strip, and the substrate is provided in a different region of the test zone and in a solution containing an analyte capable of migrating through the ligand / enzyme complex; The enzyme can be displaced and carried to the substrate region to generate a signal. Alternatively, the ligand / enzyme complex can be provided on a test rod or other solid phase, immersed in the analyte / substrate solution, and the enzyme released into the solution depending on the presence of the analyte.

  Since each molecule of the analyte potentially releases one enzyme molecule, the analyte is quantitative and the intensity of the signal generated within a given time depends on the concentration of the analyte in solution.

  Further configurations using a closed structure specimen are possible. For example, an enzyme can be activated by configuring a closed structure multispecific ligand to bind the enzyme at an allosteric site. In such embodiments, the enzyme is active in the absence of the analyte. By adding a sample, the enzyme is displaced, allosteric activation is removed, and the enzyme is inactivated.

  In the context of the above embodiment where enzyme activity is employed as a measure of analyte concentration, enzyme activation or inactivation means an increase or decrease in enzyme activity measured as the ability of the enzyme to catalyze a signal generation reaction. . For example, the enzyme catalyzes the conversion of an undetectable substrate to its detectable form. For example, horseradish peroxidase is widely used in the art with chromogenic or chemiluminescent substrates that are commercially available. The level of increase or decrease in the activity of the enzyme may be between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In the case of an increase in activity, the increase cannot exceed 100%, i.e. 200%, 300% or 500% or more, or in percentage if the reference activity of the inhibitory enzyme is not detectable.

  In a further configuration, the closed structure multispecific ligand can bind an enzyme / substrate pair rather than an enzyme. Thus, the substrate is not available for the enzyme until it is released from the closed structure multispecific ligand through analyte binding. The embodiment for this configuration is an embodiment for a configuration that binds enzymes.

  Furthermore, the test can be configured to bind fluorescent molecules such as fluorescein or other fluorophores in structures where the fluorescence disappears upon binding to the ligand. In this case, when the analyte binds to the ligand, the fluorescent molecule is displaced and a signal is generated. Alternatives to fluorescent molecules useful in the present invention include chromogenic agents, including luminescent agents such as luciferin / luciferase, and agents widely used in immunoassays such as HRP.

  The therapeutic and prophylactic use of multispecific ligands prepared according to the present invention includes administration of the ligands according to the present invention to a recipient mammal such as a human. Multispecificity can allow antibodies to bind to multimeric antigens with strong binding activity. Multispecific ligands allow cross-linking of two antigens when employing, for example, cytotoxic T cells that mediate the death of tumor cell lines.

  Substantially naïve ligands or binding proteins thereof, such as dAb monomers, having at least 90 to 95% homology are preferred for administration to mammals, with 98 to 99% or more homology especially when the mammal is human. And most preferred for pharmaceutical use. Once purified to partial or desired homology, the ligand can be used for diagnosis or therapy (including in vitro) or in the development and implementation of test procedures and immunofluorescent staining (Lefkovite and Pernis, (1979 and 1981), Immunological Methods, Volumes I and II, Academic Press (New York State).

  The ligands or binding proteins thereof, such as dAb monomers of the present invention typically contain inflammatory conditions, allergic hypersensitivity, cancer, bacterial or viral infections, and autoimmune diseases (type I diabetes, asthma, multiple sclerosis). , Rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis), will be used for prevention, suppression or treatment.

  In addition to rheumatoid arthritis, the anti-TNF-α polypeptides described herein are effective against Addison's disease (adrenal), ear autoimmune disease (ear), eye autoimmune disease (eye), autoimmune hepatitis. (Liver), autoimmune parotitis (parotid gland), Crohn's disease and inflammatory bowel disease (gut), type I diabetes (pancreas), epididymis (epididymis), glomerulonephritis (kidney) ), Graves' disease (thyroid), Guillain-Barre syndrome (neuronal cells), Hashimoto's disease (thyroid), hemolytic anemia (red blood cells), systemic lupus erythematosus (multiple tissue), male infertility (semen), multiple occurrences Multiple sclerosis (nerve cells), myasthenia gravis (neuromuscular junction), pemphigus (primarily skin), psoriasis (skin), rheumatic fever (heart and junction), sarcoid (multitissue and organ), strong Dermatosis (skin and connective tissue), Sjogren's syndrome (exocrine gland and other tissues), Self, including but not limited to spondyloarthropathies (axial skeleton and other tissues), thyroiditis (thyroid), ulcerative colitis (intestine) and vasculitis (blood vessels) (internally affected organs) Applicable to the treatment of immune diseases.

  In addition to rheumatoid arthritis and other chronic inflammatory diseases (such as Crohn's disease and psoriasis), the anti-VEGF polypeptides described herein have diabetes, acute myeloid leukemia, leukemia, and macular degeneration and diabetic retina. It can be used to treat eye diseases, including diseases.

  In this application, the term “prevention” includes the administration of a protective composition prior to induction of the disease. “Suppression” means administration of the composition after an inductive event but before the clinical manifestation of the disease.

  Animal model systems are available that can be used to investigate the effect of antibodies or their binding proteins in protecting against or treating disease. Methods for testing systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight et al. (1978), J. Exp. Med., 147: 1653; Reinersten et al., ( 1978), New Eng. J. Med., 299: 515). Myasthenia gravis (MG) in SJL / J female mice has been tested by inducing disease with soluble AchR proteins from other species (Lindstrom et al., (1988) Adv. Immurzol., 42: 233). Arthritis has been induced in susceptible strains of mice by 30 injections of type II collagen (Stuart et al. (1984), Ann. Rev. Immunol, 42: 233). A model has been described that induces adjuvant arthritis in rats susceptible to injection of mycobacterial heat shock proteins (Van Eden et al. (1988), Nature 331: 171). As described, thyroiditis is induced in mice by the administration of thyroglobulin (Maron et al. (1980), J. Exp. Med., 152: 1115). Insulin-dependent diabetes mellitus (IDDM) occurs naturally or can be induced in specific strains of mice, such as those described in Kanasawa et al. (1984), Diabetologia, 27: 113. EAE in mice and rats functions as a model for MS in humans. In this model, demyelinating disease is induced by administration of myelin basic protein (Patterson, (1986), Textbook of Immunopathology, edited by Mischer et al., Grune and Stratton (New York), pp. 179-213; McFarlin et al .; (1973) Science 179: 478; and Satoh et al. (1987) J. Immunol 138: 179).

  In general, the ligand is utilized in purified form with a pharmacologically suitable carrier. Typically these carriers include aqueous or alcohol / water solutions, emulsions or suspensions, both of which contain a salt and / or buffer medium. Parenteral vehicles include sodium chloride solution, Ringer's glucose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants for maintaining the polypeptide complex in suspension as needed may be selected from concentrating agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginate.

  Intravenous media include fluid and nutrient replenishers, and electrolyte replenishers such as Ringer dextrose-based fluids. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents and inert gases may be present (Mack, (1982) Remington's Pharmaceutical Sciences, 16th edition).

  It can be used as a composition to administer the ligands of the present invention individually or in combination with other drugs. These can include various immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin and immunotoxins. The pharmaceutical composition may be a ligand according to the invention, or even a ligand according to the invention having different specificities, such as ligands selected using different target antigens or epitopes, whether or not accumulated prior to administration. Various cocktails or other “cocktails” of other agents can be included.

  The route of administration of the pharmaceutical composition according to the present invention may be any of those well known to those skilled in the art. For treatments, including but not limited to immunotherapy, selected ligands of the invention can be administered to any patient according to standard techniques. Administration can be by any suitable manner, including parenteral administration, intravenous administration, intramuscular administration, intraperitoneal administration, transdermal administration, administration via the pulmonary route, or also by direct infusion through an appropriate catheter. is there. The dosage and frequency of administration will depend on the patient's age, sex and condition, concurrent administration of other drugs, adverse effects, and other parameters considered by the clinician.

  As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. In certain embodiments, the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Single antibody constructs are well suited for formulation as sustained release formulations due in part to their small size-the number of moles per dose is much greater than for example full size antibody administration . Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Inclusion of injectable compositions can be prolonged by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Many of the methods for preparing such dosage forms are patented or widely known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, J. Org. R. See Robinson, edited by Marcel Dekker, Inc (New York), 1978. Additional methods applicable to the sustained or extended release of polypeptides, such as the single immunoglobulin variable region polypeptides disclosed herein, are described in, for example, the United States, all of which are incorporated herein by reference. Patents 6,306,406 and 6,346,274 and, for example, US patent applications US20020182254 and US20020051808.

  The ligands described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins and lyophilization and reconstitution techniques known in the art can be employed. Lyophilization and reconstitution can result in varying degrees of antibody activity loss (eg, IgM antibodies tend to lose more activity than IgG antibodies in conventional immunoglobulins) and up-regulate usage levels to compensate One skilled in the art will understand that there are things that must be done.

  Compositions containing the present ligand or a cocktail thereof can be administered for prophylactic and / or therapeutic measures. An amount sufficient to achieve at least partial inhibition, suppression, modulation, killing or some other measurable parameter of a selected population of cells in a particular therapeutic application is defined as a “therapeutically effective dose” . The amount necessary to achieve this dose depends on the severity of the disease and the overall condition of the patient's own immune system, but generally ligands such as antibodies, receptors (eg T cell receptors) per kg body weight. ) Or the amount of its binding protein is in the range of 0.005 to 5.0 mg, with 0.05 to 2.0 mg / kg / dose being most widely used. For prophylactic use, a composition containing the ligand or a cocktail thereof can be administered at a similar or slightly lower dose.

  Treatment performed using the compositions described herein may include one or more symptoms compared to the symptoms prior to treatment or an individual (human or human) who has not been treated with the composition. “Effective” if it is reduced (eg at least 10%, or at least 1 point on the clinical assessment scale) relative to symptoms in the model animal. Symptoms obviously vary depending on the target disease or disorder, but can be measured by a normal skilled clinician or technician. The symptom can be determined by, for example, monitoring the level of one or more biochemical indicators of the disease or disorder (eg, the level of an enzyme or metabolite correlated with the disease, the number of affected cells, etc.). By monitoring clinical symptoms (eg inflammation, tumor size, etc.) or by recognized clinical assessment scales, eg, the expanded wound status scale (for multiple sclerosis), the Irvine inflammatory bowel disease questionnaire (32-point assessment Assessing function, systemic symptoms, social function and emotional state-scores range from 32 to 224, higher scores indicate better quality of life), quality of life rheumatoid arthritis scale, or It can be measured by other recognized clinical assessment scales known in the art. A continuous (eg, 1 day or more, preferably longer term) reduction of a disease or disorder symptom of at least 10% or a predetermined scale on a predetermined scale indicates an “effective” treatment. Similarly, prophylaxis performed using a composition described herein is similar to an individual (human or human) whose onset or severity of one or more symptoms has not been treated with the composition. It is “effective” when it is delayed, reduced or eliminated compared to the symptom in the animal model).

  Compositions containing a ligand or a cocktail thereof according to the present invention can be utilized in prophylactic and therapeutic environments to assist in the modification, inactivation, killing or removal of selected target cell populations in mammals. In addition, selective repertoires of the polypeptides described herein can be used selectively in vitro or in vitro to kill, extinguish or effectively remove target cell populations from non-homologous populations of cells. can do. By combining mammalian blood in vitro with ligands such as antibodies, cell surface receptors or binding proteins thereof, unwanted cells can be killed or removed from the blood and returned to the mammal according to standard techniques. .

2: Use of the half-specific bispecific ligand according to the present invention The bispecific ligand according to the method of the present invention can be used for in vivo therapeutic and prophylactic applications, in vivo diagnostic applications, and the like.

  The therapeutic and prophylactic use of bispecific ligands prepared in accordance with the present invention involves administering a ligand according to the present invention to a mammal such as a human. Bispecific antibodies according to the present invention have at least one specificity for a half-life enhancing molecule. One or more additional specificities can be directed to the target molecule. For example, a bispecific IgG can be specific for four epitopes, one of which is on a half-life enhancing molecule. Bispecificity can allow antibodies to bind to multimeric antigens with strong binding activity. Bispecific antibodies can allow, for example, cross-linking of two antigens when employing cytotoxic T cells that mediate the death of tumor cell lines.

  A substantially naïve ligand or binding protein thereof, such as a dAb monomer, having at least 90-95% homology is preferred for administration to mammals, and homology of 98-99% or more is particularly true for mammals that are human. Are most preferred for pharmaceutical use. Once purified to partial or desired homology, the ligand can be used in diagnosis or therapy (including in vitro) or in the development and implementation of test procedures and immunofluorescent staining (Lefkovite and Pernis, (1979 and 1981). ), Immunological Methods, Volumes I and II, Academic Press (New York State).

  The ligands of the invention are typically inflammatory conditions, allergic hypersensitivity, cancer, bacterial or viral infections, and autoimmune diseases (type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, clones Disease, and myasthenia gravis, including but not limited to).

  Animal model systems are available that can be used to examine the effects of bispecific ligands in protecting against or treating disease. Methods for testing systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight et al. (1978), J. Exp. Med., 147: 1653; Reinersten). (1978), New Eng. J. Med., 299: s515). Myasthenia gravis (MG) in SJL / J female mice has been tested by inducing disease with soluble AchR proteins from other species (Lindstrom et al., (1988) Adv. Immunol., 42: 233). Arthritis has been induced in susceptible strains of mice by injection of type II collagen (Stuart et al. (1984), Ann. Rev. Immunol, 42: 233). A model has been described that induces adjuvant arthritis in rats susceptible to injection of mycobacterial heat shock proteins (Van Eden et al. (1988), Nature 331: 171). As described, thyroiditis is induced in mice by the administration of thyroglobulin (Maron et al. (1980), J. Exp. Med., 152: 1115). Insulin-dependent diabetes mellitus (IDDM) occurs naturally or can be induced in specific strains of mice, such as those described in Kanasawa et al. (1984), Diabetologia, 27: 113. EAE in mice and rats functions as a model for MS in humans. In this model, administration of myelin basic protein induces five demyelinating diseases (Patterson, (1986), Textbook of Immunopathology, edited by Mischer et al., Grune and Straton (New York), pp. 179-213; McFarlin; (1973) Science 179: 478; and Satoh et al. (1987) J. Immunol 138: 179).

  The bispecific ligand according to the present invention, and a dAb monomer capable of binding to an extracellular target involved in endocytosis (eg clathrin), allows the bispecific ligand to eat and drink. It allows other specificities that can bind to intracellular targets to be delivered to the intracellular environment. This approach requires a bispecific ligand with physical properties that allow the bispecific ligand to remain functional inside the cell. Alternatively, if the final target intracellular compartment is oxidative, the fully superimposed ligand need not be disulfide free.

  In general, the bispecific ligand is utilized in purified form with a pharmacologically suitable carrier. Typically these carriers include aqueous or alcohol / water solutions, emulsions or suspensions, both of which contain a salt and / or buffer medium. Parenteral vehicles include sodium chloride solution, Ringer's glucose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants for maintaining the polypeptide complex in suspension as needed may be selected from concentrating agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginate. Intravenous media include fluid and nutrient replenishers, and electrolyte replenishers such as Ringer dextrose-based fluids. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents and inert gases may be present (Mack, (1982) Remington's Pharmaceutical Sciences, 16th edition).

  It can be used as a composition to administer the ligands of the present invention individually or in combination with other drugs. These can include various immunotherapeutic agents such as cyclosporine, methotrexate, adriamycin or cisplatin and immunotoxins. Pharmaceutical compositions can include “cocktails” of various cytotoxins or other agents along with the ligands of the present invention.

  The route of administration of the pharmaceutical composition according to the present invention may be any of those well known to those skilled in the art. For treatments including but not limited to immunotherapy, the ligands of the invention can be administered to any patient according to standard techniques. Administration can be by any suitable manner, including parenteral administration, intravenous administration, intramuscular administration, intraperitoneal administration, transdermal administration, administration via the pulmonary route, or also by direct infusion through an appropriate catheter. is there. The dosage and frequency of administration will depend on the patient's age, sex and condition, concurrent administration of other drugs, adverse effects, and other parameters considered by the clinician.

  The ligands of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional immunoglobulins and lyophilization and reconstitution techniques known in the art can be employed. Lyophilization and 30 reconstitution can result in varying degrees of antibody activity loss (eg, in conventional immunoglobulins, IgM antibodies tend to be more active than IgG antibodies), and up-regulate usage levels Those skilled in the art will understand that there may be compensation that may be required.

  Compositions containing the present ligand or a cocktail thereof can be administered for prophylactic and / or therapeutic treatments. An amount sufficient to achieve at least partial inhibition, suppression, modulation, killing or some other measurable parameter of a selected population of cells in a particular therapeutic application is defined as a “therapeutically effective dose” . The amount necessary to achieve this dose depends on the severity of the disease and the overall condition of the patient's own immune system, but generally the amount of ligand per kg body weight ranges from 0.005 to 5.0 mg. There is a 0.05 to 2.0 mg / kg dose most widely used. For prophylactic use, a composition containing the ligand or a cocktail thereof can be administered at a similar or slightly lower dose.

  Compositions containing ligands according to the present invention can be used in prophylactic and therapeutic environments to assist in the modification, inactivation, killing or removal of selected target cell populations in mammals.

  In addition, selected repertoires of the polypeptides described herein can be selectively used externally or in vitro to kill, extinguish or effectively remove target cell populations from non-homologous populations of cells. can do. By combining mammalian blood in vitro with ligands such as antibodies, cell surface receptors or binding proteins thereof, unwanted cells can be killed or removed from the blood and returned to the mammal according to standard techniques. . For purposes of illustration only, the invention will be further described in the following examples. As used herein, for the purposes of dAb nomenclature, TNF-α is referred to as TAR1, and human TNFα receptor 1 (p55 receptor) is referred to as TAR2.

3. Treatment of Rheumatoid Arthritis In a preferred embodiment, the ligands described herein can be used to treat rheumatoid arthritis.

  In one embodiment, the present invention is a method of treating rheumatoid arthritis, comprising using one or more single domain antibody polypeptide constructs, any of which antagonize binding to human TNFα receptor. Including. The invention relates to one or more single domain antibody polypeptide constructs that antagonize binding of human TNFα to a receptor and a single domain antibody in which one specificity of the ligand is directed to TNFα, A composition comprising a bispecific ligand whose specificity is a single domain antibody directed against VEGF or HSA. The present invention further encompasses bispecific ligands in which one specificity of the ligand is directed to VEGF and a second specificity is directed to HSA.

  In one embodiment, the present invention is a method of treating rheumatoid arthritis comprising administering a composition comprising one or more single domain antibody polypeptide constructs, any of which constructs being human Antagonizes TNFα receptor binding and / or prevents increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis and / or moderates TNF-α in L929 cytotoxicity studies Provide a way to reconcile. In particular, a method of treating arthritis comprises administering a composition comprising one or more single domain antibody polypeptide constructs, wherein any of the constructs antagonizes TNFα receptor binding, Administration of the product to Tg197 transgenic mice prevents an increase in the arthritic score.

a) Receptor binding studies Ligands for treating rheumatoid arthritis can interfere with the binding of TNF-α to the TNF-α receptor. The receptor may be an isolated (usually membrane bound) receptor or a receptor present on a cell in vitro or in vivo.

  A test for measuring TNF-α receptor binding and interference with such binding by the ligands described herein is described in Example 6. These include ELISA (Example 6, Section 1.3.1), BIAcore analysis (Example 6, Section 1.3.2), and isolated (or membrane associated) receptors (Example 6, Section 1). .3.3) and biochemical receptor binding studies using both receptors expressed on the surface of cultured cells (Example 6, section 1.3.3).

  As used herein, the term “antagonize binding” of a receptor refers to a given antibody polypeptide construct that interferes with the binding of TNF-α (or VEGF or other factor) to the cognate receptor. Means ability or effect. Antagonism is measured using either the in vitro cell-based test or the in vivo test described herein. Thus, the receptor can be isolated, membrane bound, or present on the cell surface. A construct interferes with binding to a cognate receptor (eg, TNFR1, TNFR2, VEGFR1, VEGFR) when there is a statistically significant decrease in binding detected in the presence of the construct compared to when the construct is absent. Or antagonize its binding. Alternatively, the construct is interfering with binding if the binding measurement in the presence of the construct is at least 10% lower than when the construct is absent.

b) L929 cytotoxicity test Ligands for the treatment of rheumatoid arthritis can interfere with the cytotoxic effects of TNF-α in the L929 cytotoxicity test. This test, based on the test described in Evans et al., 2000, Molecular Biotechnology, 15: 243-248, is described in Example 6, section 1.3.3. Anti-TNF-α ligands useful for the treatment of rheumatoid arthritis can neutralize the activity of TNF-α in this cellular test.

  As used herein, the term “neutralizing” when used with respect to an antibody or dAb polypeptide described herein, is a measurable activity or function of the target antigen. Means to interfere. A polypeptide has a measurable activity or function of a target antigen of at least 50%, preferably at least 60%, 70%, 80%, 90%, 95% or more, or 100% inhibition (ie, the effect of the target antigen). Or a “neutralizing” polypeptide when the function is reduced (inhibition rate at which function is not detected). Thus, if the target is TNF-α, ELAM-1 TNF-α using the standard L929 cell killing assay described herein or by HUVEC measuring TNF-α-induced cell activity. Neutralizing activity can be assessed by measuring the ability of the anti-TNF-α polypeptide construct to suppress induced expression.

  Further tests for antibody polypeptide interference with the receptor binding activity of TNF-α include the HeLa IL-8 test, also described in Example 6, section 1.3.3.

c) In vivo testing The Tg197 transgenic mouse arthritis model can be used to evaluate the effects of the anti-TNF-α ligands described herein. Tg197 mice are transgenic mice against the human TNF-globin hybrid gene, and heterozygotes at 4-7 weeks of age produce chronic progressive polyarthritis with histological characteristics in common with rheumatoid arthritis (Keffer et al. 1991, EMBO J. 10: 4025-4031). An arthritic phenotype can be scored by assessing joint mobility and indirect swelling. The arthritic phenotype of the joint can be scored by X-ray imaging of the joint and histopathological analysis of the knee and ankle / ankle joint fixation.

An experimental process for assessing the effect of a given antibody polypeptide construct is performed as follows.
1) To test the prevention of arthritis by antibody polypeptide constructs, animals are treated as follows.
a) Heterozygous Tg197 mice are divided into groups of 10 animals with the same number of females and males. Treatment begins at 3 weeks of age and antibody polypeptides are administered intraperitoneally weekly in PBS, or control animals receive PBS alone.
b) Weigh the mice weekly.
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) According to the system, mice are scored for macrophenotypic signs of arthritis.

  The test should be optimally performed so that individual scores are blinded to the test group. The preferred mechanism of antibody delivery for this test is IP injection. However, the test can be configured to use subcutaneous injection, IV injection (eg, via the tail vein), intramuscular injection, or oral, inhalation or topical administration.

  Treatment is effective with the Tg197 model system when the mean arthritis score in the treatment group is lower (by a statistically significant amount) than the vehicle-only control group. A treatment is also considered effective if the mean arthritis score is at least 0.5 units, at least 1.0 units, at least 1.5 units, or at least 2 units lower than vehicle-only control animals. Alternatively, treatment is effective when the average arthritis score is maintained or reduced from 0 to 0.25 throughout the course of the therapeutic treatment.

  Treatment is effective in the Tg197 model system when the mean arthritis score in the treatment group increases throughout the course of the experiment, but the onset of this increase is delayed when compared to the vehicle-only control. The onset of increase in the mean arthritis score of the treatment group when compared to the vehicle-only control is delayed by a period of at least 0.5 weeks, at least 1 week, at least 1.5 weeks, at least 2 weeks, or more than 3 weeks If it is, the process is considered valid.

  As an alternative to scoring the macro phenotype, fix the heel / foot and knee joints at various intervals during processing, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates 2 = pannus and fibrous tissue formation and localized subchondral bone erosion, 4 = histopathological analysis using extensive articular cartilage destruction and bone erosion system. A treatment is considered effective if the average histopathological score is lower (by a statistically advantageous amount) than the vehicle-only control group. The mean histopathological score is at least 0.5 units, at least 1.0 units, at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 2. A process is also considered effective if it is 5 units, at least 3.0 units, or at least 3.5 units lower. Alternatively, treatment is effective when the average histopathological score is maintained or reduced from 0 to 0.5 throughout the course of therapeutic treatment.

  2) In order to test the effect of antibody polypeptide constructs (anti-TNF-α, anti-VEGF, etc.) on established arthritis, treatment begins at 6 weeks of age, when the animals have a significant arthritic phenotype, Tests can be performed on Tg197 as described above. Scoring and effect analysis is also performed as described above. The anti-TNF-αdAb constructs described above can stop or reverse the progression of established arthritis in any of the model systems described.

  In either format, the treatment approach may be monomeric, dimeric or other multimeric forms of anti-TNF-α (eg, as described herein) anti-TNF-αdAb, monomeric, dimeric. Or other multimeric forms of anti-VEGF (eg, anti-VEGFdAbs described herein, including also camel anti-VEGFdAbs), bispecific forms of anti-TNF / anti-VEGF, and anti-HSA, PEG or others Individual or bispecific constructs carrying a half-life modifying component. Also, the anti-VEGF compositions described herein can be administered in combination with other anti-TNF compositions such as etanercept (embrel), D2E7 (Fumila) and infliximab (Remicade). The effect of the combination therapy can be assessed using, for example, cell culture and the in vivo model system described herein.

  Additional accepted animal models of arthritis include, for example, Horsfall et al., 1997, J. MoI. of Immunol. 169: 5687, and collagen-induced arthritis (CIA) and, for example, Stasluk et al., 1997, Immunol. 90:81, pristane-induced arthritis.

Tests for anti-VEGF polypeptide construct effects:
a) VEGF receptor 2 binding test This method represents a VEGF receptor binding test to determine the ability of soluble region antibodies (dAbs) to prevent binding of VEGF ₁₆₅ to VEGF receptor 2.

VEGF is a specific mitogen for endothelial cells in vitro, a potent angiogenic factor in vivo, and high levels of protein are expressed in various types of tumors. It is a 45 kDa glycoprotein that is active as a homodimer. So far, five different isoforms that occur through alternative mRNA division have been described. Of these isoforms, VEGF ₁₂₁ and VEGF ₁₆₅ are the most abundant.

  The specific action of VEGF on endothelial cells is mainly regulated by two types of receptor tyrosine kinases (RTK), VEGF R1 (Flt-1) and VEGF R2 (KDR / Flk-1). However, even though both receptors are phosphorylated upon VEGF binding, VEGF activity such as mitogenic, chemotaxis, and induction of morphological changes is mediated by VEGF R2.

This study, using recombinant human VEGF R2 / Fc chimera comprising the extracellular domain of human VEGF R2 fused to the Fc region of human IgG _1. Briefly, the receptor is captured on an ELISA plate and then the plate is blocked to prevent non-specific binding. A mixture of VEGF ₁₆₅ and dAb protein is then added, the plate is washed, and receptor-bound VEGF ₁₆₅ is detected using biotinylated anti-VEGF antibody and HRPHRP-conjugated anti-biotin antibody. The plate is developed using a colorimetric substrate and the OD is read at 450 nm. If the dAb blocks the binding of VEGF to the receptor, no color is detected.

  The test is performed as follows. Recombinant human VEGF R2 / Fc (R & D Systems, Cat. No: 357-KD-050), contained in carbonate buffer at a concentration of 0.5 μg / ml, in a volume of 100 μl per well of 96 wells Apply to test plate overnight at 4 ° C. The wells are washed 3 times with 0.05% Tween / PBS and 3 times with PBS. 200 μl of 2% BSA contained in PBS is added per well to block the plate and the plate is incubated at room temperature for a minimum of 1 hour.

  The wells are washed (as above) and then the generated dAb protein is added in 50 μl to each well. Then 50 μl of VEGF included in the dilution at a concentration of 6 ng / ml (final concentration of 3 ng / ml) is added to each well and the plate is incubated for 2 hours (supernatant test; 80 μl supernatant is added to each well) Followed by 20 μl of VEGF at a concentration of 15 ng / ml).

  The following controls: 0 ng / ml VEGF (dilution only); 3 ng / ml VEGF (R & D Systems, Cat. No: 293-VE-050); 3 ng / ml containing 0.1 μg / ml anti-VEGF neutralizing antibody ml of VEGF (R & D Systems cat # MAB293) should be included.

  The plate was washed (as above) and then 100 μl of biotinylated anti-VEGF antibody (R & D Systems, Cat No: BAF293) included in the dilution at a concentration of 0.5 μg / ml was added, and 2 at room temperature. Incubate for hours.

  The wells are washed (as above) and then 100 μl of HRP-conjugated anti-biotin antibody (diluted 1: 5000 with diluent; Stratech, Cat No: 200-032-096) is added. The plate is then incubated for 1 hour at room temperature.

  Wash the plate (as above) to remove any traces of Tween 20 to limit background in the next peroxidase test and to prevent bubbles in the test plate wells that would result in an incorrect OD reading. To.

  100 μl SureBlue 1-component TMB microwell peroxidase solution is added to each well and the plate is left at room temperature for up to 20 minutes. When the bound HRP tag complex reacts with the substrate, a dark blue soluble product is generated. The reaction is stopped by adding 100 μl of 1M hydrochloric acid (blue turns yellow). The OD at 450 nm of the plate should be read on a 96 well plate reader within 30 minutes of adding the acid. The OD at 450 nm is proportional to the amount of bound streptavidin-HRP complex.

  The results from the expected controls are as follows: 0 ng / ml VEGF gave a low signal of less than 0.15 OD, 3 ng / ml VEGF gave a signal above 0.5 OD, and 3 ng / ml preincubated with 0.1 μg / ml neutralizing antibody. VEGF gives a signal of less than 0.2 OD.

b) VEGF receptor 1 binding test This test measures the binding of VEGF165 to VEGF R1 and the ability of dAbs to block this interaction.

Here, using recombinant human VEGF R1 / Fc chimera comprising the extracellular region of human VEGF R1 fused to an Fc region of human IgG _1. The receptor is captured on an ELISA plate and then the plate is blocked to prevent non-specific binding. A mixture of VEGF ₁₆₅ and dAb protein is then added, the plate is washed, and receptor-bound VEGF ₁₆₅ is detected using biotinylated anti-VEGF antibody and HRPHRP-conjugated anti-biotin antibody. The plate is developed using a colorimetric substrate and the OD is read at 450 nm. If the dAb blocks the binding of VEGF to the receptor, no color is detected.

  The test is performed as follows. Recombinant human VEGF R1 / Fc (R & D Systems, Cat. No: 321-FL-050), contained in carbonate buffer at a concentration of 0.1 μg / ml, in a volume of 100 μl per well, 96-well nunk manoxy soap Apply to test plate overnight at 4 ° C. The wells are washed 3 times with 0.05% Tween / PBS and 3 times with PBS.

  200 μl of 2% BSA contained in PBS is added per well to block the plate and the plate is incubated at room temperature for a minimum of 1 hour.

  The wells are washed (as above) and then the generated dAb protein is added in 50 μl to each well. Then 50 μl of VEGF included in the dilution at a concentration of 1 ng / ml (final concentration 500 pg / ml) is added to each well and the plate is incubated for 1 hour (supernatant test; 80 μl supernatant is added to each well And then add 20 μl of VEGF at a concentration of 2.5 ng / ml).

  The following controls should be included: 0 ng / ml VEGF (dilution only); 500 pg / ml VEGF; and 500 pg / ml VEGF (R & D Systems cat # MAB293) with 1 μg / ml anti-VEGF neutralizing antibody .

  The plate is washed (as above) and then 100 μl biotinylated anti-VEGF antibody included in the dilution at a concentration of 50 ng / ml is added and incubated for 1 hour at room temperature.

  The wells are washed (as above) and then 100 μl of HRP conjugated anti-biotin antibody (diluted 1: 5000 with diluent) is added. The plate is then incubated for 1 hour at room temperature.

  Wash the plate (as above) to remove any traces of Tween 20 to limit background in the next peroxidase test and to prevent bubbles in the test plate wells that would result in an incorrect OD reading. To.

  100 μl SureBlue 1-component TMB microwell peroxidase solution is added to each well and the plate is left at room temperature for up to 20 minutes. When the bound HRP tag complex reacts with the substrate, a dark blue soluble product is generated. The reaction is stopped by adding 100 μl of 1M hydrochloric acid (blue turns yellow). The OD at 450 nm of the plate should be read on a 96 well plate reader within 30 minutes of adding the acid. The OD at 450 nm is proportional to the amount of bound streptavidin-HRP complex.

  The results from the expected controls are as follows: 0 ng / ml VEGF gives a low signal of less than 0.15 OD, 500 pg / ml VEGF gives a signal above 0.8 OD, 500 pg / ml VEGF pre-incubated with 1 μg / ml neutralizing antibody. Gives a signal of less than 0.3 OD.

c) Cell-based test for VEGF activity This biological test measures the ability of antibody polypeptides (eg, dAbs) and other inhibitors to neutralize VEGF-induced proliferation of HUVE cells. HUVE cells loaded in 96 well plates are incubated for 72 hours with pre-equilibrated VEGF and dAb proteins. Next, the number of cells is measured using a cell viability dye.

The test is performed as follows. HUVE cells are trypsinized from a submerged 175 cm ² flask. The medium is aspirated and the cells are washed with 5 ml trypsin and then incubated with 2 ml trypsin for 5 minutes at room temperature. Gently remove the cells from the bottom of the flask by tapping on the hand. Then 8 ml of triggering medium is added to the flask and the cells are pipetted to disperse all clumps. Viable cells are counted using trypan blue dye.

The cells are spun down and washed twice with the triggering medium, the cells are spun down, and the medium is aspirated after each wash. After the last aspiration, the cells are diluted to 10 ⁵ cells / ml (in induction medium) and loaded into a 96 well plate in an amount of 100 μl per well (10000 cells / well). Plates are incubated for more than 2 hours at 37 ° C. to allow cells to adhere.

60 μl of induction medium containing 60 μl dAb protein and 40 ng / ml VEGF ₁₆₅ (final concentration 10 ng / ml) is added to the v-bottom 96-well plate and sealed with film. The dAb / VEGF mixture is then incubated for 0.5-1 hour at 37 ° C.

  The dAb / VEGF plate is removed from the incubator and 100 μl of solution is added to each well of the HUVEC containing plate (final volume 200 μl). The plate is then returned to the 37 ° C. incubator for at least 72 hours.

  Control wells include wells containing cells but no VEGF; cells, wells containing positive control neutralizing anti-VEGF antibody and VEGF; and control wells containing cells and VEGF only.

  Cell viability is assessed by adding 20 μl of cell titer 96 reagent per well and the plate is incubated at 37 ° C. for 2-4 hours until brownish. The reaction is stopped by adding 20 μl per well of 10% (w / v) SDS. The absorbance at 490 nm is then read using a Wallac microplate reader.

  The absorbance of control wells without VEGF is subtracted from all other values. Absorbance is proportional to cell number. Control wells containing a control anti-VEGF antibody should also show minimal cell proliferation. Wells containing only VEGF should show minimal cell growth.

d) In vivo testing for VEGF activity The effects of anti-VEGF polypeptide constructs (monomers, multimers or bispecific or multispecific) can be tested in a Tg197 transgenic mouse model of arthritic disease. Administration and scoring is basically performed as described for anti-TNF-α polypeptide constructs.

4). Treatment of Crohn's Disease The anti-TNF-α polypeptides described herein can be used to treat human Crohn's disease. In one embodiment, the present invention provides a method of treating Crohn's disease or other inflammatory bowel disease (IBD) involving TNF-α. The method comprises administering a composition comprising one or more single region antibody polypeptide constructs, wherein any of the constructs antagonizes binding of human TNFα to the receptor and / or of IBD. When administered to mice of the Tnf ^ΔARE transgenic mouse model, it prevents an increase in acute or chronic inflammatory bowel score and / or neutralizes TNF-α in the L929 cytotoxicity test. In particular, a method of treating Crohn's disease or other inflammatory bowel disease comprises administering a composition comprising one or more single domain antibody polypeptide constructs, which constructs against human TNFα receptors. Antagonizing binding, administration of the composition to Tnf ^ΔARE transgenic mice prevents or results in an increase in acute or chronic inflammatory bowel score.

The Tnf ^ΔARE transgenic mouse model of Crohn's disease was first described in Kontoiannis et al., 1999, Immunity 10: 387-398. Kontoiannis et al., 2002, J. MoI. Exp. Med. 196: 1563-1574. These mice have a targeted deletion mutation in the 3 ′ AU rich element (ARE) of TNF-α mRNA. AU-rich elements are involved in maintaining low mRNA stability, and when they are disrupted, overexpression of mouse TNF-α occurs in these animals. These animals develop an IBD phenotype very similar to Crohn's disease that begins between 4 and 8 weeks of age. Basic histopathologic features are PMN / macrophage and lymphocyte exudate-dominated ciliary blunting and submucosal inflammation resulting in heterogeneous transmural inflammation and the appearance of lymphocyte aggregates and trace granulomas (Kontoiannis et al., 2002, supra). Since these animals also develop an arthritic phenotype, they can also be used to individually evaluate the effects of anti-TNF-α treatment in RA.

  If the treatment is to be evaluated for its effect in preventing IBD, treatment is initiated, for example, at 3 weeks of age, with the first weekly IP administration of a given antibody polypeptide construct. To some extent, the administration frequency can be selected by those skilled in the art depending on the results of the initial test. The animals can then be monitored for bowel disease according to the standard scale described in Kontoiannis et al., 2002, supra. Paraffin-embedded intestinal tissue of the ileum is evaluated histologically blindly according to the following scale. Acute and chronic inflammation is assessed as follows with a minimum of 8 high power fields (hpf). For acute inflammation scores, 0 = 0 to 1 polymorphonuclear (PMN) cells (PMN / hpf) per hpf; 1 = 2 to 10 PMN / hpf (intramucosal); 2 = 11 to 20 PMN / hpf (intramucosal); 3 = 21-30 PMN / hpf (intramucosal) or 11-20 PMN / hpf (spreading below the mucosal lamina); Chronic inflammation score: 0 = 0-10 mononuclear leukocytes (ML) (ML / hpf) (intramucosal) per hpf; 1 = 11-20 ML / hpf (intramucosal); 2 = 21-30 ML / hpf (mucosal) Within) or 11-20 ML / hpf (spreading below the mucosal muscular plate); 3 = 31-40 ML / hpf (intramucosal) or 21-30 ML / hpf (spreading below the mucosal muscular plate or follicular hyperplasia); > 40 ML / hpf (intramucosal), or 30 ML / hpf (spreading or follicular hyperplasia under mucosal muscularis) The total disease score for each mouse is calculated by adding the acute inflammation or chronic inflammation scores for each mouse.

  In order to evaluate the effect of the treatment on the confirmed disease, it is possible to start the treatment from 6 to 8 months after birth and score similarly.

  A treatment is considered effective if the average histopathological disease score in the treated animal is lower (by a statistically significant amount) than the vehicle control group. The mean histopathological score is at least 0.5 units, at least 1.0 units, at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3 compared to the vehicle-only control group. Treatment is considered effective even if it is 0 units, or at least 3.5 units lower, or the average histopathological score is maintained or reduced from 0 to 0.5 throughout the course of therapeutic treatment In addition, the processing is effective.

  Other models of IBD include, for example, DSS (cold dextran sulfate) for colitis in BALB / c mice. The DSS model was first described in Okayasu et al., 1990, Gastroenterology 98: 694-702, and Kojoharoff et al., 1997, Clin Exp. Immunol. 107: 353-358 (see also WO 2004/041862 designating the United States, which is incorporated herein by reference). Treat BALB / c mice weighing 21-22g and administer DSS at a concentration of 5% w / v in drinking water in a cycle of 7 days treatment and 12 days recovery interval without DSS To induce chronic colitis. The fourth recovery period can be extended to 12 to 21 days to indicate a chronic inflammatory condition rather than an acute condition modeled by shorter recovery. Following the final recovery period, treatment with an antibody polypeptide, eg, a TNF-α polypeptide as described herein, is performed. Initially weekly dosing is recommended, but can be adjusted by one skilled in the art as needed (especially to evaluate dosage forms with different half-life modifying components). At regular intervals during the treatment, the animals are sacrificed, the intestines are dissected and histopathological scores are evaluated as described herein, or as described in Kojoharoff et al., 1997, supra.

  Other animal models of inflammatory bowel disease include chronic intestinal inflammation induced by rectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS: Neuroth et al., 1995, J. Exp. Med. 182: 1281). See also US Pat. No. 6,764,838, which is incorporated herein by reference). Histopathological scoring can be performed using the same criteria as described above.

Comparison with Other Anti-TNF-α Agents Disclosed herein are TNF-α dAb constructs effective for the treatment of RA, Crohn's disease and other TNF-α mediated diseases. In one aspect, the effect of the anti-TNF-α dAb construct is from etanercept (ENBREL), infliximab (REMICADE) and D2E7 (HUMIRA; see US Pat. No. 6,090,382, incorporated herein by reference). More than the effect of the drug selected from the group consisting of.

  Recombinant clinical trials of soluble human TNFR (p75) conjugated to the Fc part of human IgG1 (sTNFR (p75): Fc, ENBREL, Imnex), its administration significantly and rapidly reduced RA disease activity (Moreland et al., 1997, N. Eng. J. Med., 337: 141-147). In addition, preliminary safety data from pediatric clinical trials for TNFR (p75): Fc indicate that this drug is generally well tolerated in patients with juvenile rheumatoid arthritis (JRA) (Garrison). 1998, Am. College of Rheumatology meeting, Nov. 9, 1998, abstract 584).

  As described above, ENBREL is a dimeric fusion protein consisting of the extracellular ligand binding portion of human 75 kilodalton (p75) TNFR (GenBank accession number P20333) bound to the Fc portion of human IgG1. The FBR component of ENBREL contains a CH2 region, a CH3 region and a hinge region, but does not contain an IgG1 CH1 region. ENBREL is produced in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of about 150 kilodaltons (Smith et al., 1990, Science, 248: 1019-1023; Mohler et al., 1993, J. Immunol. 151: 1548-1561; US Pat. No. 5,395,760 (Immunex Corporation, Seattle, WA); incorporated herein by reference; US Pat. No. 5,605,690 (Immunex Corporation, incorporated herein by reference) Seattle, Washington))).

  Monoclonal antibodies directed against TNF-α (infliximab, REMICADE, St. Col) have demonstrated clinical efficacy in the treatment of RA, whether administered with or without methotrexate (Elliot et al., 1993, Arthritis Rheum, 36: 1681-1690; Elliot et al., 1994, Lancet, 344: 1051-1110). These data demonstrate a significant reduction of the Paulus 20% and 50% criteria at weeks 4, 12, and 26. This treatment is administered intravenously and the anti-TNF monoclonal antibody disappears from the blood over a period of 2 months. Repeated administration appears to reduce the duration of the effect. Patients can generate antibodies against anti-TNF antibodies that limit the efficacy and duration of this treatment (Kavanaugh et al., 1998, Rheum. Dis. Clim., North Am., 24: 593-614). Administration of methotrexate in combination with infliximab helps to prevent the development of anti-infliximab antibodies (Maini et al., 1998, Arthritis Rheum., 41: 1552-1563). Infliximab has also demonstrated clinical efficacy in the treatment of inflammatory bowel disease Crohn's disease (Baert et al., 1999, Gastroenterology, 116: 22-28).

  As stated in the background section, infliximab is a chimeric monoclonal IgG antibody having a human IgG4 constant region and a mouse variable region. Infliximab polypeptides are described in US Pat. Nos. 5,698,195 and 5,656,272, which are incorporated herein by reference.

  To compare the effects with these or other anti-TNF-α compositions, anti-TNF-α dAb constructs can be used in combination with existing compositions to produce the receptor binding assays described herein, cell based Any one of the test and the in vivo test may be performed. Thus, this approach identifies anti-TNF-α dAb constructs that exhibit an effect of suppressing the effect of TNF-α in any of the tests that is equivalent to (or statistically significant) that of the comparative composition. Examples of such constructs and analyzes that show equivalent or better effects are given in the examples.

Example 1
Selection of bispecific scFv antibody (K8) directed against human serum albumin (HSA) and β-galactosidase (β-gal) In this example, the VK variable region bound to the germline (dummy) VH region Double repertoire directed against β-gal and HSA, with a repertoire of VH variable regions selected for binding to p-gal and a repertoire of VH variable regions bound to germline (dummy) VK regions selected A method for producing a sex ligand will be described. The selected variable VH HSA and V _K β-gal regions are then combined and antibodies are selected for binding to β-gal and HSA. HSA is a half-life increasing protein found in human blood.

In this experiment, four human phage antibody libraries were used.
Library 1 Germline V _K / DVT V _H 8.46 × 10 ⁷
Library 2 Germline V _K / NNK V _H 9.64 × 10 ⁷
Library 3 Germline VH / DVT V _K 1.47 × 10 ⁸
Library 4 Germline V _H / NNK V _K 1.45 × 10 ⁸

All libraries have V _H (V3-23 / DP47 and J _H 4b) and V _K (O12 / O2 / DPK9 and J _K ) with side chain diversity introduced into the complementarity determining regions (CDR2 and CDR3). Based on a single human framework for 1).

Library 1 and Library 2, relative to contain _{V K} sequences, the sequence of _{V H} is, H50, H52, H52a, H53 , H55, H56, H58, H95, H96, at the location of the H97 and H98 Diversified (DVT or NNK coded respectively) (Figure 1). Library 3 and Library 4, whereas containing _{V H} sequence, the sequence of _{V K} is, L50, L53, L91, H92 , H93, H94 and diversified by which (respectively DVT at a position L96 Or NNK coded) (FIG. 1). These libraries are in the phagemid pIT2 / ScFv format (FIG. 2) and are pre-selected for binding to universal ligands, ie protein A and protein L, so that the majority of clones in the unselected library are functional. Has been. The size of the above library corresponds to the size after the pre-selection. Prior to selection for antigen, library 1 and library 2 are mixed to generate a single V _H / dummy V _K library, library 3 and library 4 are mixed, and single V _K / dummy A _VH library was formed.

Three rounds of selection for β-gal were performed using the V _K / dummy V _H library, and three rounds of selection were performed for HSA using the V _H / dummy V _K library. In the case of β-gal, the phage titer increased from 1.1 × 10 ^{6 in the} first round to 2.0 × 10 ⁸ in the third round. In the case of HSA, the phage titer increased from 2 × 10 ^{4 in the} first round to 1.4 × 10 ⁹ in the third round. Their selection was done using Griffith, except that KM13 helper phage (containing pIII protein with protease cleavage between D2 and D3 regions) was used and the phage was eluted with 1 mg / ml trypsin in PBS. Et al. (1993). Addition of trypsin cleaves pIII protein derived from helper phage (not protein derived from phagemid) and elutes bound scFv phage by cleavage at the c-myc tag (FIG. 2), thereby functional scFvs. Are further enriched and correspondingly reduced in background (Kristensen & Winter, Folding & Design 3: 321-328, Jul 9, 1998). Selection was performed using an immunotube coated with HSA or β-gal at a concentration of 100 μg / ml.

  To check for binding, colonies from the third round of each selection were screened by monoclonal phage ELISA. Harrison et al., Methods Enzymol. , 1996; 267: 83-109. A 96-well ELISA plate was coated with 100 μl of HSA or β-gal at a concentration of 10 μg / ml contained in OBS at 4 ° C. overnight. A standard ELISA protocol was performed using detection of bound phage with anti-M13-HRP complex. Selection of clones gave an ELISA signal greater than 1.0 with 50 μl of supernatant.

Next, using the QIAprep spin miniprep kit (Qiagen), DNA prep from the V _H / dummy V _K library selected for HSA and the V _K / dummy V _H library selected for β-gal Manufactured.

In order to take advantage of most of the diversity, DNA prep was prepared from each of the three rounds of selection and then summarized for each antigen. The DNA prep was then digested with SalI / NotI overnight at 37 ° C. Following gel purification of the fragments, the _{V K} chains from the selected VK / dummy _{V H} library for beta-gal, instead of the dummy _{V K} chains of _{V H} / dummy _{V K} library selected for HSA Combined to make 3.3 × 10 ⁹ clones.

  This library was then selected for HSA (first round) and β-gal (second round) (HSA / β-gal selection), or β-gal (first round) and HSA (second round) (Β-gal / HSA selection). The selection was made as described above. In each case after the second round, 48 clones were tested for binding to HSA and β-gal by monoclonal phage ELISA (described above) and soluble scFv fragment ELISA. Soluble antibody fragments were prepared as described in Harrison et al. (1996), using 2% Tween / PBS as a blocking buffer, except for detecting bound scFvs with protein L-HRP. A general ELISA protocol was performed (Hoogenboom et al. (1991), Nucleic Acids Res., 19: 4133). Three clones with HSA / β-gal selection (E4, E5 and E8) and two clones with K-gal / HSA selection (K8 and K10) were able to bind both antigens. ScFvs from these clones were PCR amplified and using primers LMB3 and pHENseq, Ignatovich et al. (1999), J. MoI. Mol. Biol. 1999 Nov. 26; 294 (2): 457-65. Sequence analysis revealed that all clones were identical. Therefore, only one clone encoding the bispecific antibody (K8) was selected for the next test (FIG. 3).

(Example 2)
Characterization of the binding properties of the K8 antibody First, the binding properties of the K8 antibody were characterized by monoclonal phage ELISA. In a 96 well plate, alkaline phosphate (APS), bovine serum albumin (BSA), peanut agglutinin, lysozyme and cytochrome c (for cross-reactivity testing), 100 μl HSA at a concentration of 10 μg / ml in PBS and β-gal 10 μg / ml was applied at 4 ° C. overnight. Phagemids from K8 clones were released with KM13 as described in Harrison et al. (1996) and supernatants containing phage (50 μl) were tested directly. A standard ELISA protocol was performed using detection of bound phage with anti-M13-HRP complex (Hoogenboom et al., 1991). The bispecific K8 antibody was confirmed to bind to HSA and β-gal when displayed on the phage surface with an absorption signal greater than 1.0 (FIG. 4). Strong binding to BSA was also observed (Figure 4).

  Since HSA and BSA have 76% homology at the amino acid level, it is not surprising that the K8 antibody recognized both of these structurally related proteins. No cross-reactivity with other proteins was detected (Figure 4).

  Second, the binding properties of the K8 antibody were tested in a soluble scFv ELISA.

  Production of soluble scFv fragments was induced by IPTG as described by Harrison et al. (1996). To measure the expression level of K8scFv, a protein A sepharose column was used to obtain a supernatant from a 50 ml derivative as described in Harlow and Lane, Antibodies: a Laboratory Manual, (1988), Cold Spring Harbor. Soluble antibody fragments were purified. OD280 was then measured and protein concentration was calculated as described in Sambrook et al. (1989). K8scFv was produced in the supernatant at a concentration of 19 mg / l.

  A soluble scFv ELISA was then performed using known concentrations of the K8 antibody fragment. A 96-well plate was coated with 100 μl HSA, BSA and β-gal at a concentration of 10 μg / ml, and protein A at a concentration of 1 μg / ml. 50 μl of a serial dilution of K8scFv was applied, and the bound antibody fragment was detected with protein L-HRP. The ELISA results confirmed the bispecificity of the K8 antibody (FIG. 5).

binding to beta-gal is dictated by the _{V K} domain, binding to HAS / BSA in order to confirm that dictated by the _{V H} region of K8scFv antibody excised VK region from K8scFv DNA by SalI / NotI digestion, dummy V was coupled to the SalI / NotI digested pIT2 vector containing the _H chain (Figures 1 and 2). The binding characteristics of the resulting clone K8V _K / dummy V _H were analyzed by soluble scFv ELISA. As described in Harrison et al. (1996), IPTG induced the production of soluble scFv fragments and supernatants containing scFvs (50 μl) were tested directly. Soluble scFv ELISA was performed as described in Example 1 and scFvs was detected with protein L-HRP. ELISA results showed that this clone was able to bind β-gal whereas binding to BSA disappeared (FIG. 6).

(Example 3)
Selection of Single V _H Region Antibody Antigens A and B and Single V _K Region Antibody directed against Antigens C and D In this example, a single V _H region antibody directed against antigens A and B, And single V _K region antibodies directed against antigens C and D by selecting a repertoire of naive single antibody variable regions for binding to these antigens in the absence of complementary variable regions. The method will be described.

Selection and characterization of binding clones is performed as previously described (see Example 5, PCT / GB02 / 003014). The following 4 clones are selected for further testing.
VH1-anti-AV _H
VH2-anti-BV _H
VK1-anti-CV _K
VK2-Anti-DV _K

The procedure described in Examples 1 to 3 is carried out in the same way, and a dimer molecule comprising a combination of a V _H region (ie V _H ligand) and a combination of a V _L region (V _L -V _L ligand) Can be generated.

Example 4
Bispecific ScFv antibodies (VH1 / VH2 directed against antigens A and B and VK1 / VK2 directed against antigens C and D)
In this embodiment, by combining the _{V K} and _{V H} single domain selected for each of the antigens in ScFv vector was derived for bispecific ScFv antibody (antigen A and B VH1 / VH2 , And VK1 / VK2) directed against antigens C and D). To make bispecific antibody VH1 / VH2, VH1 single region was excised from variable region vector 1 (FIG. 7) by NcoI / XhoI digestion and ligated to NcoI / XhoI digested variable region vector 2 (FIG. 7). Thus, VH1 / variable region vector 2 is prepared. Using primers, the VH2 single region is PCR amplified from the variable region vector to introduce a SalI restriction site at the 5 ′ end and a NotI restriction site at the 3 ′ end. The PCR product is then digested with SalI / NotI and ligated to SalI / NotI digested VH1 / variable region vector 2 to create VH1 / VH2 / variable region vector 2.

  Similarly, VK1 / VK2 / variable region vector 2 is prepared. The bispecificity of the generated VH1 / VH2ScFv and VK1 / VK2ScFv is tested in a soluble ScFv ELISA as previously described (see Example 6, PCT / GB02 / 003014). A competitive ELISA is performed as previously described (see Example 8, PCT / GB02 / 003014).

Expected results-VH1 / VH2 ScFv can bind antigens A and B simultaneously.
-VK1 / VK2 ScFv can bind antigens C and D simultaneously.
-VH1 / VH2 ScFv binding is competitive (when bound to antigen A, VH1 / VH2 ScFv is unable to bind antigen B).
-VK1 / VK2 ScFv binding is competitive (when bound to antigen C, VK1 / VK2 ScFv is unable to bind antigen D).

(Example 5)
Construction of bispecific VH1 / VH2 Fabs and analysis of their binding properties To make VH1 / VH2 Fabs, VH1 single region was ligated into NcoI / XhoI digested CH vector (FIG. 8) and VH1 / CH And ligate the VH2 single region to the SalI / NotI digested CK vector (FIG. 9) to make VH2 / CK. Plasmids from VH1 / CH and VH2 / CK are used to co-transform competitor E. coli cells as previously described (see Example 8, PCT / GB02 / 003014).

  A clone containing the VH1 / CH and VH2 / CK plasmids is then induced with IPTG to produce soluble VH1 / VH2 Fab as described above (see Example 8, PCT / GB02 / 003014).

  Similarly, VK1 / VK2 Fab is generated.

  As described above, the binding properties of the produced Fab are tested by competitive ELISA (see Example 8, PCT / GB02 / 003014).

Expected results-VH1 / VH2 Fab is capable of binding antigens A and B simultaneously.
-VK1 / VK2 Fab can bind antigens C and D simultaneously.
-VH1 / VH2 Fab binding is competitive (when bound to antigen A, VH1 / VH2 Fab is unable to bind antigen B).
-VK1 / VK2 Fab binding is competitive (when bound to antigen C, VK1 / VK2 Fab is unable to bind antigen D).

(Example 6)
Chelated dAb dimers Overview A flexible polypeptide linker is used to make VH and VK homodimers in the dAb-linker-dAb format. A dAb linker-dAb format vector containing glycine-serine linker 3U: (Gly ₄ Ser) ₃ , 5U: (Gly ₄ Ser) ₅ , 7U: (Gly ₄ Ser) ₇ of different lengths was prepared. Dimer libraries using the guide dAbs upstream of the linker, ie TAR1-5 (VK), 5TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK), and after the linker A corresponding second dAbs library was created. Using this method, new dimeric dAbs were selected. The effect of dimerization on antigen binding was measured by ELISA and BIAcore tests, and cell neutralization and receptor binding tests. Dimerization of both TAR1-5 and TAR1-27 significantly improved binding affinity and neutralization levels.

1.0 Methods 1.1 Library Generation 1.1.1 Vectors pEDA3U, pEDA5U and pEDA7U vectors were designed to introduce different linker lengths compatible with the dAb-linker-dAb format. For pEDA3U, the sense and antisense 73 base pair oligo linkers were run in a slow annealing program (95 ° C.-5 min, 80 ° C.—in a buffer (pH 7.4) containing 0.1 M NaCl, 10 mM Tris HCl. 10 min, 70 ° C-15 min, 56 ° C-15 min, 42 ° C until use) and cloned using XhoI and NotI restriction sites.

The linker included three (Gly ₄ Ser) (SEQ ID NO: 7) units and a stuffer region housed between the SalI and NotI cloning sites (Scheme 1). To reduce the likelihood of monomeric dAbs being selected for phage display, include a frameshift mutation that excludes the region from the frame when three stop codons, a SacI restriction site, and a second dAb are not present The staffer area was designed. For pEDA5U and 7U, depending on the length of the linker required, overlapping oligo-linkers were designed for each vector, annealed and extended with Klenow. The fragment was then purified and digested using the appropriate enzyme before cloning using the XhoI and NotI restriction sites.

1.1.2 Library Preparation The N-terminal V gene corresponding to the guide dAb was cloned upstream of the linker using NcoI and XhoI restriction sites. Although the VH gene has an existing compatible site, cloning of the VK gene required the introduction of a suitable restriction site. This is a 30-cycle PCR amplification using a 2: 1 mixture of SuperTaq (HTBiotechnology Ltd) and pfu turbo (Stratagene) with modified PCR primers (VK-DLIBF: 5′cggccatggcgtcaacggacat-3 ′; VKXhotIRgtgtgtgtgtgtgtgtgtgtgt This is achieved by using 3 ′). This maintained the 5NcoI site at the 5 ′ end, while destroying the adjacent SalI site and introducing the XhoI site at the 3 ′ end. Five guide dAbs were cloned into each of the three dimer vectors (TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH), TAR2-6 (VK) and TAR2-7 (VK). )). All constructs were verified by sequence analysis.

Cloning the guide dAbs upstream of the linker in each of the vectors (pEDA3U, 5U and 7U) (TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK)) A corresponding library of second dAbs was cloned after the linker. To achieve this, a VK library for human TNF-α (approximately 1 × 10 ⁶ diversity after round 1) when TAR1-5 or TAR1-27 is a guide dAbs, or TAR2-5 or TAR2 Complementary dAbs from phage recovered from round 1 selection of VH or VK libraries for human p55TNF receptor (both approximately 1 × 10 ⁵ diversity after round 1) when -6 are each guide dAbs The library was PCR amplified. For VK libraries, PCR amplification was performed using primers in 30 cycles of PCR amplification using a 2: 1 mixture of SuperTaq and pfu turbo. In order to introduce a SalI restriction site at the 5 'end of the gene, the VH library was PCR amplified using primers. The dAb library PCR was digested with appropriate restriction enzymes, ligated to the corresponding vector downstream of the linker using SalI / NotI restriction sites, and electroporated into freshly prepared competitor TG1 cells.

The titers achieved for each library are as follows:

1.2 Selection 1.2.1 TNF-α
Selection was performed using human TNFa passively applied to immunotubes. Briefly, 1-4 ml of the required antigen was applied to the immunotube overnight. The immunotube was then washed 3 times with PBS, blocked with 2% milk powder in PBS for 1-2 hours and washed 3 more times with PBS. The phage solution is diluted with 2% milk powder in PBS and incubated for 2 hours at room temperature. The tube is then washed with PBS and the phage eluted with 1 mg / ml trypsin-PBS. Three selection approaches for the TAR1-5 dimer library were examined. In the immunotube, human TNFa coated at a concentration of 1 μg / ml or 20 μg / ml was used and washed 20 times with PBS 0.1% Tween for the first round of selection. TG1 cells are infected with the eluted phage and titered (eg Marks et al., J Mol Biol., 1991, Dec 5; 222 (3): 581-97, Richmann et al., Biochemistry., 1993, Aug 31; 32 (34): 8848-55).

The recovered titers were as follows:

The second round of selection was performed using three different methods.
1. Wash 20 times in immunotube, incubate overnight, then wash 10 more times.
2. In an immunotube, after washing 20 times, incubate with (1 μg / ml TNF-α) in washing buffer at room temperature for 1 hour and further wash 10 times.
Selection for streptavidin beads using 3.33 pmoles of biotinylated human TNF-α (Henderikx et al., 2002, “Selection of antibodies to biotinylated antigen. Antibody phage display: methods and protocols. Selection of antigenized antigens. (Antibody Page Display: Methods and protocols) "edited by O'Brien and Atkin, Humana Pers). Single clones from round 2 selection were collected in 96 well plates and crude supernatant prep was made in 2 ml 96 well plate format.

  TAR1-27 was selected as described above with the following changes. In the immunotube, human TNF-α coated at a concentration of 1 μg / ml or 20 μg / ml was used and washed 20 times with PBS 0.1% Tween for the first round of selection. In the immunotube, after washing 20 times and incubating overnight, another 20 washes were performed for the second round of selection. Single clones from round 2 selection were collected in 96-well plates and crude supernatant prep was made in a 2 ml 96-well plate format.

The TAR1-27 titers are as follows:

1.2.2 TNF receptor 1 (p55 receptor; TAR2)
Selection was performed as described above for only the TAR2h-5 library. In an immunotube, use 1 μg / ml human p55TNF receptor or 10 μg / ml human p55TNF receptor, wash 20 times with PBS 0.1% Tween, incubate overnight, then wash 20 more times. Three rounds of selection were made. Single clones from round 2 and 3 selections were collected in 96 well plates and crude supernatant prep made in 2 ml 96 well plate format.

The 0 TAR2h-5 titers are as follows:

1.3 Screening Single clones from round 2 or 3 selection were picked from each of 3U, 5U and 7U libraries with different selection methods as appropriate. Clones were grown overnight at 37 ° C. in 2 × TY containing 100 μg / ml ampicillin and 1% glucose. A 1/100 dilution of this culture is inoculated in 2 ml 96 well plate format into 2 ml 2 × TY containing 100 μg / ml ampicillin and 0.1% glucose and shaken until the OD600 is about 0.9. While growing at 37 ° C. Subsequently, overnight culture was promoted at 30 ° C. with 1 mM IPTG.

  The supernatant was clarified by centrifuging at 4000 rpm for 15 minutes in a Sorval plate centrifuge. The supernatant prep was used for the initial screening.

1.3.1 ELISA
The binding activity of the dimeric recombinant protein was compared with that of the monomer by protein A / L ELISA or antigen ELISA. Briefly, antigen or protein A / L was applied to a 96 well plate and placed at 4 ° C. overnight. Plates are washed with 0.05% Tween-PBS and blocked with 2% Tween-PBS for 2 hours. Samples are added to the plate and incubated for 1 hour at room temperature. Plates are washed and incubated with secondary reagents for 1 hour at room temperature. Wash plate and develop TMB substrate. Protein A / L HRP or India-HRP was used as a secondary reagent. For antigen ELISA, the antigen concentration used was 1 μg / ml in PBS for human TNFa and human TNF receptor 1. Due to the presence of the guide dAb, in most cases the dimer gave an ELISA signal, so the off-rate measurement was considered by BIAcore.

1.3.2 BIAcore
BIAcore analysis was performed on TAR1-5 and TAR2h-5 clones. For screening, human TNF-α was bound to a CM5 chip at high density (approximately 10,000 RU).

  50 μl of human TNFa (50 μg / ml) was bound to the chip at 5 μl / min in acetate buffer (pH 5.5). Chip regeneration following analysis using standard methods is not possible due to instability of human TNF-α. Therefore, after analyzing each sample, the chip was washed with buffer for 10 minutes.

For TAR1-5, clone supernatants from round 2 selection were screened by BIAcore. Forty-eight clones were screened from each of the 3U, 5U and 7U libraries using the following selection method.
R1: 1 μg / ml human TNF-α immunotube, R2 1 μg / ml human TNF-α immunotube, washed overnight.
R1: 20 μg / ml human TNF-α immunotube, R2 20 μg / ml human TNF-α immunotube, washed overnight.
R1: 1 μg / ml human TNF-α immunotube, 33 pmol biotinylated human TNF-α on R2 beads
R1: 20 μg / ml human TNF-α immunotube, 33 picomolar biotinylated human TNF-α on R2 beads

  For screening, the human p55TNF receptor was bound to a CM5 chip at high density (approximately 4000 RU). 100 μl of human p55TNF receptor (10 10 μg / ml) was bound to the chip at 5 μl / min in acetate buffer (pH 5.5). Standard regeneration conditions (glycine pH 2 or pH 3) were examined, but in each case the antigen was removed from the surface of the chip, so for TNF-α, the chip was buffered with 10 buffer after analysis of each sample. Washed for minutes.

  For TAR2-5, clone supernatants by round selection were screened.

48 clones from each of 3U, 5U and 7U were screened using the following selection method.
R1: 1 μg / ml human p55TNF receptor immunotube, R2 1 μg / ml human p55TNF receptor immunotube, washed overnight.
R1: 10 μg / ml human p55TNF receptor immunotube, R2 10 μg / ml human p55TNF receptor immunotube, washed overnight.

1.3.3 Receptor and Cell Test The ability of the dimer to neutralize in the receptor test was induced as follows.
Receptor binding Anti-TNF dAbs were tested for their ability to inhibit TNF binding to recombinant TNF receptor 1 (p55). Briefly, Maxisorp plates were incubated overnight with 30 mg / ml anti-human Fc mouse monoclonal antibody (Zymed (San Francisco, USA)). Wells were washed with phosphate buffered salt (PBS) containing 0.05% Tween 20, then blocked with 1% BSA in PBS before 100 ng / ml TNF receptor 1 Fc fusion protein (R & D Systems). (Minneapolis, USA)). Anti-TNF dAb was mixed with TNF and added to the washed wells to a final concentration of 10 ng / ml. After detecting TNF binding with 0.2 mg / ml biotinylated anti-TNF antibody (HyCult biotechnology (Uben, The Netherlands)), diluted 1: 500 with horseradish peroxidase tagged streptavidin (Amersham Biosciences (UK)) and then TMB Incubated with substrate (KPL (Gaithersburg, USA)). The reaction was stopped by adding HCl and the absorbance at 450 nm was read. Anti-TNF dAb activity resulted in a decrease in TNF binding, resulting in a decrease in absorbance compared to the TNF-only control.

  L929 cytotoxicity test for ability to neutralize cytotoxic activity of TNF against mouse L929 fibroblasts Anti-TNF dAb was also tested (Evans, T. (2000), Molecular Biotechnology, 15, 243-248). . Briefly, L929 cells loaded in microtiter plates were incubated overnight with anti-TNF dAb, 100 pg / ml TNF and 1 mg / ml actinomycin D (Sigma, Poole, UK). After incubation with [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (Promega, Madison, USA) Cell viability was measured by reading the absorbance at 490 nm. Anti-TNF dAb activity resulted in a decrease in TNF cytotoxicity, resulting in an increase in absorbance compared to the TNF-only control.

  In the initial screen, the supernatant prepared for the BIAcore analysis described above was also used for receptor testing. Further analysis of the selected dimer was performed using the purified protein in receptor and cell studies.

HeLa IL-8 Test Anti-TNFR1 or anti-TNFαdAbs were tested for their ability to neutralize the induction of IL-8 secretion by TNF in HeLa cells (Akeson, which describes the induction of IL-8 by IL-1 in HUVEC, L. et al. (1996), Journal of Biological Chemistry, 271, 30517-30523; here we examine induction by human TNFα and use HeLa cells instead of HUVEC cell lines). Briefly, HeLa cells loaded in microtiter plates were incubated overnight with dAb and 300 pg / ml TNF. After incubation, the supernatant was aspirated from the cells and the IL-8 concentration was measured via a sandwich ELISA (R & D Systems). Anti-TNFR1 dAb activity resulted in a decrease in IL-8 secretion into the supernatant compared to the TNF-only control.

  The L929 test is used throughout the following experiment. However, it is preferred to use the HeLa IL-8 test to measure anti-TNF receptor 1 (p55) ligands. The presence of mouse p55 in the L929 test is a definitive limiting factor for its use.

1.4 Sequence analysis Dimers that proved to have interesting properties in BIAcore and receptor test screens were sequenced. The sequences are detailed in the following sequence listing, drawings and examples.

1.5 Setting 1.5.1 TAR1-5-19 Dimer TAR1-5 dimers that were shown to have good neutralizing properties were reset and analyzed in cell and receptor studies. The TAR1-5 guide dAb was replaced with the affinity matured clone TAR1-5-19. To accomplish this, TAR1-5 was cloned from individual dimer pairs and replaced with TAR1-5-19 amplified by PCR. In addition, TAR1-5-19 homodimers were also composed of 3U, 5U and 7U vectors. The N-terminal replica of the gene was amplified by PCR, cloned as described above, and the C-terminal gene fragment was cloned using SalI and NotI restriction sites.

1.5.2 Mutagenesis The amber stop codon present in dAb2, one of the C-terminal dAbs in the TAR1-5 dimer pair, was mutated to glutamine by site-directed mutagenesis.

1.5.3 Fabs
The dimer containing TAR1-5 or TAR1-5-19 is reset to the Fab expression vector. Using SfiI and NotI restriction sites, dAbs were cloned into expression vectors containing CK or CH genes and verified by sequence analysis. The CK vector is derived from a pUC-based ampicillin resistant vector and the CH vector is derived from a pACYC chloramphenicol resistant vector. For Fab expression, dAb-CH and dAb-CK constructs were co-transformed into HB2151 cells and grown in 2 × TY containing 0.1% glucose, 100 μg / ml ampicillin and 10 μg / ml chloramphenicol. .

1.5.3 Hinge dimerization The dimerization of dAbs by cystine bond formation was investigated. A short amino acid sequence EPKSGDKTHTCPPCP (SEQ ID NO: 11) A modified form of the human IgGC1 hinge was created in the C-terminal region of the dAb. An oligo linker encoding for this sequence was synthesized and annealed as described above. The linker was cloned into the pEDA vector containing TAR1-5-19 using XhoI and NotI restriction sites. Dimerization is performed in situ in the periplasm.

1.6 Expression and Purification 1.6.1 Expression Supernatant was prepared in a 2 ml 96 well plate format for initial screening as previously described. Following the initial screening process, the selected dimers were further analyzed. As a supernatant, the dimer construct was expressed in TOP10F ′ or HB2151 cells. Briefly, individual colonies from freshly striped plates were grown overnight at 37 ° C. in 2 × TY containing 100 μg / ml ampicillin and 1% glucose. A 1/100 dilution of this culture was inoculated into 2 × TY containing 100 μg / ml ampicillin and 0.1% glucose and grown at 37 ° C. with shaking until the OD600 was approximately 0.9. The culture was then induced overnight at 30 ° C. with 1 mM IPTG. Cells were removed by centrifugation and the supernatant was purified with protein A or L agarose.

  Fab and cysteine hinge dimers were expressed as periplasmic proteins in HB2152 cells.

A 1/100 dilution of an overnight culture was inoculated into 2 × TY containing 0.1% glucose and appropriate antibiotics and grown at 37 ° C. with shaking until the OD600 was approximately 0.9. . The culture was then induced with 1 mM IPTG at 30 ° C. for 3-4 hours. Cells were harvested by centrifugation and the pellet was resuspended in periplasmic preparation buffer (30 mM Tris HCl (pH 8.0), 1 mM EDTA, 20% sucrose). Following centrifugation, the supernatant was retained and the pellet was resuspended in 5 mM MgSO ₄ . The supernatant was harvested again by centrifugation, accumulated and purified.

1.6.2 Protein A / L Purification Optimization of purification of dimeric protein from protein L agarose (Affitech (Norway)) or protein A agarose (Sigma (UK)) was investigated. The protein was eluted by batch or column elution using a peristaltic pump. Three buffers, 0.1M phosphate-citrate buffer (pH 2.6), 0.2M glycine (pH 2.5) and 0.1M glycine (pH 2.5) were examined.

  Optimal conditions were determined to be peristaltic pump conditions using 0.1 M glycine (pH 2.5) for 10 column volumes. Purification with protein A was performed under peristaltic pump conditions using 0.1 M glycine (pH 2.5).

1.6.3 FPLC purification Further purification was performed by FPLC analysis on an AKTA Explorer 100 system (Amersham Biosciences Ltd). TAR1-5 and TAR1-5-19 dimers were fractionated by cation exchange chromatography (1 ml Resource S-Amersham Biosciences Ltd) eluted with a 0-1 M NaCl gradient in 50 mM acetate buffer (pH 4). The hinge dimer was purified by ion exchange (1 ml Resource Q Amersham Biosciences Ltd) eluting with a 0-1 M NaCl gradient in 25 mM Tris HCl, pH 8.0. Fabs were purified by size exclusion chromatography using a Superose 12 (Amersham Biosciences Ltd) column treated in PBS containing 0.05% Tween at a flow rate of 0.5 ml / min. Following purification, the Vivaspin 5K cutoff concentrator Samples were concentrated using (Vivascience Ltd).

2.0 Results 2.1 TAR1-5 dimer 6 × 96 clones were picked from the second round of selection including all libraries and selection conditions. Supernatant prep was made and tested in antigen and protein L ELISA, BIAcore and receptor tests. For ELISA, positive clones were identified from each selection method and distributed into 3U, 5U and 7U libraries. However, since guide dAbs always exist, it was impossible to discriminate between high affinity and low affinity by this method. Therefore, BIAcore analysis was performed.

BIAcore analysis was performed using 2 ml of supernatant. BIAcore analysis revealed that the dimer Koff rate was significantly improved compared to monomeric TAR1-5. Compared to the dimer Koff rate in the range of 10 ⁻³ to 10 ⁻⁴ M, the monomer Koff rate was in the range of 10 ⁻¹ M. Sixteen clones were selected that appeared to be very slow off-rate, and these were obtained from 3U, 5U and 7U libraries and sequenced. In addition, the supernatant was analyzed for the ability to neutralize human TNF-α in a receptor test.

The six lead clones (d1-d6 below) neutralized in these tests have been sequenced. The results show that of the 6 clones obtained, there are only 3 different second dAbs (dAb1, dAb2 and dAb3), but if the second dAb is observed more than once, it has a different length. Combined with a linker.
TAR1-5d1: 3U linker second dAb = dAb1-1 μg / ml Ag immunotube, overnight wash TAR1-5d2: 3U linker second dAb = dAb2-1 μg / ml Ag immunotube, overnight wash TAR1-5d3: 5U linker second dAb = dAb2-1 μg / ml Ag immunotube, overnight wash TAR1-5d4: 5U linker second dAb = dAb3-20 μg / ml Ag immunotube, overnight wash TAR1-5d5: 5U linker second DAb = dAb 1-20 μg / ml Ag immunotube, overnight wash TAR1-5d6: 7 U linker Second dAb = dAb1-R1: 1 μg / ml Ag immunotube, overnight wash, R2: beads

  Six lead clones were further examined. Protein was produced from periplasm and supernatant, purified with protein L agarose and examined in cell and receptor tests. The level of neutralization varied (Table 1). Optimal conditions for protein preparation were determined. The highest yield of protein produced from HB2151 cells as supernatant (about 10 mgs / L culture) was obtained. The supernatant was incubated with protein L agarose for 2 hours at room temperature or overnight at 4 ° C. The beads were washed with PBS / NaCl and loaded onto the FPLC column using a peristaltic pump. The beads were washed with 10 column volumes of PBS / NaCl and eluted with 0.1 M glycine (pH 2.5). In general, the dimeric protein is eluted after the monomer.

  The TAR1-5dl-6 dimer was purified by FPLC. Three species were obtained by FPLC purification and identified by SDS PAGE. One species corresponds to the monomer and the other two species correspond to dimers of different sizes. The larger of these two species is probably due to the presence of a C-terminal tag. These proteins were examined in a receptor test. The data shown in Table 1 represents the optimal results obtained from two dimeric species (Figure 11). Three second dAbs from dimer pairs (ie dAb1, dAb2, and dAb3) were cloned as monomers and examined in ELISA and cell and receptor studies. All three dAbs bind specifically to TNF by antigen ELISA and do not cross-react with plastic or BSA. As a monomer, dAbs are neutralized in cell or receptor tests.

2.1.2 TAR-5-19 dimer TAR1-5-19 was substituted for TAR1-5 in 6 lead clones. Analysis of all TAR1-5-19 dimers in cell and receptor studies was performed using total protein (only purified protein L) unless otherwise specified (Table 2). TAR1-5-19d4 and TAR1-5-19d3 have the best ND ₅₀ (about 5 nM) in the cell test, which is consistent with the receptor test results and the TAR1-5-19 monomer ( ND ₅₀ approximately 30 nM). The purified TAR1-5 dimer showed results that varied in the receptor and cell specimens, but the TAR1-5-19 dimer was more consistent. Variation was shown when using different elution buffers during protein purification. Elution using 0.1 M phosphate-citrate buffer (pH 2.6) or 0.2 M glycine (pH 2.5) removed all proteins from protein L in most cases, but functionality was improved. It became lower.

TAR1-5-19d4 was expressed in a fermentor and purified by cation exchange FPLC to produce a completely naive dimer. For TAR1-5d4, FPLC generation yielded three species corresponding to the monomer and two dimer species. The amino acid sequence of this dimer was determined. The TAR1-5-19 monomer and TAR1-5-19d4 were then examined in a receptor test and the IC ₅₀ obtained for the monomer was 30 nM, the IC ₅₀ obtained for the dimer. Was 5 nM.

  FIG. 10 shows the results of a receptor test comparing TAR1-5-19 monomer, TAR1-5-19d4, and TAR1-5d4.

TAR1-5-19 homodimers were made with 3U, 5U and 7U vectors, expressed and purified with protein L. These proteins were examined in cell and receptor tests and the resulting IC50 (for receptor test) and ND ₅₀ (for cell test) were determined (Table 3, FIG. 12).

2.2 Fab
TAR1-5 and TAR1-5-19 dimers were also cloned into the Fab format, expressed and purified with protein L agarose. Fab was evaluated in the receptor test (Table 4). The results showed that for both TAR1-5-19 and TAR1-5 dimers, the neutralization levels were similar to the original Gly ₄ Ser linker dimer from which they were derived. . TAR1-5-19 Fab expressing TAR1-5-19 in both CH and CK was expressed, protein L produced, and evaluated in a receptor test. The resulting IC ₅₀ was about 1 nM.

2.3 TAR1-27 dimer 3x96 clones were picked from round 2 selection encompassing all libraries and selection conditions. A 2 ml supernatant prep was made for analysis in ELISA and biological tests. Antigen ELISA resulted in 71 positive clones. The crude supernatant receptor test produced 42 clones with inhibitory properties (TNF binding 0-60%). In most cases, the inhibitory properties correlated with a strong ELISA signal. 42 clones were sequenced, 39 of these having a second dAb sequence. The 12 dimers that gave the best inhibitory properties were further analyzed.

Twelve neutralizing clones were expressed as 200 ml supernatant prep and purified with protein L. These were evaluated with protein L and antigen ELISA, BIAcore and receptor tests. In all cases, strong positive ELISAs were obtained. BIAcore analysis revealed that all clones had fast on and off rates. The off-rate was improved compared to monomeric TAR1-27, but the off-rate of TAR1-27 dimer (Koff is in the range of about 10 ⁻¹ to 10 ⁻² M) was investigated earlier. Faster than the TAR1-5 dimer (Koff is in the range of 10 ⁻³ to 10 ⁻⁴ M). Since the stability of the purified dimer was questionable, the addition of 5% glycol, 0.5% Triton X100 or 0.5% NP40 (Sigma) was added to the two TAR1-27 to improve stability. It was included in the purification of dimers (d2 and dl6). The addition of NP40 or Triton X100 improved the yield of the purified product by a factor of about 2. Both dimers were evaluated in the receptor test. TAR1-27d2 gave an IC ₅₀ of approximately 30 nM under all purification conditions. TAR1-27d16 showed no neutralizing effect when purified without the use of a stabilizer, but an IC ₅₀ of about 50 nM was obtained when purified under stabilizing conditions. No further analysis was performed.

2.4 TAR1-5 dimer 3 × 96 clones were picked from the second round of selection including all libraries and selection conditions. 2 ml of supernatant was made for analysis. Protein A and antigen ELISA were performed per plate. Thirty interesting clones were identified as having good off-rate by BIAcore (Koff is in the range of 10 ⁻² to 10 ⁻³ M). Clones were sequenced and 13 unique dimers were identified by sequence analysis.

^＊二量体２及び二量体３は、同じ第２のｄＡｂ（ｄＡｂ２と呼ばれる）を有するが、リンカー長が異なる（ｄ２＝（Ｇｌｙ_４Ｓｅｒ）_３、ｄ３＝（Ｇｌｙ_４Ｓｅｒ）_３）ことに留意されたい。ｄＡｂ１は、二量体１、５及び６に対するパートナーｄＡｂである。ｄＡｂ３は、二量体４に対するパートナーｄＡｂである。パートナーｄＡｂのいずれも単独で中和しない。ＦＰＬＣ精製は、特に指定のない限り陽イオン交換によって行われる。ＥＰＬＣによって得られた各二量体に対する最適な二量体化学種がこれらの試験で決定された。 Table 1: TAR1-5 dimer

^* Dimer 2 and Dimer 3 have the same second dAb (referred to as dAb2) but different linker lengths (d2 = (Gly ₄ Ser) ₃ , d3 = (Gly ₄ Ser) ₃ ) Please note that. dAb1 is the partner dAb for dimers 1, 5, and 6. dAb3 is the partner dAb for dimer 4. None of the partner dAbs alone neutralizes. FPLC purification is performed by cation exchange unless otherwise specified. The optimal dimer species for each dimer obtained by EPLC was determined in these tests.

Table 2: TAR1-5-19 dimer

Table 3: TAR1-5-19 homodimer

Table 4: TAR1-5 / TAR1-5-19 Fab

PCR Construction of TAR1-5-19CYS Dimer See Example 8 describing the dAb trimer. Trimers produce a mixture of monomers, dimers and trimers.

Expression and Purification of TAR1-5-19CYS Dimer As outlined in Example 8, the dimer was purified from the culture supernatant by capture on protein L agarose.

Separation of TAR1-5-19CYS monomer from TAR1-5-19CYS dimer Prior to cation exchange separation, using a PD-10 column (Amersham Pharmacia) according to the manufacturer's guidelines, the monomer / The dimer mixed sample was buffer-exchanged into 50 mM sodium acetate buffer (pH 4.0). The sample was then loaded onto a 1 mL Resource S cation exchange column (Amersham Pharmacia) pre-equilibrated with 50 mM sodium acetate (pH 4.0). Monomers and dimers were separated using the following salt gradient in 50 mM sodium acetate (pH 4.0).
150 to 200 mM sodium chloride for 15 column volumes 200 to 450 mM sodium chloride for 10 column volumes 450 to 100 mM sodium chloride for 15 column volumes

  Components containing only the dimer were identified using SDS-PAGE, then pooled and the pH increased to 8 by adding 1/5 volume of 1M Tris (pH 8.0).

In Vitro Functional Binding Test: TNF Receptor Test and Cell Test The affinity of the dimer for human TNFFcz was measured using the TNF receptor and cell test. The IC50 in the receptor test was about 0.3-0.8 nM. The ND50 in the cell test was about 3-8 nM.

Other possible TAR1-5-19CYS dimer formats PEG dimers and custom synthesized maleimide dimers Nektar (Shearwater) is a small linker that separates dAbs, both attached to PEGs ranging in size from 5 to 40 kDa A series of dimaleimide PEGs [mPEG2- (MAL) 2 or mPEG- (MAL) 2] are provided that allow the monomer to be formatted as a dimer. 5 kDa mPEG- (MAL) 2 (ie, [TAR1-5-19] -cis-maleimide-PEGx2 in which maleimides are linked to each other in a dimer) has an affinity from off to 1-3 nM in the TNF receptor test. It was shown to have sex. It is also possible to generate dimers using TMEA (Tris [25 maleimidoethyl] amine) (Pierce Biotechnology) or other bifunctional linkers.

  It is also possible to generate disulfide dimers using chemical coupling procedures using 2,2'-dithiodipyridine (Sigma Aldrich) and reducing monomers.

Addition of polypeptide linker or hinge to the C-terminus of dAb Small linker, ie (Gly ₄ Ser) _n (n = 1 to 10, eg 1, 2, 3, 4, 5, 6 or 7) or immunoglobulin (eg IgG hinge A region or random peptide sequence (eg, selected from a library of random peptide sequences) can be created between the dAb and the terminal cysteine residue. This can then be used to make a dimer as described above.

(Example 8)
dAb trimerization Overview dAb trimerization requires a free cysteine at the C-terminus of the protein. The cysteine residue reduced to give a free thiol can then be used to specifically bind the protein to a trimeric maleimide molecule, such as TMEA (Tris [2 maleimidoethyl] amine).

ＰＣＲ反応（体積５０μＬ）を、２００μＭのｄＮＴＰ、０．４μＭの各プライマー、５μＬの１０×ＰｆｕＴｕｒｂｏ緩衝液（Ｓｔｒａｔａｇｅｎｅ）、１００ｎｇの鋳型プラスミド（ＴＡＲ１−５−１９をコードする）、１μＬのＰｆｕＴｕｒｂｏ酵素（Ｓｔｒａｔａｇｅｎｅ）に設定し、無菌水を使用して体積を５０μＬに調整した。以下のＰＣＲ条件を用いた。：９４℃２分間の初期変性工程、次いで９４℃３０秒間、６４℃３０秒間及び７２℃３０秒間のサイクル。７２℃５分間の最終伸長工程も含めた。ＰＣＲ産物を精製し、ＳａｌＩ及びＢａｍＨＩで消化し、セインレスキクション酵素で切断されたベクターにライゲートした。ＤＮＡシークエンシングによって正確なクローンを検証した。 PCR construction of TAR1-5-19CYS For cloning PCR TAR1-5-19 was specifically linked to SalI and BamHI sites and the following oligonucleotides were used to introduce a C-terminal cysteine residue.

PCR reaction (50 μL volume) was performed using 200 μM dNTP, 0.4 μM of each primer, 5 μL of 10 × Pfu Turbo buffer (Stratagene), 100 ng template plasmid (encoding TAR1-5-19), 1 μL of PfuTurbo enzyme ( The volume was adjusted to 50 μL using sterile water. The following PCR conditions were used. : 94 ° C for 2 minutes initial denaturation step, then 94 ° C for 30 seconds, 64 ° C for 30 seconds and 72 ° C for 30 seconds. A final extension step at 72 ° C. for 5 minutes was also included. The PCR product was purified, digested with SalI and BamHI, and ligated into a vector cut with a cein restriction enzyme. Correct clones were verified by DNA sequencing.

Expression and purification of TAR1-5-19CYS The TAR1-5-19CYS vector was transformed into chemically competent cells BL21 (DE3) pLysS (Novagen) according to the manufacturer's protocol. Cells carrying the dAb plasmid were selected to use 100 μg / mL carbenicillin and 37 μg / mL chloramphenicol. The culture was set up in a 2 L flask with baffle containing 500 mL of culture (Sigma-Aldrich), 100 μg / mL carbenicillium and 37 μg / mL chloramphenicol. The culture was treated at 30 ° C. and 200 rpm with an O.D. D. It was grown to 600 and then induced with 1 mM IPTG (Melford Laboratories Isopropyl-β-D-thiogalactopyranoside). dAb expression was maintained at 30 ° C. for 12-16 hours. It was confirmed that most of the dAb was present in the medium. Therefore, the cells were separated from the culture medium by centrifugation (8000 × g, 30 minutes) and the supernatant was used to purify the dAb. 30 mL of protein L agarose (Afftech) was added per liter of supernatant and the dAb was batch bound with stirring for 2 hours. The resin was allowed to settle by gravity for an additional hour before the supernatant was aspirated. The agarose was then loaded onto an XK50 column (Amersham Pharmacia) and washed with 10 column volumes of PBS. The bound dAb was eluted with 100 mM glycine (pH 2.0) and then the protein-containing components were neutralized by adding 1.5 volumes of 1M Tris (pH 8.0). 20 mg of naive protein containing 50 to 50 ratio of monomer and dimer per liter of culture supernatant was isolated.

Trimerization of TAR1-5-19CYS 2.5 ml of 100 M TAR1-5-19CYS was reduced with 5 mM dithiothreitol and left at room temperature for 20 minutes. The sample was then buffer exchanged using a PD-10 column (Amersham Pharmacia). The column was pre-equilibrated with 5 mM EDTA, 50 mM sodium phosphate (pH 6.5), the sample was loaded and eluted according to the manufacturer's guidelines. Samples were placed on ice until needed. TMEA (Tris [2-maleimidoethyl] amine) was purchased from Pierce Biotechnology. A 20 mM stock solution of TMEA was made with 100% DMSO (dimethyl sulfoxide). It was confirmed that when the concentration of TMEA was greater than 3: 1 (dAb: TMEA molar ratio), rapid precipitation and crosslinking of the protein occurred. Also, the rate of precipitation and crosslinking increased with increasing pH. Therefore, 100 μM reduced TAR1-5-19CYS was used, 25 μM TMEA was added to trimerize the protein, and the reaction was allowed to proceed for 2 hours at room temperature. It was confirmed that the addition of additives such as glycerol or ethylene glycol to 20% (v / v) significantly reduced the trimer precipitation as the coupling reaction proceeded. After coupling, SDS-PAGE analysis showed that monomers, dimers and trimers were present in the solution.

Purification of trimer TAR1-5-19CYS 40 μL of 40% glacial acetic acid per mL of TMEA-TAR1-5-19cys reaction was added to reduce the pH to 4. The sample was then loaded onto a 1 mL Resource S cation exchange column (Amersham Pharmacia) pre-equilibrated with 50 mM sodium acetate (pH 4.0). The dimer and trimer were partially separated using a salt gradient of 340 to 450 mM sodium chloride, 50 mM sodium acetate (pH 4.0) for 30 column volumes. Components containing only trimers were identified using SDS-PAGE, then accumulated and the pH was raised to 8 by adding 1/5 volume of 1 M Tris (pH 8.0). To prevent trimer precipitation during the concentration step (using 5K cutoff Viva spin concentrator (Vivascience)), 10% glycerol was added to the sample.

In Vitro Functional Binding Test: TNF Receptor Test and Cell Test The affinity of the trimer for human TNF-α was measured using the TNF receptor and cell test. The IC50 in the receptor test was 0.3 nM. The ND50 in the cell test ranged from 3 to 10 nM (eg 3 nM).

Other possible TAR1-5-19CYS trimer formats The following reagents can also be used to set TAR1-5-19CYS to a trimer.
PEG Trimers and Custom Synthetic Maleimide Trimers Nektar (Shearwater) provides a series of multi-arm PEGs that can be chemically modified at the end of PEG. Thus, using a PEG trimer with a maleimide functional group at the end of each arm allows the dAb to be trimerized in a manner similar to the trimerization described above using THEA. PEG may also be advantageous in preventing aggregation problems by increasing the solubility of the trimer. Thus, each dAb has a C-terminal cysteine attached to the maleimide functional group, and it is possible to generate a dAb in which the maleimide functional group is attached to the PEG trimer.

Addition of polypeptide linker or hinge to C-terminus of dAb Small linker, ie (Gly ₄ Ser) _n (n = 1 to 10, eg 1, 2, 3, 4, 5, 6 or 7), immunoglobulin (eg IgG Hinge regions) or random peptide sequences (eg, selected from a library of random peptide sequences) can be made between the dAb and the terminal cysteine residue. When used to make multimers (eg dimers or trimers), this also introduces a higher degree of flexibility and greater distance between individual monomers, and targets such as human TNF The binding properties for multi-subunit targets such as -α can be improved.

Example 9
Selection of single domain antibody (dAb) directed against human serum albumin (HSA) and mouse serum albumin (MSA) In this example, a single domain antibody (dAb) directed against serum albumin is selected. A method for manufacturing will be described. The selection of dAbs for both mouse serum albumin (MSA) and human serum albumin (HSA) is described. Each VH (Figure 13: V3-23 / DP47 and array references dummy VH based on JH4b) and _{V K:} single human framework for (FIG. 15 012/02 / DPK9 and array references dummy _{V K} based on JK1) Three human phage display antibody libraries based on work and incorporating the side chain diversity encoded by the NNK codon in the complementarity determining regions (CDR1, CDR2 and CDR3) were used in this experiment.

Library 1 (V _H ):
Position: Diversity at H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98.
Library size: 6.2 × 10 ⁹
Library 2 (V _H ):
Position: Diversity at H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b.
Library size: 4.3 × 10 ⁹
Library 3 (V _K ):
Position: Diversity at L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96.
Library size: 2 × 10 ⁹

The V _H and V _K libraries were preselected for binding to the universal gene ligand proteins A and L so that the majority of clones in the unselected library were functional. The size of the library shown above corresponds to the size after preliminary selection. Two rounds of selection were performed on serum albumin using each of the libraries individually. For each selection, an antigen contained in 4 ml PBS at a concentration of 100 μg / ml was placed in an immunotube (nunc). In the first round of selection, each of the three libraries was individually panned against HSA (Sigma) and MSA (Sigma). In the second round of selection, the phage from each of the six first round selections are (i) panned again against the same antigen (eg, first round MSA, second round MSA) and (ii) reciprocal antigen ( For example, a total of 12 second rounds were selected by panning the first round MSA and the second round HSA). In each case, 48 clones were tested for binding to HSA and MSA after the second round of selection. Soluble dAb fragments were obtained from Harrison et al., Methods Enzymol. , 1996; 267: 83-109, as described for the scFv fragment, using 2% Tween PBS as a blocking buffer and binding dAb to protein L-HRP (Sigma) (for V _K s) and except that it was detected by protein a-HRP (Amersham Phamacia Biotech) ( for _{V H} s), according to standard ELISA protocol (Hoogenboom et al., (1991), nucleic Acids Res , 19:. 4133).

DAbs that gave a background signal indicating binding to MSA, HSA, or both were tested in ELISA soluble form for binding to plastic only, all of which were specific for serum albumin. Subsequent sequencing of the clones (see table below) revealed that 21 unique dAb sequences were identified. The minimum similarity (at the amino acid level) between selected VK dAb clones was 86.25% ((69/80) × 100; the result when all of the diversified residues were different ( For example clones 24 and 34)). The minimum similarity between the selected V _H dAb clones was 94% ((127/136) × 100).

Next, serum albumin dAbs were tested for their ability to incorporate biotinylated antigen from solution. Except that the application of the 1 [mu] g / ml (for _{V K} clones) protein L and 1 [mu] g / ml protein A (for _{V H} clones) to ELISA plate, in accordance with (above) ELISA protocol. Soluble dAbs were taken up from the solution as per protocol and detected with biotinylated MSA or HSA and streptavidin HRP. Biotinylated MSA and HSA were prepared according to the manufacturer's instructions with the aim of achieving an average of two biotins per serum albumin molecule. Twenty four clones were identified that incorporated biotinylated MSA from solution in the ELISA. Two of these (clones 2 and 38 below) were also incorporated biotinylated HSA. The dAb was then tested for its ability to bind MSA applied to a CM5BIAcore chip. Eight clones were identified that bound MSA on BIAcore.

  In all cases, the frameworks were identical to the frameworks in the corresponding dummy sequences as shown in the table above for diversity in the CDRs.

  Of the 8 clones that bound MSA on BIAcore, 2 clones (clone MSA16 and MSA26) that are highly expressed in E. coli were selected for further testing (see Example 10).

  The complete nucleotide and amino acid sequences for MSA 16 and 26 are shown in FIG.

(Example 10)
Affinity and serum half-life measurement of MSA-conjugated dAbs MSA16 and MSA26 in mice dαbs MSA16 and MSA26 are expressed in the periplasm of E. coli, batch adsorbed to protein L-agarose affinity resin (Affitech (Norway)), followed by pH 2 Purified using an elution with 2 glycine. The purified dAb was then analyzed by inhibition BIAcore to determine the _Kd . Briefly, purified MSA16 and MSA26 were tested to determine the concentration of dAb required to achieve a 200 RU response on a BIAcoreCM5 chip coated with high density MSA. Once the required concentration of dAb was determined, a series of concentrations of MSA near the expected K _d were mixed with the dAb and incubated overnight. The dAb MSA coated BIAcore chips in each of these mixtures were then measured at a high flow rate of 30 μl / min. Using the obtained curve to create a Clotz plot, the estimated Kd was 200 nM for MSA16 and 70 nM for MSA26 (FIGS. 17A and 17B).

  Next, clones MSA16 and MSA26 were cloned into an expression vector having an HA tag (nucleic acid sequence: TATCCTTATGATGTTCCTGATTATGCA (SEQ ID NO: 91) and amino acid sequence: YPYDVPDYA (SEQ ID NO: 92)), and an amount of 2-10 mg was expressed in E. coli. Purified from the supernatant with protein L-agarose affinity resin (Affitech (Norway)) and eluted with glycine at pH 2.2. The serum half-life of dAb in mice was determined. MSA2630 and MSA16 were administered to CD1 mice as a single intravenous injection at a concentration of about 1.5 mg / kg.

  Serum levels were analyzed by goat anti-HA (Abcam, UK) capture and protein L-HRP (Invitrogen) detection ELISA blocked with 4% Marvel. Washed with 0.05% Tween PBS. A standard curve of known concentration of dAb was set up in the presence of 1 × mouse serum for comparison with the test sample. Modeling with a two-compartment model, MSA-26 is 0.16 hours t1 / 2α, 14.5 hours t1 / 2β, and 465 hours · mg / ml area under the curve (AUC) (data not shown) MSA-16 was shown to have 0.98 hours t1 / 2α, 36.5 hours t1 / 2β, and 913 hours mg / ml AUC (FIG. 18). Both MSA clones had a much longer half-life compared to HEL4 (anti-hen egg white lysozyme dAb) with t1 / 2α of 0.06 hours and t1 / 2β of 0.34 hours.

(Example 11)
Production of V _H -V _H and V _K -V _K Bispecific Fab-like Fragments This example describes a method for producing V _H and V _K bispecifics as Fab-like fragments. Prior to constructing each of the described Fab-like fragments, dAbs that bind to the target of interest were first selected from a dAb library similar to the library described in Example 9. A V _H dAb, HEL4, that binds to chicken egg lysozyme (Sigma) was isolated, and a second V _H dAb (TAR2h-5) that binds to the TNF-α receptor (R and D systems) was also isolated. These sequences are shown in the sequence listing. A V _K dAb that binds TNF-α (TAR1-5-19) was isolated by selection and affinity maturation, the sequence of which is also shown in the sequence listing. The second V _K dAb (MSA26) described in Example 9 whose sequence is shown in FIG. 17B was also used in these experiments.

DNA from the expression vector containing the above four dAbs was digested with the enzymes SalI and NotI to excise the DNA encoding for the dAb. The digest was run on an agarose gel and the band was excised, followed by gel purification using a Qiagen gel purification kit (Qiagen (UK)) to purify the band of the expected size (300-400 bp). . The DNA encoding dAb was then inserted into the CH or CK vector (FIGS. 8 and 9) shown in the following table.

  VHCH and VHCκ constructs were cotransformed into HB2151 cells. Separately, VκCH and VκCκ constructs were cotransformed into HB2151 cells. Each culture of the co-transformed cell line was grown overnight (5% glucose, 10 μg / ml chloramphenicol, and 100 μg / ml ampicillin to maintain antibiotic selection for both CH and Cκ plasmids. Containing 2 × TY). Overnight cultures are used to inoculate fresh medium (2 × TY, 10 μg / ml chloramphenicol and 100 μg / ml ampicillin) before expressing their CH and Cκ constructs by addition of IPTG. To OD 0.7-0.9.

  The expressed Fab-like fragment was then purified from the periplasm by protein A purification (for co-transformed VHCH and VHCκ) and MSA affinity resin purification (for co-transformed VκCH and VκCκ).

By treating the V _H -V _H dual specific gel on protein were assayed for the expression of the VHCH and VHCκ bispecific. The gel was blotted and the expected size band for the Fab fragment could be detected by Western blot, indicating that both the VHCH and VHCκ portions of the Fab-like fragment were present. Next, to determine if two halves of bispecificity are present in the same Fab-like fragment, egg lysozyme (HEL) included in sodium bicarbonate buffer at a concentration of 3 mg / ml is used. 100 μl per well ELISA plate was applied and placed at 4 ° C. overnight. The plate was then blocked with 2% Tween PBS (as described in Example 1) and then incubated with the VHCH / VHCκ bispecific Fab-like fragment. Detection of binding to bispecific HEL was performed via non-cognate chains using 9el0 (monoclonal antibody that binds myc tag, Roche) and anti-mouse IgG-HRP (Amersham Pharmacia Biotech). The signal for the VHCH / VHCκ bispecific Fab-like fragment was 0.154 compared to the 0.069 background signal for VHCκ expressed alone. This demonstrates that the Fab-like fragment has binding specificity for the target antigen.

V _K -V _K Bispecificity After purifying co-transformed V _K CH and V _K C _K bispecific Fab-like fragments with MSA affinity resin, the resulting protein was used to obtain 1 μg / The ELISA plate coated with ml TNF-α and the ELISA plate coated with 10 μg / ml MSA were probed. As expected, there was a signal above the background when detected with protein L-HRP on both ELISA plates (data not shown). This indicates that the component of the protein that can bind to MSA (and thus purified on an MSA affinity column) can also bind to TNF-α in a subsequent ELISA, and the antibody antibody bispecificity. Was confirmed. This protein component was then used in the subsequent two experiments. First, an ELISA plate coated with 1 μg / ml TNF-α was probed with bispecific V _K CH and V _K C _K Fab-like fragments and at a concentration calculated to give a similar signal in ELISA Probed with a control TNF-α conjugated dAb. ELISA plates were probed in the presence and absence of 2 mg / ml MSA using both bispecific and control dAbs. The signal in the bispecific well was reduced by more than 50%, but the signal in the dAb well was not reduced at all (see FIG. 19a). In addition, the same protein was included in the receptor test with MSA and the receptor test without MSA, and the competition with MSA was also shown (see FIG. 19c). This demonstrates that binding to MSA bispecificity competes with binding to TNF-α.

(Example 12)
Generation of V _K -V _K Bispecific Cys Binding Bispecificity with Specificity for Mouse Serum Albumin and TNFα In this example, both mouse serum albumin and TNF-α are bound by chemical coupling via disulfide bonds. A method for producing a specific bispecific antibody fragment will be described. Both MSA16 (according to Example 1) and TAR1-5-19dAb were cloned again into a pET-based vector with a C-terminal cysteine and no tag. Two dAbs were expressed at the 4-10 mg level and purified from the supernatant using protein L agarose affinity resin (Affitech (Norway)). The cysteine tag dAb was then reduced with dithiothreitol. TAR1-5-19dAb was then coupled with dithiodipyridine to prevent disulfide bond re-formation resulting in the formation of PEP1-5-19 homodimers. Two different dAbs were then mixed at pH 6.5 to promote disulfide bond formation and formation of TAR1-5-19, MSA16 cis-linked heterodimers. This method for producing a complex of two dissimilar proteins was originally described by King et al. (King TP, Li Kochoumian Biochemistry. 1978, 17: 1499-506, “Protein complexes by intermolecular disulfide bond formation. (Preparation of protein conjugates via intermolecular disbonded bond formation). Heterodimers were separated from monomeric species by cation exchange. Separation was confirmed by the presence of a band of the expected size on the SDS gel. The heterodimer species obtained was tested in the TNF receptor test and was confirmed to have an IC50 for neutralizing about 18 nM TNF. The receptor test was then repeated using a constant concentration of heterodimer (18 nM) and a series of dilutions of MSA and HSA. The presence of a range of concentrations (up to 2 mg / ml) of HSA did not cause a decrease in the dimer's ability to suppress TNF-α.

  However, the addition of MSA decreased the ability of the dimer to suppress TNF-α depending on the dose (FIG. 20). This demonstrates that MSA and TNF-α compete for binding to the cis-bound TAR1-19, MSA16 dimer.

Data Summary An appendix 4 provides a summary of data obtained in the experiments described in the previous examples.

(Example 13)
Summary of Nucleic Acid and Polypeptide Sequences for Anti-TNF-α dAbs Throughout the course of testing for anti-TNF-α dAbs described herein, several different dAbs that bind human and / or mouse TNF-α are identified. Identified. The sequence and further information are given below.

Clone binding mouse TNF-α The nucleotide and amino acid sequences for the four anti-mouse TNF-α dAbs are shown below. Two of these (TAR1-2m-9 and TAR1-2m-30) inhibit the activity of mouse TNF-α, while two (TAR1-2m-1 and TAR1-2m-2) bind. , Do not suppress.

TAR1-2m-9
TAR-2m-9 is a Vk clone with an IC50 of 6 μM and an ND50 of 5 μM. IC50 and ND50 are not improved by protein L cross-linking. This clone has no effect on human TNF-α (evaluated for species cross-reactivity in two concentrations of cell test), but has similar neutralizing activity on rat TNF-α.
Amino acid sequence (CDR3 is shown in bold) (SEQ ID NO: 93):

TAR1-2m-30:
TAR1-2m-30 is a Vk clone with an ND50 of 10 μM. ND50 is not improved by protein L cross-linking. This clone has no effect on human TNF-α (species cross-reactivity was assessed in two concentrations of cell test) and is slightly less effective on rat TNF when compared to mice.
Amino acid sequence (CDR3 is shown in bold) (SEQ ID NO: 95):

TAR1-2m-1:
This clone binds mouse TNF-α but does not suppress receptor binding activity.
Amino acid sequence (SEQ ID NO: 97):

TAR1-2m-2:
This clone binds mouse TNF-α but does not suppress receptor binding activity.
Amino acid sequence (SEQ ID NO: 99):

DAb clones that bind human TNF-α A list of nucleotide sequences of dAbs identified for binding of human TNF-α is shown below. The corresponding amino acid sequence is shown in FIG.

Additional anti-human TNF-αdAb clones include the following:
Several clones were affinity matured. Clone TAR1-100-47 is an affinity matured clone with an ND50 of 30-50 nM in the L929 cell test and an ND50 of 3-5 nM when crosslinked with protein L. TAR1-100-47 cross-reacts with rhesus monkey TNF. Its amino acid sequence and the amino acid sequences of some other clones are shown below. TAR1-2-100 and TAR1-2109 are parental clones used for library construction. Good TAR1 clones in this group have the following consensus sequences:
D / E30, W32, R94 and F96 shown in bold in TAR1-100-47.

The sequences of TAR1-5-19 anti-human TNF-αdAb configured for various formats in these examples are as follows:

(Example 14)
Effect test of PEGylated TAR1-5-19 in arthritis prevention model Tg197 mice are transgenic mice against human TNF-globin hybrid gene, and heterozygotes at 4-7 weeks of age are common tissues with rheumatoid arthritis Develop chronic multiple progressive arthritis with clinical features [Keffer, J. et al. , Probet, L .; Cazlaris, H .; Georgopoulos, S .; Kaslaris, E .; Kiussis, D .; Kollias, G .; (1991), “Transgenic mice expressing human tumor necrosis factor: Transgenic mice expressing factor: a predictive genetic model of art. J”. 10: 4025-4031].

  PEGylated dAb (2x20k branched PEG format with two sites for attachment of dAb [ie 40K mPEG2 MAL2] and dAb is TAR1-5-19 cis) in prevention of arthritis in Tg197 model In order to test the effect of, heterozygous transgenic mice were divided into groups of 10 males and females. Treatment started at 3 weeks of age and test items were injected intraperitoneally weekly. An overview of TAR1-5-19 monomer expression and PEGylation is described in Section 1.3.3, Example 1. All protein preparations were included in phosphate buffered saline and tested for acceptable levels of endotoxin.

  The test was performed blind. Animals are weighed weekly, 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe Macroscopic phenotypic signs of arthritis were scored according to the system of arthritis (significant movement disorder).

  The results of the study clearly demonstrated that 10 mg / kg PEGylated TAR1-5-19 inhibited the development of arthritis and that there was a significant difference in the arthritis score between saline control and treated groups. The 1 mg / kg dose of PEGylated TAR1-5-19 also resulted in a statistically significantly lower intermediate arthritis score compared to the saline control group (p <0. 0 using the standard approximation for the Wilcoxon test). 05%).

(Example 15)
Efficacy test of PEGylated TAR1-5-19 in arthritis treatment model To test the effect of PEGylated dAb in arthritis treatment model in Tg197 mouse model, heterozygous transgenic mice were equated with 10 males and females Divided into groups. Treatment began at 6 weeks of age when the mice had a significant arthritic phenotype. The treatment of 4.6 mg / kg intraperitoneal injection of the test item was performed twice weekly. Sample preparation and disease scoring are as described in Example 14.

  Arthritis scoring clearly demonstrated that PEGylated TAR1-5-19 inhibited the progression of arthritis in the treatment model. The 4.6 mg / kg dose of PEGylated TAR1-5-19 resulted in a statistically significantly lower intermediate arthritis score compared to the saline control group at 9 weeks (standard approximation to the Wilcoxon test). When used, p <0.01%).

(Example 16)
DAb effect in slow release format To test the effect of dAb in slow release format, dAbs with small PEG molecules (PEG is 4x5k with 4 sites for attachment of dAbs with C-terminal cis residues, dAb was TAR1-5-19 [ie 20K PEG 4 arm MAL] was loaded into a 0.2 ml peristaltic pump. The peristaltic pump had a release rate of 0.2 ml in 4 weeks and was implanted subcutaneously into mice at 6 weeks in the above-described therapeutic Tg197 model. The arthritic score for these animals increased at a clearly slower rate compared to animals implanted with saline-filled pumps. This demonstrates that the dAb is effective when delivered in a slow release format.

(Example 17)
Half-life stabilized anti-human TNF-α dAb prevents the development of RA in the Tg197 mouse model. The dAb monomer TAR1-5-19 described herein contains passively applied TNF-α. Affinity matured dAb monomer derived from a dAb initially selected for use. Early clones have an ND50 in the L929TNF cytotoxic neutralization test greater than 5 μM. When set up as an Fc fusion as described herein, the TAR1-5-19 clone has an ND50 in the L929 test of less than 5 nM.

  After injection into mice, the serum half-life of the TAR1-5-19dAb Fc fusion was examined. The results are shown in FIG. If the TAR1-5-19 dAb monomer had a t1 / 2β of about 20 minutes, the same Fc fusion version of the dAb had a t1 / 2β greater than 24 hours and increased serum half-life Was more than 70 times.

  The TAR1-5-19dAb Fc fusion construct was tested in the Tg197 mouse model of RA described above. The mice were divided into 5 groups, each group containing 10 females and males. Treatment with twice weekly IP injections of TAR1-5-19dAb Fc fusion, ENBREL or saline was started at 3 weeks of age when RA symptoms have not yet been demonstrated. The study was conducted over 7 weeks. As shown in FIG. 25, two doses were administered: 1 mg / kg and 10 mg / kg of TAR1-5-19 dAb Fc fusion. Negative control animals received a negative control anti-β-gal Fc fusion twice weekly at a dose of 10 mg / kg, and one group was treated twice weekly with saline injection. For comparison, one group received 10 mg / kg ENBREL twice weekly.

  Animals were evaluated for arthritis scores as described herein in a blind manner. At the end of the 7-week treatment course, animals that received a 10 mg / kg dose of TAR1-5-19dAb Fc fusion twice weekly had a lower arthritic score than animals that received ENBREL at a dose of 10 mg / kg. Arthritis disease was substantially completely prevented compared to untreated animals or animals given a negative control dAb Fc fusion.

  TNF-α is associated with cachexia. Animals were weighed throughout the course of anti-TNF-αdAb treatment. The body weight of the animals given the TAR1-5-19 dAb Fc fusion was significantly larger than the animals given the negative control dAb Fc fusion and the animals not receiving treatment, and the body weight of the animals receiving the ENBREL injection It was similar.

  In summary, 10 mg / kg TAR1-5-19 completely prevented the development of arthritis in the Tg197 model. This response was dose dependent, with some effects attributed to the 1 mg / kg dose, and the response was superior to the response observed with a similar dose of the existing anti-TNF-α acting ENBREL This study demonstrates the effect of dAbs as therapeutics in clinically acceptable models of human disease.

  Histopathological analysis of the fixed part of the animal's joint is consistent with these data (not shown).

(Example 18)
In vivo testing for different extended half-life formats Three different extended half-life anti-TNF-αdAb formats were investigated for their effect on arthritis scores. These formats consist of anti-TNF-αdAb Fc fusions (two anti-human TNF-αdAbs homodimerized by fusion to the human IgG C _H2 / C _H 3 region), two different PEG-conjugated anti-TNF- αdAb constructs (homodimers formed by cis maleimide linkage of 2 identical dAbs to 2 × 20K branched PEG and homo tetramers formed by cis maleimide linkage to 4 identical dAbs to 4 × 10K branched PEG ), As well as two identical anti-TNF-α dAbs followed by an anti-mouse serum albumin dAb, a bispecific anti-TNF-α / anti-SA dAb.

  In individual studies, starting at 3 weeks of age and continuously over 7 weeks, the pharmaceutical composition is administered weekly at doses of 10 mg / kg or 1 mg / kg as shown in FIG. However, it was administered twice a week.

  PEGylated anti-TNF dAb homodimer was effective at 10 mg / kg in the weekly infusion protocol for complete prevention of arthritis based on arthritis scores. The current anti-TNF-α drug used for comparison had a lower arthritis score compared to untreated animals but was statistically significantly higher than the score achieved with the PEGylated dAb construct. Anti-TNF-α / anti-SA bispecific and Fc fusion showed an effect compared to untreated.

  In the regimen of weekly infusions of 1 mg / kg, none of the treatments were 100% effective in preventing the onset of the disease, but PEGylated anti-TNF-αdAb constructs were treated with untreated and current anti-TNF-α drugs. In comparison, the effect of preventing the progression of disease symptoms was high. In this dosing regimen, anti-TNF-αdAb Fc fusions and bispecific constructs were also more effective than current drugs.

  In summary, weekly dosing therapy trials using three different forms of half-life extended dAbs further validate the effects of treatment in clinically acceptable models of human disease.

(Example 19)
Effect of anti-human TNF-αdAb in the Tg197 mouse RA model compared to existing anti-TNF-α therapeutics for established disease In this study, various forms and dosage regimens of anti-TNF-αdAb constructs for established disease were tested. The effect was compared to the same molar dose of the current anti-TNF-α therapeutics ENBREL, HUMIRA and REMICADE in the Tg197RA model. The administration of the therapeutic agent to the animals was started not from 3 weeks but from 6 weeks when the symptoms of joint inflammation had already been shown. Symptoms were monitored histologically (9 weeks) and blinded arthritis scores (weekly).

Various formats and dosages for twice weekly administration are shown in FIG. The format is Fc fusion (2 replicas of TAR1-5-19dAb dimerized by fusion to human IgG1 C _H2 / C _H3 region), TAR1-5-19 dAb PEG dimer (2 × 20K branch) Homodimer formed by cis-maleimide linkage of two identical dAbs to branched PEG), TAR1-5-19 dAb PEG tetramer (cis-maleimide linkage of four identical dAbs to 4 × 20K branched PEG) And a TAR1-5-19dAb / anti-mouse SA bispecific (two identical anti-TNF-αdAbs followed by a linear fusion of anti-mouse serum albumin dAbs). . Dosing therapy is shown schematically in FIG. Evaluation of continuous administration of 4x5k PEGylated TAR1-5-19 construct via transplanted peristaltic pump was also performed.

  The results of the study showed that none of the current biologics significantly reversed the arthritis score at 9 weeks. Both TAR formats stabilized the arthritis score more or less when compared to saline controls, which was statistically significant. Furthermore, there were signs of disease reversal when compared to the 6 week score.

  In addition, the arthritic joint at 9 weeks was examined for histopathological disease state, and the disease severity after treatment in the TAR format was reduced when compared to the 6 week joint. This confirms that the TAR format can induce reversal of the arthritic phenotype of the established disease.

  These studies demonstrate the established arthritic disease effects of the tested anti-TNF-αdAb constructs, including the ability of TNFα-dAbs to at least partially reverse the disease process.

(Example 20)
Effect of anti-TNF dAb as a fusion with antiserum albumin dAb Test of the effect of TAR1-5-19 / antiserum albumin dAb fusion in an arthritis prevention model Tg197 mice are transgenic mice against the human TNF-globin hybrid gene And heterozygotes at 4-7 weeks of age develop chronic multiple progressive arthritis with histological features in common with rheumatoid arthritis [Keffer, J. et al. , Probet, L .; Cazlaris, H .; Georgopoulos, S .; Kaslaris, E .; Kiussis, D .; Kollias, G .; (1991), “Transgenic mice expressing human tumor necrosis factor: Transgenic mice expressing factor: a predictive genetic model of art. J”. 10: 4025-4031].

  To test TAR1-5-19 / anti-serum albumin dAb fusions (TAR1-5-19, TAR1-5-19 and in-line trimer of anti-mouse serum albumin dAb 3dAb) in the prevention of arthritis in the Tg197 model The heterozygous transgenic mice were divided into groups of 10 males and females. Treatment started at 3 weeks of age and test items were injected intraperitoneally weekly. The TAR1-5-19 / antiserum albumin dAb fusion was expressed in E. coli with a C-terminal hexahistidine tag and purified by Ni affinity chromatography, IEX and gel filtration. All protein preparations were included in phosphate buffered saline and tested for acceptable levels of endotoxin.

  The test was performed blind. Animals are weighed weekly, 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe Macroscopic phenotypic signs of arthritis were scored according to the system of arthritis (significant movement disorder).

  The results of the study clearly demonstrate that 10 mg / kg TAR1-5-19 / antiserum albumin dAb fusion suppresses the development of arthritis and that there is a significant difference in the arthritis score between saline control and treated groups. It was. The 1 mg / kg dose of TAR1-5-19 / antiserum albumin dAb fusion also resulted in a statistically significantly lower intermediate arthritis score compared to the saline control group (when using standard approximations to the Wilcoxon test) P <2%).

B. Efficacy test of TAR1-5-19 / anti-serum albumin dAb fusion in therapeutic model of arthritis To test the effect of TAR1-5-19 / anti-serum albumin dAb fusion in therapeutic model of arthritis in Tg197 model The zygotic transgenic mice were divided into groups of 10 males and females. Treatment began at 6 weeks of age when the animals had a significant arthritic phenotype. Treatment of 2.7 mg / kg intraperitoneal injection of test item was performed twice weekly. Sample preparation and disease scoring are as described above.

  Arthritis scoring clearly demonstrated that the TAR1-5-19 / antiserum albumin dAb fusion inhibits the progression of arthritis in the treatment model. The TAR1-5-19 / antiserum albumin dAb fusion at the 2.7 mg / kg dose resulted in a statistically significantly lower intermediate arthritis score at 9 weeks compared to the saline control group (standard for Wilcoxon test). P <0.05% when using approximate values).

  This clearly shows that anti-TNF dAbs can be effective in the form with anti-SA dAbs and that anti-SA dAbs have extended the half-life of anti-TNF dAbs over the expected half-life for anti-TNF dAb alone. To prove.

(Example 21)
Investigation of the effects of anti-TNF-αdAbs described herein on arthritis and histopathological scores in the Tg197 mouse model of RA. Effects of anti-TNF-αdAbs on arthritis and histopathological scores in the Tg197 model of RA. Two further tests were conducted to investigate.

  In the first study, administration of the TAR1-5-19dAb Fc fusion described above was started twice weekly at a dose of 10 mg / kg from 3 weeks before the onset of RA symptoms. Results were judged in comparison to saline, ENBREL and control Fc fusion dAb infusions with the same schedule.

  The TAR1-5-19dAb Fc fusion was more effective than ENBREL in preventing the development of RA symptoms as judged by the arthritis score and from analysis of the tissue slides.

  In a second study, the effect of weekly administration of 10 or 1 mg / kg anti-TNF-αdAb Fc fusion, PEG dimer and bispecific anti-TNF / anti-SA starting at 3 weeks of age is examined . Compare with ENBREL and HUMIRA.

  Arthritis scores for all TAR formats given at a dose of 1 mg / kg or 10 mg / kg were reduced compared to saline controls. In addition, there was evidence that the onset of the disease was delayed. PEGylated and anti-SA bispecific formats were more effective in reducing the severity of arthritis compared to HUMIRA and ENBREL. In addition, analysis of joint tissue at 10 weeks showed that the TAR format was more effective and reduced disease severity compared to saline controls.

  In summary, the TAR1-5-19 anti-TNF-dAb, PEGylation and anti-SA bispecific format in Fc fusions, whether administered before or after the onset of joint inflammation symptoms, is a RA in the Tg197 model. All effective against symptoms. The most effective anti-TNF dAb format is equivalent to or more effective than HUMIRA, and the most effective anti-TNF dAb format is significantly more effective than ENBREL in all tests.

(Example 22)
Anti-human VEGF dAb
TAR15 (anti-human VEGF)
VK dAbs that bind human VEGF are described below. RBA refers to the receptor 2 binding test described herein.

The TAR15-1 clone has a Kd of 50-80 nM when tested at various concentrations on a low density BIAcore chip. Another VK clone was contacted to the low density chip at one concentration (50 nM). Different clones show different kinetic profiles.
Amino acid sequence:
Common sequence: W28, G30, E32, S34, H50 and Y93.

Additional TAR15 anti-human VEGF dAb clones have consensus sequences: W28, G30, E32, S34, H50 and Y93 as shown in TAR15-10 below.

VH dAbs that bind human VEGF are described below. These clones result in a reduction (over 50%) of the supernatant RBA (R2).

VH clones were contacted with a BIAcore low density VEGF chip at one concentration (50 nM). Different clones give different kinetic profiles.
Amino acid sequence:

(Example 23)
Further Testing with Anti-VEGF dAbs Anti-VEGF dAbs described herein can be used in combination with anti-TNF-, including, for example, Fc fusions, Fabs, PEGylated forms, dimers, tetramers and anti-SA bispecific forms. It is possible to test for effects in the various formats described above for αdAbs. Anti-VEGF dAbs can be evaluated not only with the anti-TNF-α dAbs described herein, but with other anti-TNF-α preparations such as HUMIRA, ENBREL and / or REMICADE.

  For example, further studies can be performed to examine the effects of photo-VEGF dAbs on arthritis and histopathological scores in the Tg197 model of RA.

  For example, the TAR1-5-19Fc fusion described above at 1 mg / kg or 10 mg / kg once or twice weekly from 3 weeks of age (before the onset of RA symptoms) or 6 weeks of age (after the onset of symptoms) Intraperitoneal administration of a TAR15dAb Fc fusion similar to was initiated and continued for over 7 weeks. The results are preferably judged in comparison with equal molar amounts of saline, control Fc fusion (anti-β-gal), TAR1-5-19 alone, ENBREL, REMICADE and / or HUMIRA.

Animals are scored for the macro phenotypic indicators (eg, arthritis score) and histopathological scores described above. The effect is
i) Do not cause disease symptoms (as evidenced by arthritis or histopathological scores) when starting administration to animals from 3 weeks of age,
ii) When administration is started from 3 weeks of age, the severity of the disease symptoms appearing is lower than that of control animals,
iii) When administration is started from 6 weeks of age, it does not progress to a more severe disease or the progression rate is low compared to control animals,
iv) Symptom reversal (again, based on arthritis score or histopathological score) at 7, 8, 9, 10, 11, 12 or 14 weeks when administration to animals is started at 6 weeks of age To be demonstrated by any of them.

  Similar tests can be performed with each of the different formats described above, eg, Fab, PEGylated forms, dimers, tetramers and anti-SA bispecific forms.

  An anti-VEGF dAb, such as a TAR15 dAb, can also be administered to the Tg197 mouse model in combination with HUMIRA, ENBREL and / or REMICADE. The test is performed in the same manner as described above for the test of VEGF dAb alone, and the effect is measured in the same manner.

(Example 24)
Evaluation of anti-TNF-αdAb in Crohn's disease model To evaluate the effect of anti-TNF-αdAb (and / or anti-VEGF dAb) in Crohn's disease, it was first described in Kontoiannis et al., 1999, Immunity, 10: 387-398. A Tnf ^ΔARE transgenic mouse model of Crohn's disease is used. These animals develop an IBD phenotype with similarity to Crohn's disease that begins between 4 and 8 weeks of age. Thus, anti-TNF-a dAbs such as various forms of TAR1-5-19 (Fc fusion, Fab, PEGylation (dimer, tetramer pair etc.), bispecificity by VEGF, bispecificity by anti-SA Gender etc.) at 3 weeks of age (to test disease prevention) or 6 weeks of age (to test stabilization, prevention of progression or reversal of disease symptoms) and are described herein. Animals are scored according to body weight and histologically. In the initial test, intraperitoneal administration of 1 mg / kg and 10 mg / kg is used and adjusted according to the results of these initial tests. The test composition can be administered once or twice weekly or continuously using, for example, a peristaltic pump. Alternatively, it is also possible to apply an orally administered preparation by, for example, a gavage using zantac or an enteric preparation. The test will continue for 7 weeks or more after it has been started.

The effect in the Tnf ^ΔARE model of Crohn's disease is
i) Do not cause disease symptoms when administration to animals is started from 3 weeks of age,
ii) When administration is started from 3 weeks of age, the severity of the disease symptoms appearing is lower than that of control animals,
iii) When administration is started from 6 weeks of age, it does not progress to a more severe disease or the progression rate is low compared to control animals,
iv) indicated by any reversal of symptoms at any of 7, 8, 9, 10, 11, 12, or 14 weeks when administration to animals is initiated at 6 weeks of age.

  In particular, treatment is considered effective if the average histopathological disease score in the treated animals is lower (by a statistically significant amount) than the vehicle control group. Mean histopathological score is at least 0.5 units, at least 1.0 units, at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3 compared to the vehicle-only control group A process is also considered valid if it is 0.0 units, or at least 3.5 units lower. Alternatively, the treatment is effective when the average histopathological score is maintained from 0 to 0.5 or less throughout the course of treatment.

  For the RA model, the effect of combination therapy with dAbs specific for VEGF or other anti-TNF-a compositions (eg, ENBREL, REMICADE and / or HUMIRA) is also evaluated in this model.

(Example 25)
Bispecific IgG directed against human TNF-α and human VEGF
In the modified IgG-like bispecific format described below, two different specific dAbs are fused to the heavy and light chain constant regions, respectively. When coexpressed in a cell, two variable regions capable of binding to two therapeutic targets (eg, a target specific for TNF-α and a target specific for VEGF) are A bi-arm IgG-like molecule present in each arm is generated.

  DNA construct. The mammalian expression vector used was based on Invitrogen pcDNA3.1 backbone, which promotes gene expression in mammalian cells via the CMV immediate early promoter. For heavy chain expression, a cassette consisting of a human CD33 signal peptide and a human IgG1 heavy chain constant region is inserted into the NheI and XbaI restriction sites of the vector pcDNA3.1 (+) and is used in VEGF using HindIII and NotI restriction sites. A variable region specific for and expressed as part of the heavy chain polypeptide was cloned into this cassette between the CD33 signal peptide and the IgG1 heavy chain constant region. For light chain expression, a cassette consisting of the CD33 signal peptide and the human C kappa constant region is inserted into the NheI and XhoI restriction sites of the vector pcDNA3.1zeo (+) and used to bind TNF-α using HindIII and NotI restriction sites. A variable region that was specific for and expressed as part of the light chain polypeptide was cloned into this cassette between the CD33 signal peptide and the Ckappa constant region.

  Protein expression and purification. Heavy chain and light chain expression vector DNA was prepared using Qiagen EndoFree plasmid mega kit according to manufacturer's instructions, HEK293 (obtained from European cell culture collection) or Cos-7 cells (obtained from American Type Culture Collection) Roche transfection reagent Fugene 6 was used to transfect according to the manufacturer's instructions. After 5 days, the culture supernatant was harvested by centrifugation and the screened bispecific antibody was purified using two-step affinity purification. First, the culture supernatant was supplemented with phosphate buffered saline (PBS) to a final concentration of 1.5 × PBS, and the antibody was incorporated onto Amersham streamline protein A resin. The resin was washed with 2 × PBS followed by 10 mM Tris (pH 8) and the bound antibody was eluted using 0.1 M glycine (pH 2). The eluate was neutralized by adding 25% volume of M Tris (pH 8) and the recombinant antibody was loaded onto Affitech protein L agarose resin. The resin was washed again with 2 × PBS, then with 10 mM Tris (pH 8), and 0.1 M glycine (pH 2) was used to elute the bound recombinant antibody and 25% volume of 1 M Tris. The eluate was neutralized by adding (pH 8).

  Analysis of recombinant antibodies. Using the absorbance reading at 280 nm, the purified recombinant antibody was quantitated spectrophotometrically and analyzed by SDS-PAGE using Invitrogen NuPAGE 4-12% Bis-Tris gel and SilverQuest staining according to the manufacturer's instructions. . FIG. 29 shows a kappa variable region specific for human VEGF fused to a human IgG1 heavy chain constant region and a kappa variable region specific for human TNF-α fused to a human C kappa constant region. It is a figure which shows the SDS-PAGE analysis of the bispecific antibody containing this. Lane 1 is filled with Invitrogen multi-mark molecular weight marker, Lane 2 is filled with bispecific antibody contained in 1 × Invitrogen NuPAGE LDS sample buffer, and Lane 3 is filled with 1 × Invitrogen NuPAGE supplemented with 10 mM β-mercaptoethanol. The bispecific antibody included in the LDS sample buffer was loaded. In lane 3, the heavy chain is seen as a 50 kDa band and the light chain is seen as a 25 kDa band.

  Dual specificity test. The bispecificity of the expressed antibody was demonstrated by measuring the drag of each purified antibody batch in both the human TNF cell test and the human VEGF receptor binding test.

  The human TNF cell-based test used was the L929 cytotoxicity test described in Evans (2000, Molecular Biotechnology, 15, 243-248). Briefly, L929 cells loaded in microtiter plates were incubated overnight with a bispecific antibody, 100 pg / ml TNF and 1 mg / ml actinomycin D (Sigma, Poole, UK). [After incubation with 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfonyl) -2H-tetrazolium (Promega, Madison, USA) Cell viability was measured by reading the absorbance at 490 nm. Anti-TNF activity resulted in a decrease in TNF cytotoxicity, resulting in higher absorbance compared to the TNF-only control.

Basically, VEGF activity was measured using VEGFR2, as described in the section titled “Preparation of immunoglobulin-based multispecific ligands”. Briefly, a 96-well Nunc Maxisorp test plate was loaded with recombinant human VEGF R2 / Fc (R & D Systems, Cat. No: 357-KD-050) contained in carbonate buffer at a concentration of 0.5 μg / ml. It was applied over the night. The wells were washed repeatedly with 0.05% Tween / PBS and then PBS. The plate was blocked by adding 2% BSA contained in PBS. Wells were washed (as described above) and then purified bispecific antibody was added to each well. VEGF, which was included in the dilution at a concentration of 6 ng / ml (final concentration 3 ng / ml), was then added to each well and the plate was incubated at room temperature for 2 hours. Plates were washed as above, then biotinylated anti-VEGF antibody (R & D Systems, Cat No: BAF293) included in the dilution at a concentration of 0.5 μg / ml was added and incubated for 2 hours at room temperature. After washing the wells as described above, HRP-conjugated anti-biotin antibody (diluted 1: 5000 with diluent; Stratech, Cat No: 200-032-096) was added. The plate was then incubated for 1 hour at room temperature. The plate was washed as above to remove any traces of Tween 20. For detection, 100 μl of SureBlue 1-component TMB microwell peroxidase solution was added to each well. After stopping the reaction by adding 1 M hydrochloric acid, the OD ₄₅₀ was read using a plate reader.

  FIG. 30 shows a kappa variable region specific for human VEGF fused to a human IgG1 heavy chain constant region and a kappa variable region specific for human TNF-α fused to a human C kappa constant region. It is a figure which shows the result with respect to the bispecific antibody containing this. Bispecific antibodies (representing anti-TNF-αx anti-VEGF) bound both human TNF-α and human VEGF. The antibody is bivalent against both targets. The ND50 for TNF-α is significantly lower (24 nM) than the ND50 for anti-TNF-α monomer (200 nM) fused to C kappa in the bispecific molecule as a variable region. The EC50 for VEGF is much lower (75 pM) than that for anti-VEGF monomer (12 nM, not shown) fused to the heavy chain constant region in the bispecific molecule as a variable region and is oligomerized by protein L cross-linking. Lower than the EC50 for the anti-VEGF monomer (straight line with squared data points, 990 pM).

Constructs of the present embodiment, two copies of the V _H or V _L single domain antibody binds two copies of the V _H or V _L single domain antibody that binds a first epitope and a second epitope Is a tetravalent bispecific antigen-binding polypeptide construct. Each of the two copies of the single region antibody that binds the first epitope is fused to an IgG heavy chain constant region, and each of the two copies of the single region antibody that binds the second epitope is light Fused to the chain constant region.

Those skilled in the art will be able to use, for example, other anti-TNF-α and anti-VEGF antibody sequences, eg, any of the sequences described herein, to construct additional two similar to the constructs described in this example. Bispecific tetravalent polypeptide constructs can be generated. In other embodiments, _Cκ or _Cλ light chain constant regions can be used, and IgG heavy chain constant regions other than IgG1 can also be used. Of particular interest for use in the development of this type of construct is a single-region anti-TNF that prevents an increase in arthritic score when administered as a dAb monomer to a Tg197 transgenic mouse model of arthritis. -Α antibody clone, as well as a single domain anti-VEGF antibody clone that prevents an increase in arthritis score when administered to mice in the collagen-induced arthritis mouse model as a dAb monomer. Also, the monomer of the single region anti-TNF-α antibody clone neutralizes human TNF-α in the L929 cytotoxicity test, and the monomer of the single region anti-VEGF antibody clone It is preferred to antagonize VEGF receptor binding in the VEGF receptor 2 binding test described in. The single region antibody clone used preferably binds each epitope with a _Kd of less than 100 nM. The bispecific tetravalent construct also binds the respective epitope with a K _d of less than 100 nM, resulting in increased arthritis scores in either or both of the Tg197 and CIA models of arthritis described herein. It is preferable to prevent.

  The construct can be used for the treatment of rheumatoid arthritis in the same manner as other constructs described herein in terms of administration, dosage, and effect monitoring. Constructs as described above, for example by addition of a PEG component and / or further fusion of a binding component specific to a serum protein such as HSA, such as an additional single domain antibody, such as increasing circulating half-life. It is possible to modify the half-life of

  All publications, patents and published patent applications mentioned in this specification, and references cited in such publications, are hereby incorporated by reference. Those skilled in the art will appreciate that various modifications and changes can be made to the described method and system of the present invention without departing from the scope or spirit of the invention. Although the invention has been described with reference to specific preferred embodiments, it is to be understood that the claimed invention is not unduly limited to such specific embodiments. Indeed, various modifications to the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be included within the scope of the following claims.

Annex 1: Polypeptide α-1 glycoprotein (orosomucoid) (AAG) which improves in vivo half-life
α-1 Antichymotrimicin (ACT)
α-1 Antitrypsin (AAT)
α-1 microglobulin (protein HC) (AIM)
α-2 Macroglobulin (A2M)
Antithrombin III (ATIII)
Apolipoprotein A-1 (ApoA-1)
Apoliprotein B (ApoB)
β-2-microglobulin (β2M)
Ceruloplasmin (Cp)
Complement component (C3)
Complement component (C4)
C1 esterase inhibitor (C1INH)
C-reactive protein (CRP)
Cysteine C (cis C)
Ferritin (FER)
Fibrinogen (FIB)
Fibronectin (FN)
Haptoglobin (Hp)
Hemopectin (HPX)
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Immunoglobulin light chain (kappa / lambda)
Lipoprotein (a) [Lp (a)]
Mannose binding protein (MBP)
Myoglobin (Myo)
Plasminogen (PSM)
Prealbumin (transthyretin) (PAL)
Retinol binding protein (RBP)
Rheumatoid factor (RF)
Serum amyloid A (SAA)
Soluble transferrin receptor (sTfR)
Transferrin (Tf)

Annex 2

Annex 3: Tumor combinations

Annex 4:

VH / HSA diversification at the H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98 positions present in the antigen binding site of VH HSA (respectively DVT or NNK encoded) FIG. The sequence of VK is diversified at L50 and L53. Amino acid sequence, SEQ ID NO: 219; missing repotide sequence, top chain, SEQ ID NO: 220. Library 1: Germline VK / DVT VH; Library 2: Germline VK / NNK VH; Library 3: Germline VH / DVT VK; Library 4: Germline VH / NNK VK. Phage display / ScFv format. These libraries were preselected for binding to the gene ligand proteins A and L so that the majority of the clones and the selected library were functional. Libraries were selected with HSA (first round) and β-gal (second round) or HSA β-gal selection, or β-gal (first round) and HSA (second round) β-gal selection. The soluble scFv of these clones of PCR is amplified with that sequence. One clone encoding bispecific antibody K8 was selected for further work. Nucleotide sequence: SEQ ID NO: 221; Amino acid sequence: SEQ ID NO: 222. It is a figure which shows alignment of VH chain and VK chain. _VH dummy, SEQ ID NO: 1; K8, SEQ ID No. 223 (VH) and 232 (VK); VH2, SEQ ID NO: 224; VH4, SEQ ID NO: 225; VHC11, SEQ ID NO: 226; VHA10sd, SEQ ID NO: 227; VHA1sd, SEQ ID NO: 228; VHA5sd, SEQ ID NO: 229; VHC5sd, SEQ ID NO: 230; VHC11sd, SEQ ID NO: 231; _{V k} dummy, SEQ ID NO: 3; E5sd, SEQ ID NO: 233; C3, SEQ ID NO: 234. FIG. 5 shows the characterization of the binding properties of K8 antibody, the binding properties of K8 antibody characterized by monoclonal phage ELISA. The bispecific K8 antibody was confirmed to bind HSA and β-gal and displayed on the surface of the phage with an absorption signal greater than 1.0. No cross-reactivity with other proteins was detected. FIG. 2 shows a soluble scFv ELISA realized using known concentrations of K8 antibody fragments. 100 μg HSA, BSA, and β-gal were applied to a 96-well plate at a concentration of 10 μg / ml, and 100 μg / ml protein A was applied at a concentration of 1 μg / ml. 50 μg of a serial dilution of K8scFv was applied and bound antibody fragments were detected with protein L-HRP. The ELISA results demonstrate the bispecificity of the K8 antibody. FIG. 5 shows the binding characteristics of clone K8VK / dummy VH analyzed using a soluble scFv ELISA. Production of soluble scFv fragments was induced by IPTG as described by Harrison et al., Methods Enzymol. A soluble scFv ELISA was realized as described in Example 1 and bound scFvs were detected with the protein L-HRP. The ELISA results revealed that the clones can still bind β-gal while the bound BSA is destroyed. It is a figure which shows the arrangement | sequence of variable region vector 1 and 2. Nucleotide sequence: SEQ ID NO: 221; Amino acid sequence: SEQ ID NO: 222. FIG. 6 is a map of CH vectors used to construct VH1 / VH2 multispecific ligands. Figure 3 is a map of VK vectors used to construct VK1 / VK2 multispecific ligands. TNF receptor test comparing TAR1-5 dimer 4, TAR1-5-19 dimer 4 and TAR1-5-19 monomer. TNF receptor test comparing mers 1-6 to TAR1-5. All dimers are EPLC generated and the results for the optimal dimer species are shown. TNF receptor test of TAR1-5 19 homodimer in different formats: dAb linker with 3U, 5U or 7U linker-dAb format, Fab format and cysteine hinge linker format. Dummy VH sequence for library 1. VH framework sequence based on germline DP47-JH4b. The position where NNK randomization (N = A or T or C or G nucleotide; K = G or T nucleotide) was incorporated into Library 1 is indicated by bold underlined text. Amino acid sequence, SEQ ID NO: 1; nucleotide sequence, top chain, SEQ ID NO: 2. Dummy VH array for library 2. VH framework sequence based on germline sequence DP47-JH4b. The position where NNK randomization (N = A or T or C or G nucleotides; K = G or T nucleotides) was incorporated into Library 2 is shown in bold underlined text. Amino acid sequence, SEQ ID NO: 235; nucleotide sequence, top chain, SEQ ID NO: 236. Dummy VH sequence for library 3. Sequence of VH framework 5 based on germline sequence DPK9-JK1. The position where NNK randomization (N = A or T or C or G nucleotide; K = G or T nucleotide) was incorporated into Library 3 is indicated by bold underlined text. Amino acid sequence, SEQ ID NO: 3; nucleotide sequence, top chain, SEQ ID NO: 4. Nucleotide and amino acid sequences of anti-MSA dAbs MSA16 and MSA26. Amino acid sequence, SEQ ID NO: 237; nucleotide sequence, SEQ ID NO: 238. MSA26: amino acid sequence, SEQ ID NO: 239; nucleotide sequence, SEQ ID NO: 240. Inhibition of BIAcore of MSA16. Purified dAb MSA16 was analyzed by inhibition BIAcore to determine _Kd . Briefly, dAbs were tested to determine the concentration of dAb required to achieve a 200 RU response on a BIAcoreCM5 chip coated with high density MSA. Once the required dAb concentration was determined, MSA antigens in the concentration range around the expected _Kd were premixed with the dAb and incubated overnight. The binding of dAb MSA to each BIAcore chip in each premix was then measured at a high flow rate of 30 μl / min. Inhibition of BIAcore of MSA26. Purified dAb MSA26 was analyzed by inhibition BIAcore to determine _Kd . Briefly, dAbs were tested to determine the concentration of dAb required to achieve a 200 RU response on a BIAcoreCM5 chip coated with high density MSA. Once the required dAb concentration was determined, MSA antigens in the concentration range around the expected _Kd were premixed with the dAb and incubated overnight. The binding of dAb MSA to each BIAcore chip in each premix was then measured at a high flow rate of 30 μl / min. Serum level of MSA16 after injection. Serum half-life of dAb MSA16 in mice was determined. MSA16 was administered to CD1 mice as a single in vivo injection at a concentration of about 1.5 mg / kg. Modeling with a two-compartment model showed that MSA16 had 0.98 hours t1 / 2β, 36.5 hours t1 / 2β, and 913 hours mg / ml AUC. MSA16 had a significantly longer half-life compared to HEL4 (anti-hen egg white lysozyme dAb) with t1 / 2β of 0.06 hours and t1 / 2β of 0.34 hours. ELISA (a) and TNF receptor test (c) showing inhibition of TNF binding by Fab-like fragments containing MSA26Ck and TAR1-5-19CH. Addition of MSA along with Fab-like fragments reduces the level of inhibition. Probing ELISA plates coated with 1 ug / ml TNF-α with bispecific VK CH and VK CK Fab-like fragments, and concentrations of control TNF-α binding calculated to give similar signals in ELISA Exploration with dAb. Both bispecific and control dAbs were used to probe ELISA plates in the presence and absence of 2 mg / ml MSA. The signal in the bispecific well was reduced by more than 50%, but the signal in the dAb was not reduced at all (see Figure 19a). The same bispecific protein was introduced into the receptor test with or without MSA and also showed competition by MSA (see 19c). This demonstrates that MSA binding to bispecificity competes with binding to TNF-α. TNF receptor test showing inhibitors of TNF binding by disulfide bond heterodimers of TAR1-5-19dAb and MSA16dAb. When MSA is added along with the dimer, the level of inhibitor decreases with dose. TNF receptor testing was performed in the presence of a constant concentration of heterodimer (18 nM) and serial dilutions of MSA and HSA. The presence of a range of concentrations (up to 2 mg / ml) of HSA did not cause a decrease in the dimer's ability to suppress TNF-α. However, the addition of MSA caused a reduction in the dimer's ability to suppress TNF-α depending on the dose. This demonstrates that MSA and TNF-α compete for binding to the cysteine bond TAR1-5-19, MSA16 dimer. MSA and HSA alone had no effect on TNF binding levels in the study. FIG. 2 shows the polynucleotide and amino acid sequence of human germline framework segment DP47 (see FIG. 1). The amino acid sequence is SEQ ID NO: 1, and the polynucleotide sequence of the top strand is SEQ ID NO: 2. It is a figure which shows the polynucleotide and amino acid sequence of human germline framework segment DPK9 (refer FIG. 1). The amino acid sequence is SEQ ID NO: 3, and the polynucleotide sequence of the top strand is SEQ ID NO: 4. It is a figure which shows the amino acid sequence with respect to the TAR1 clone described in this specification. TAR1-5, SEQ ID NO: 241; TAR1-27, SEQ ID NO: 242; TAR1-261, SEQ ID NO: 243; TAR1-398, SEQ ID NO: 244; TAR1-701, SEQ ID NO: 245; TAR1-5-2, SEQ ID NO: 246; TAR1-5-3, SEQ ID NO: 247; TAR1-5-4, SEQ ID NO: 248; TAR1-5-7, SEQ ID NO: 249; TAR1-5-8, SEQ ID NO: 250; TAR1-5-10, SEQ ID NO: 251; TAR1-5-11, SEQ ID NO: 252; TAR1-5-12, SEQ ID NO: 253; TAR1-5-13, SEQ ID NO: 254; TAR1-5-19, SEQ ID NO: 191; TAR1-5-20, SEQ ID NO: 255; TAR1-5-21, SEQ ID NO: 256; TAR1-5-22, SEQ ID NO: 257; TAR1-5-23, SEQ ID NO: 258; TAR1 5-14, SEQ ID NO: 259; TAR1-5-25, SEQ ID NO: 260; TAR1-5-26, SEQ ID NO: 261; TAR1-5-27, SEQ ID NO: 262; TAR1-5-28, SEQ ID NO: 263; TAR1- 5-29, SEQ ID NO: 264; TAR1-5-34, SEQ ID NO: 265; TAR1-5-35, SEQ ID NO: 266; TAR1-5-36, SEQ ID NO: 267; TAR1-5-464, SEQ ID NO: 268; TAR1- 5-463, SEQ ID NO: 269; TAR1-5-460, SEQ ID NO: 270; TAR1-5-461, SEQ ID NO: 271; TAR1-5-479, SEQ ID NO: 272; TAR1-5-477, SEQ ID NO: 273; TAR1- 5-478, SEQ ID NO: 274; TAR1-5-476, SEQ ID NO: 275; TAR1-5-490, SEQ ID NO: 276; TAR1 -1, SEQ ID NO: 277; TAR1h-2, SEQ ID NO: 278; TAR1h-3, SEQ ID NO: 279. FIG. 9 shows a comparison of serum half-life of TAR1-5-19 in dAb monomer format or Fc fusion format after single intravenous injection. FIG. 5 shows a summary of the dosage and timing of dAb constructs administered in a series of Tg197 model studies using TAR1-5-19. FIG. 5 shows a summary of weekly doses of different forms of TAR1-5-19dAb (Fc fusion, PEGylation, anti-TNF / anti-SA bispecificity) used for investigation in the Tg197 mouse RA model. Formats of anti-TNF dAb constructs administered in the Tg197 mouse RA model study (Fc fusion, PEG dimer, PEG tetramer, anti-TNF /) compared to the effects of anti-TNF dAb constructs and current anti-TNF products (Anti-SA bispecificity), delivery mode and dose summary. FIG. 7 shows administration and scoring methods for investigations examining the effects of anti-TNF dAbs on established disease symptoms in the Tg197 mouse RA model. And V _kappa variable region specific for human VEGF fused to a human IgG1 constant region, IgG-like including a specific V _kappa variable region against human TNF-alpha fused to a human C _kappa constant region FIG. 6 shows SDS PAGE gel analysis for bispecific antibodies. Lane 1: Invitrogen multi-mark MW marker. Lane 2: anti-TNFx anti-VEGF bispecific antibody in 1X non-reducing additive. Lane 3: anti-TNFx anti-VEGF bispecific antibody in 1X addition solution containing 10 mM β-mercaptoethanol. It is a figure which shows the result of the test which investigates the inhibitory effect of the anti- TNFx anti-VEGF bispecific antibody in the test of TNF- (alpha) activity and a VEGF receptor. Results of L929TNF-α cytotoxin neutralization test. Curve showing square data points (control anti-TNF-α antibody). Curve showing upward triangle data points (anti-TNFx anti-VEGF bispecific antibody). Curve showing downward triangle data points (anti-TNF-α monomer). It is a figure which shows the result of the test which investigates the inhibitory effect of the anti- TNFx anti-VEGF bispecific antibody in the test of TNF- (alpha) activity and a VEGF receptor. Results of human VEGF receptor 2 binding test. Curve showing square data points (anti-TNFx anti-VEGF bispecific antibody). Curve showing upward triangle data points (anti-VEGF control). Curve showing downward triangle data points (negative control).

Claims (202)

  Use of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human TNFα in vitro for the preparation of a medicament for the treatment, prevention, inhibition of progression or delay of onset of rheumatoid arthritis .   Including single domain antibody polypeptide constructs that antagonize binding to a receptor for human TNFα in vitro to prepare a medicament for inducing a statistically significant change in one or more indicators of rheumatoid arthritis Use of the composition.   The use according to claim 1 or 2, wherein the composition prevents an increase in arthritic score when administered to a mouse of a Tg197 transgenic mouse model of arthritis.   4. Use according to claim 3, wherein the administration results in a statistically significant change in one or more indicators of RA.   The one or more indicators of RA include blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, X-ray imaging of one or more joints, and one or more 5. Use according to claim 4, comprising any of histopathological analysis of the fixed part of the joint.   The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, and the Tg197 transgenic mice The macrophenotypic signs of arthritis were scored for: 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis ( Use according to claim 4, scored according to a system of swelling, joint deformation), 3 = severe arthritis (significant movement disorder).   The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, the Tg197 transgenic mice The histopathological signs of arthritis are scored for the macrophenotypic signs of the arthritis, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates, 2 5. Use according to claim 4, scored using a system of pannus and fibrous tissue formation and localized subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = extensive articular cartilage destruction and bone erosion . Administration of the composition to Tg197 transgenic mice is as follows:
a) weekly administration of said composition by intraperitoneal injection to heterozygous Tg197 transgenic mice;
b) measuring the weight of the mouse in step a) weekly;
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) 4. The use of claim 3, comprising scoring the mice weekly for macrophenotypic signs of arthritis according to the system.   4. Use according to claim 3, wherein the composition is administered before the onset of an arthritic condition.   4. Use according to claim 3, wherein the composition is administered first when the mouse is 3 weeks old.   4. Use according to claim 3, wherein the composition is administered first when the mouse is 6 weeks old.   The use according to any one of claims 1 to 11, wherein the composition has an effect in a Tg197 transgenic mouse arthritis test which is more than a drug selected from the group consisting of etanercept, infliximab and D2E7.   13. Use according to any of claims 1 to 12, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that the treatment results in an arthritic score of 0 to 0.5.   14. Use according to any of claims 1 to 13, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score of 0 to 1.0.   15. Use according to any of claims 1 to 14, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that the treatment results in an arthritic score of 0 to 1.5.   16. Use according to any of claims 1 to 15, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritis score of 0 to 2.0.   A method of treating rheumatoid arthritis, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human TNFα. A method wherein rheumatoid arthritis is treated.   A method of treating rheumatoid arthritis, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human TNFα. Wherein the single domain antibody polypeptide construct inhibits binding of human TNFα to the TNFα receptor, thereby treating the rheumatoid arthritis.   The method according to claim 17 or 18, wherein the treatment comprises suppressing progression of the rheumatoid arthritis.   19. A method according to claim 17 or 18, wherein the treatment comprises preventing or delaying the onset of rheumatoid arthritis.   19. The method according to claim 17 or 18, or the use according to claim 3 or 4, wherein said composition prevents an increase in arthritis score when administered to a mouse of a Tg197 transgenic mouse model of arthritis.   24. The method of claim 21, wherein the administration results in a statistically significant change in one or more indicators of RA.   The one or more indicators of RA include blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, X-ray imaging of one or more joints, and one or more 23. The method of claim 22, comprising any of histopathological analysis of the joint fixation part.   The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, and the Tg197 transgenic mice The macrophenotypic signs of arthritis were scored for: 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis ( 23. Method according to claim 22, scored according to a system of swelling, joint deformation), 3 = severe arthritis (significant movement disorder).   The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in Tg197 transgenic mice, the composition is administered to Tg197 transgenic mice, and the Tg197 transgenic mice The histopathological signs of arthritis are scored for the macrophenotypic signs of the arthritis, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates, 2 23. The method of claim 22, scored using a system of pannus and fibrous tissue formation and localized subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = extensive articular cartilage destruction and bone erosion . Administration of the composition to Tg197 transgenic mice is as follows:
a) weekly administration of said composition by intraperitoneal injection to heterozygous Tg197 transgenic mice;
b) measuring the weight of the mouse in step a) weekly;
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) 24. The method of claim 21, comprising scoring the mice weekly for macrophenotypic signs of arthritis according to the system of   24. The method of claim 21, wherein the composition is administered before an onset of joint inflammation is indicated.   24. The method of claim 21, wherein the composition is administered first when the mouse is 3 weeks old.   24. The method of claim 21, wherein the composition is administered first when the mouse is 6 weeks old.   30. The method or use according to any of claims 1 to 29, wherein the composition has an effect in a Tg197 transgenic mouse arthritis test that is equal to or greater than a drug selected from the group consisting of etanercept, infliximab and D2E7.   31. The method or use of any of claims 1 to 30, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score of 0 to 0.5.   32. The method or use of any of claims 1-31, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score of 0 to 1.0.   33. The method or use of any of claims 1-32, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritic score of 0 to 1.5.   34. The method or use of any of claims 1-33, wherein the composition has an effect in the Tg197 transgenic mouse arthritis test such that treatment results in an arthritis score of 0 to 2.0.   35. The method or use of any of claims 1-34, wherein the single region antibody polypeptide construct comprises a human single region antibody polypeptide.   36. The method or use of any of claims 1-35, wherein the human single region antibody polypeptide binds TNFα.   37. The method or use of any of claims 1-36, wherein the single domain antibody polypeptide construct binds human TNFα with a Kd of less than 100 nM. 38. The method or use of any of claims 1-37, wherein the single domain antibody polypeptide construct binds human TNFα with a _Kd ranging from 100 nM to 50 pM. 39. The method or use of any of claims 1-38, wherein the single domain antibody polypeptide construct binds human TNFα with a _Kd of 30 nM to 50 pM. 40. The method or use of any of claims 1-39, wherein the single domain antibody polypeptide construct binds human TNFα with a _Kd of 10 nM to 50 pM. 41. The method or use of any of claims 1 to 40, wherein the single domain antibody polypeptide construct binds human TNFα with a _Kd ranging from 1 nm to 50 pM.   42. The method or use of any of claims 1-41, wherein the single domain antibody polypeptide construct antagonizes human TNFα as measured in a standard L929 cell test.   43. The method or use of any of claims 1-42, wherein the single domain antibody polypeptide construct specifically binds human TNF- [alpha] that binds to a cell surface receptor.   44. The method or use of any of claims 1-43, wherein the single domain antibody polypeptide construct has an in vivo tα half-life in the range of 15 minutes to 12 hours.   45. The method or use of any of claims 1-44, wherein the single domain antibody polypeptide construct has an in vivo tα half-life in the range of 1 to 6 hours.   46. The method or use of any of claims 1-45, wherein the single domain antibody polypeptide construct has an in vivo tα half-life in the range of 2 to 5 hours.   47. The method or use of any of claims 1-46, wherein the single domain antibody polypeptide construct has an in vivo tα half-life in the range of 3 to 4 hours.   48. The method or use of any of claims 1-47, wherein the single domain antibody polypeptide construct has an in vivo tβ half-life in the range of 12 to 60 hours.   49. The method or use of any of claims 1-48, wherein the single domain antibody polypeptide construct has an in vivo tβ half-life in the range of 12 to 48 hours.   50. The method or use of any of claims 1-49, wherein the single domain antibody polypeptide construct has an in vivo tβ half-life in the range of 12 to 26 hours.   51. The method or use of any of claims 1-50, wherein the single domain antibody polypeptide construct has an in vivo AUC half-life of 15 mg min / ml to 150 mg min / ml.   52. The method or use according to any of claims 1 to 51, wherein the single domain antibody polypeptide construct has an in vivo AUC half-life of 15 mg / ml to 100 mg / ml.   53. The method or use of any of claims 1 to 52, wherein the single domain antibody polypeptide construct has an in vivo AUC half-life of 15 mg min / ml to 75 mg min / ml.   54. The method or use of any of claims 1 to 53, wherein the single domain antibody polypeptide construct has an in vivo AUC half-life of 15 mg / ml to 50 mg / ml.   55. The method or use of any of claims 1 to 54, wherein the single domain antibody polypeptide construct is conjugated to a PEG molecule.   56. The method or use of claim 55, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa.   56. The method or use of claim 55, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa.   56. The method or use of claim 55, wherein the PEG-binding protein binds on average to 1-20 polyethylene glycol molecules.   59. The method or use of any of claims 1 to 58, wherein the antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human TNFα.   60. The method or use of any of claims 1 to 59, wherein the antibody construct comprises a homodimer of a single immunoglobulin variable region polypeptide that binds human TNFα.   61. The method or use of any of claims 1-60, wherein the antibody construct comprises a homotrimer of a single immunoglobulin variable region polypeptide that binds human TNFα.   62. The method or use of any of claims 1 to 61, wherein the antibody construct comprises a homotetramer of a single immunoglobulin variable region polypeptide that binds human TNFα.   64. The method or use according to any of claims 1 to 62, wherein the antibody construct further comprises an antibody polypeptide specific for an antigen other than TNFα.   64. The method or use of claim 63, wherein the antibody polypeptide specific for an antigen other than TNFα comprises a single region antibody polypeptide.   65. The method or use of claim 63 or 64, wherein binding of said antigen other than TNFα by said antibody polypeptide specific for an antigen other than TNFα increases the in vivo half-life of said antibody polypeptide construct.   66. The method or use of claims 63 to 65, wherein the antigen other than TNFα comprises a serum protein.   The serum protein is selected from the group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. Item 67. The method or use according to Item 66.   64. The method or use of claim 63, wherein the antigen other than TNFα comprises HSA.   69. The method of any one of claims 17 to 68, wherein the treatment further comprises administration of at least one additional therapeutic agent. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of: The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 92% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 94% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto. The single region antibody polypeptide construct is a clone

70. The method or use according to any one of claims 1 to 69, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 99% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor of human TNFα for the preparation of a medicament for the treatment, prevention, inhibition of progression or delay of onset of rheumatoid arthritis comprising:
Said composition prevents an increase in arthritic score when administered to mice in a Tg197 transgenic mouse model of arthritis;
The single domain antibody polypeptide construct binds human TNFα with a Kd of less than 100 nM;
Use of a composition wherein the single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test. A method of treating rheumatoid arthritis, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding to a receptor for human TNFα. Including
The composition prevents an increase in arthritic score when administered to a Tg197 transgenic mouse model of arthritis,
The single domain antibody polypeptide construct binds human TNFα with a Kd of less than 100 nM;
The single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test;
The method wherein the rheumatoid arthritis is treated. The single domain antibody polypeptide construct comprises:
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
80. The method or use according to any one of claims 1 to 79, wherein the polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds the first and second epitopes.   81. The method or use of claim 80, wherein the first and / or the second epitope is a TNF- [alpha] epitope.   A single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor, wherein said single domain antibody polypeptide construct prevents an increase in arthritis score when administered to a mouse in a Tg197 transgenic mouse model of arthritis, Is a composition that neutralizes human TNFα as measured in a standard L929 cell test.   A single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor, wherein said single domain antibody polypeptide construct prevents an increase in arthritis score when administered to a mouse in a Tg197 transgenic mouse model of arthritis, Is a composition that suppresses the progression of rheumatoid arthritis.   A single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor, wherein said single domain antibody polypeptide construct prevents an increase in arthritis score when administered to a mouse in a Tg197 transgenic mouse model of arthritis, Is a composition that binds human TNFα with a Kd of less than 100 nM.   A composition comprising a single region antibody polypeptide construct that antagonizes binding of human TNFα to a receptor, wherein the composition prevents administration of an arthritic score when administered to a mouse of the Tg197 transgenic mouse model of arthritis, The single region antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test, the single region antibody polypeptide construct inhibits the progression of rheumatoid arthritis, and the single region antibody polypeptide The construct is a composition that binds human TNFα with a Kd of less than 100 nM. The single domain antibody polypeptide construct comprises:
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
86. The composition of any one of claims 82 to 85, wherein the polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds the first and second epitopes.   87. The composition of claim 86, wherein the first and / or second epitope is a TNF- [alpha] epitope. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 92% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising the amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 94% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising the amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto. The single region antibody polypeptide construct is a clone

88. A composition according to any one of claims 82 to 87, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 99% identity thereto.   Use of a composition comprising a single region antibody polypeptide construct that antagonizes the binding of human VEGF to the VEGF receptor for the preparation of a medicament for the treatment, prevention, prevention of progression or delay of onset of rheumatoid arthritis.   A method of treating rheumatoid arthritis, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to the VEGF receptor. And wherein said rheumatoid arthritis is treated.   99. The use of claim 96, or the method of claim 97, wherein the composition prevents an increase in arthritis score when administered to mice in a collagen-induced arthritis (CIA) mouse model. Administration of the composition to the mouse comprises
a) weekly administration of the composition to CIA model mice by intraperitoneal injection;
b) measuring the weight of the mouse in step a) weekly;
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) 99. The method or use of claim 98, comprising scoring the mouse for a macrophenotypic indication of arthritis according to the system.   99. The method according to any one of claims 97 to 99, wherein the treatment comprises inhibiting progression of the rheumatoid arthritis.   99. A method according to any one of claims 97 to 99, wherein the treatment comprises preventing or delaying the onset of rheumatoid arthritis.   99. The method or use according to any one of claims 97 to 99, wherein the administration results in a statistically significant change in one or more indicators of RA.   The one or more indicators of RA include blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, X-ray imaging of one or more joints, and one or more 103. The method or use of claim 102, comprising any of histopathological analysis of a fixed part of the joint. The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in mice of a collagen-induced arthritis mouse model;
The composition is administered to the mouse;
The mice are scored for the macrophenotypic signs of arthritis;
Said macro phenotypic signs of arthritis are: 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe 105. The method or use of claim 102, scored according to a system of arthritis (significant movement disorder). The one or more indicators of RA include a decrease in macrophenotypic signs of histopathological signs of arthritis in mice of a collagen-induced arthritis mouse model;
The composition is administered to the mouse;
The mice are scored for the histopathological signs of arthritis;
Said histopathological signs of arthritis are realized in the joints, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates, 2 = pannus and fibrous tissue formation and localization 103. Method or use according to claim 102, scored using a system of articular cartilage destruction and bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = extensive articular cartilage destruction and bone erosion. The bispecific antibody construct is:
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
135. The use or method of claim 134, wherein the polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds the first and second epitopes.   107. The method or use of claim 106, wherein the first and / or the second epitope is a VEGF epitope.   108. The method or use of any one of claims 96 to 107, wherein the single domain antibody polypeptide construct comprises a human single domain antibody polypeptide.   109. The method or use of claim 108, wherein the human single region antibody polypeptide binds VEGF.   110. The method or use according to any one of claims 96 to 109, wherein the single domain antibody polypeptide construct binds human VEGF with a Kd of less than 100 nM.   110. The method or use according to any one of claims 96 to 109, wherein the single domain antibody polypeptide construct binds human VEGF with a Kd in the range of 100 nM to 50 pM.   111. The method or use according to any one of claims 96 to 109, wherein the single domain antibody polypeptide construct binds human VEGF with a Kd of 30 nM to 50 pM.   110. The method or use according to any one of claims 96 to 109, wherein the single domain antibody polypeptide construct binds human VEGF with a Kd of 10 nM to 50 pM.   110. The method or use according to any one of claims 96 to 109, wherein the single domain antibody polypeptide construct binds human VEGF with a Kd in the range of 1 nm to 50 pM.   115. The method or use according to any one of claims 96 to 114, wherein the single domain antibody polypeptide construct neutralizes human VEGF as measured in a VEGF receptor 1 test or a VEGF receptor 2 test.   116. The method or use of any one of claims 96 to 115, wherein the single domain antibody polypeptide construct specifically binds human VEGF that binds to a cell surface receptor.   117. The method or use of any one of claims 96 to 116, wherein the single domain antibody polypeptide construct is conjugated to a PEG molecule.   118. The method or use of claim 117, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa.   118. The method or use of claim 117, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa.   120. The method or use according to any one of claims 117 to 119, wherein the PEG-conjugated single region antibody polypeptide construct is conjugated on average to two or more polyethylene glycol molecules.   120. The method or use according to any one of claims 117 to 119, wherein the PEG-conjugated single domain antibody polypeptide construct is conjugated to 2 to 20 PEG molecules on average.     122. The method or use according to any one of claims 96 to 121, wherein the antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human VEGF.   122. The method or use according to any one of claims 96 to 121, wherein the antibody construct comprises a homodimer of a single immunoglobulin variable region polypeptide that binds human VEGF.   122. The method or use of any of claims 96 to 121, wherein the antibody construct comprises a homotrimer of a single immunoglobulin variable region polypeptide that binds human VEGF.   122. The method or use according to any one of claims 96 to 121, wherein the antibody construct comprises a homotetramer of a single immunoglobulin variable region polypeptide that binds human VEGF.   126. The method or use according to any one of claims 96 to 125, wherein the construct further comprises an antibody polypeptide specific for an antigen other than VEGF.   127. The method or use of claim 126, wherein said antibody polypeptide specific for an antigen other than VEGF comprises a single region antibody polypeptide.   128. The method or use of claim 126 or 127, wherein binding of the non-VEGF antigen by the antibody polypeptide specific for an antigen other than VEGF increases the in vivo half-life of the antibody polypeptide construct.   129. The method or use according to any one of claims 126 to 128, wherein the antigen other than VEGF comprises a serum protein.   The serum protein is selected from the group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. 129. The method or use according to item 129.   129. The method or use according to any one of claims 126 to 129, wherein the antigen other than VEGF comprises HSA.   132. The method of any one of claims 97 to 131, wherein the treatment further comprises administration of at least one additional therapeutic agent.   135. The method of claim 132, wherein the therapeutic agent is selected from the group consisting of etanercept, infliximab, and D2E7.   The therapeutic agents include corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDS), acetylsalicylic acid, pyrazolone, fenamic acid salt, diflunisal, acetic acid derivative, propionic acid derivative, oxicam, mefenamic acid, ponster, meclofenamic acid salt, meclomene, phenyl Butazone, Butazolidine, Diflunisal, Drobid, Diclofenac, Voltaren, Indomethacin, Indocin, Sulindac, Crinolyl, Etodolac, Rosin, Ketorolac, Tradol, Nabumetone, Lerafen, Tolmetine, Trectin, Ibuprofen, Motuline, Fenoprofen, Narphone, Flurbipro Fen, Ante, Carprofen, Rimadil, Ketoprofen, Ordis, Naproxen, Anaprox, Naprosin, Piroxicam and Felden The method of claim 132 selected from the group consisting. The single region antibody polypeptide construct is a clone

135. The method or use of claim 134, comprising the amino acid sequence of CDR3 of an antibody polypeptide selected from the group consisting of: The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 85% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 90% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 92% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 94% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 96% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 98% identity thereto. The single region antibody polypeptide construct is a clone

135. The method or use according to any one of claims 96 to 134, comprising an amino acid sequence of an antibody polypeptide selected from the group consisting of, or an amino acid sequence having at least 99% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising a CDR3 sequence selected from the group consisting of: A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 85% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 90% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of or a sequence having at least 92% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 94% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 96% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 98% identity thereto. A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human VEGF to a receptor, the single domain antibody polypeptide construct comprising:

A composition comprising an amino acid sequence selected from the group consisting of: or a sequence having at least 99% identity thereto. The single domain antibody polypeptide construct comprises:
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
161. The composition of any one of claims 143 to 150, wherein the polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds the first and second epitopes.   152. The composition of claim 151, wherein the first and / or the second epitope is a VEGF epitope.   A single domain antibody polypeptide construct that antagonizes the activity of human TNFα and antagonizes binding to the receptor of human VEGF for the preparation of a medicament for the treatment, prevention, prevention of progression or delay of onset of rheumatoid arthritis Use of a composition comprising.   A single domain antibody poly-antagonizing the binding of human TNFα to the receptor and antagonizing the binding of human VEGF to the receptor for the preparation of a medicament for the treatment, prevention, prevention of progression or delay of onset of rheumatoid arthritis Use of a composition comprising a peptide construct.   A method of treating rheumatoid arthritis, comprising simulating a single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor and antagonizes binding of a human VEGF receptor to an individual in need thereof. Administering a therapeutically effective amount of the composition, whereby said rheumatoid arthritis is treated.   156. Use according to claim 153 or 154, or method according to claim 155, wherein said polypeptide construct is a bispecific antibody construct. The bispecific antibody construct is:
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
135. The use or method of claim 134, wherein the polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds the first and second epitopes.   158. The method or use according to any one of claims 153 to 157, wherein the composition prevents an increase in arthritic score when administered to a mouse of a Tg197 transgenic mouse model of arthritis. Administration of the composition to Tg197 transgenic mice is as follows:
a) weekly administration of said composition by intraperitoneal injection to heterozygous Tg197 transgenic mice;
b) measuring the weight of the mouse in step a) weekly;
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) 159. The method or use of claim 158, comprising scoring the mouse weekly for macrophenotypic signs of arthritis according to the system of   159. The method or use of claim 158, wherein the composition has an effect in a Tg197 transgenic mouse arthritis test that is greater than or equal to a drug selected from the group consisting of etanercept, infliximab and D2E7.   158. The method or use according to any one of claims 153 to 157, wherein the composition prevents an increase in arthritis score when administered to a mouse of a collagen-induced arthritis (CIA) mouse model. Administration of the composition to the mouse comprises
a) weekly administration of the composition to CIA model mice by intraperitoneal injection;
b) measuring the weight of the mouse in step a) weekly;
c) 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe arthritis (significant movement disorder) 163. The method or use of claim 161, comprising scoring the mouse for a macrophenotypic indication of arthritis according to the system.   159. The method or use of claim 158, wherein the administration results in a statistically significant change in one or more indicators of RA.   138. The method according to any one of claims 131 or 134 to 137, wherein the treatment comprises inhibiting progression of the rheumatoid arthritis.   164. The method of any one of claims 155 to 163, wherein the treatment comprises preventing or delaying the onset of rheumatoid arthritis.   The one or more indicators of RA include blood sedimentation (ESR), rich joint index and morning stiffness duration, joint mobility, joint swelling, X-ray imaging of one or more joints, and one or more 166. A method or use according to claim 163, comprising any of histopathological analysis of the fixed part of the joint. The one or more indicators of RA include a reduction in the macrophenotypic signs of arthritis in Tg197 transgenic mice;
The composition is administered to a Tg197 transgenic mouse;
The Tg197 transgenic mice are scored for the macrophenotypic signs of arthritis;
Said macro phenotypic signs of arthritis are: 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe 166. The method or use of claim 163, scored according to a system of arthritis (significant movement disorder). The one or more indicators of RA include a reduction in the macrophenotypic signs of arthritis in Tg197 transgenic mice;
The composition is administered to a Tg197 transgenic mouse;
The Tg197 transgenic mice are scored for the macrophenotypic signs of arthritis;
Said histopathological signs of arthritis are realized in the joints, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates, 2 = pannus and fibrous tissue formation and localization 141. Method or use according to claim 140, scored using a system of articular cartilage destruction and bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = extensive articular cartilage destruction and bone erosion.   164. The method or use of claim 161, wherein the administration results in a statistically significant change in one or more indicators of RA. The one or more indicators of RA include a decrease in the macrophenotypic signs of arthritis in mice of a collagen-induced arthritis mouse model;
The composition is administered to the mouse;
The mice are scored for the macrophenotypic signs of arthritis;
Said macro phenotypic signs of arthritis are: 0 = not arthritis (normal appearance and flexion), 1 = mild arthritis (joint distortion), 2 = moderate arthritis (swelling, joint deformation), 3 = severe 170. The method or use of claim 169, scored according to a system of arthritis (significant movement disorder). The one or more indicators of RA include a decrease in macrophenotypic signs of histopathological signs of arthritis in mice of a collagen-induced arthritis mouse model;
The composition is administered to the mouse;
The mice are scored for the histopathological signs of arthritis;
Said histopathological signs of arthritis are realized in the joints, 0 = no detectable pathology, 1 = presence of synovial hyperplasia and polymorphonuclear infiltrates, 2 = pannus and fibrous tissue formation and localization 170. The method or use of claim 169, scored using a system of articular cartilage destruction and bone erosion, 3 = articular cartilage destruction and bone erosion, 4 = extensive articular cartilage destruction and bone erosion.   172. A method or use according to any one of claims 153 to 171 wherein the single region antibody polypeptide construct comprises a human single region antibody polypeptide.   173. The method or use of claim 172, wherein the human single region antibody polypeptide construct binds TNFα and VEGF.   175. The method or use of claim 172, wherein said single domain antibody polypeptide component that antagonizes the activity of human VEGF neutralizes human VEGF as measured in a VEGF receptor 1 test or a VEGF receptor 2 test.   175. The method or use according to any one of claims 153 to 174, wherein said single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test.   175. A method or use according to any one of claims 153 to 175, wherein said single domain antibody polypeptide construct is conjugated to a PEG molecule.   177. The method or use of claim 176, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 24 kDa and the total PEG size is 20 to 60 kDa.   177. The method or use of claim 176, wherein the PEG-conjugated single region antibody polypeptide construct has a hydrodynamic size of at least 200 kDa and the total PEG size is 20 to 60 kDa.   179. The method or use of any one of claims 176 to 178, wherein the antibody polypeptide construct is conjugated to two or more polyethylene glycol molecules on average.   179. The method or use of any one of claims 176 to 178, wherein the antibody polypeptide construct is conjugated to 2 to 20 PEG molecules on average.   153. The antibody construct comprises two or more single immunoglobulin variable region polypeptides that bind human TNFα and / or two or more single immunoglobulin variable region polypeptides that bind human VEGF. The method or use according to any one of the above.   153. The antibody construct comprises a homodimer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homodimer of a single immunoglobulin variable region polypeptide that binds human VEGF. 183. A method or use according to any one of 1 to 180.   153. The antibody construct comprises a homotrimer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homotrimer of a single immunoglobulin variable region polypeptide that binds human VEGF. 183. A method or use according to any one of 1 to 180.   153. The antibody construct comprises a homotetramer of a single immunoglobulin variable region polypeptide that binds human TNFα and / or a homotetramer of a single immunoglobulin variable region polypeptide that binds human VEGF. 183. A method or use according to any one of 1 to 180.   181. The method or use according to any one of claims 153 to 180, wherein the construct further comprises an antibody polypeptide specific for an antigen other than TNFα or VEGF.   186. The method or use of claim 185, wherein said antibody polypeptide specific for an antigen other than TNFα or VEGF comprises a single region antibody polypeptide.   187. The method of claim 185 or 186, wherein binding of said antigen other than TNFα or VEGF by said antibody polypeptide specific for an antigen other than TNFα or VEGF increases the in vivo half-life of said antibody polypeptide construct or use.   188. The method or use according to any one of claims 185 to 187, wherein the antigen other than TNFα or VEGF comprises a serum protein.   The serum protein is selected from the group consisting of fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen, serum amyloid protein A, heptaglobin, protein, ubiquitin, uterine globulin and β-2-microglobulin. 188. Method or use according to item 188.   188. The method or use according to any one of claims 185 to 189, wherein the antigen other than TNFα comprises HSA.   195. The method of any one of claims 155 to 190, wherein the treatment further comprises administration of at least one additional therapeutic agent.   Said further therapeutic agents are corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDS), acetylsalicylic acid, pyrazolone, fenamic acid, diflunisal, acetic acid derivatives, propionic acid derivatives, oxicam, mefenamic acid, ponster, meclofenamic acid, meclomene, Phenylbutazone, Butazolidine, Diflunisal, Drobid, Diclofenac, Voltaren, Indomethacin, Indocin, Sulindac, Crinolyl, Etodolac, Rosin, Ketorolac, Tradol, Nabumetone, Leraphene, Tolmethine, Trectin, Ibuprofen, Motuline, Fenoprofen, Narphone, Flurbi Profen, Ante, Carprofen, Rimadil, Ketoprofen, Ordis, Naproxen, Anaprox, Naprosin, Piroxicam and Fe The method of claim 191 which is selected from the group consisting den.   A bispecific antigen-binding polypeptide comprising a first antibody single region polypeptide that binds TNF-α and a second antibody single region polypeptide that binds VEGF. The construct is
a) a first replica of a first fusion protein comprising a single region antibody polypeptide that binds a first epitope fused to an IgG heavy chain constant region;
b) a second replica of said first fusion protein;
c) a first replica of a second fusion protein comprising a single region antibody polypeptide that binds a second epitope fused to a light chain constant region;
d) a second replica of said second fusion protein,
The first and second replicas of the first fusion protein are disulfide bonded to each other via respective IgG heavy chain constant regions;
The light chain constant region of the first replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the first replica of the first fusion protein;
The light chain constant region of the second replica of the second fusion protein is disulfide bonded to the IgG heavy chain constant region of the second replica of the first fusion protein;
197. The bispecific antigen binding polypeptide of claim 193, wherein said polypeptide construct comprises a tetravalent bispecific antigen binding polypeptide construct that binds said first and second epitopes.   166. The bispecific antigen binding polypeptide of claim 165, wherein said first epitope is a TNF- [alpha] epitope and said second epitope is a VEGF epitope.   166. The bispecific antigen binding polypeptide of claim 165, wherein said first epitope is a VEGF epitope and said second epitope is a TNF-α epitope.   Increased arthritis score when administered to mice in the Tg197 transgenic mouse model of arthritis, comprising a single domain antibody polypeptide construct that antagonizes binding of human TNFα to the receptor and antagonizes binding of human VEGF to the receptor Wherein the single domain antibody polypeptide construct inhibits the progression of rheumatoid arthritis.   A composition comprising a single domain antibody polypeptide construct that antagonizes binding of human TNFα to a receptor and antagonizes binding of human VEGF to a receptor, said composition comprising a Tg197 transgenic mouse model of arthritis When administered to mice, it prevents an increase in arthritis score, the single domain antibody polypeptide construct binds human TNFα with a Kd of less than 100 nM, and the single domain antibody polypeptide construct is a progression of rheumatoid arthritis. The composition which suppresses.   Increased arthritis score when administered to mice in the Tg197 transgenic mouse model of arthritis, comprising a single domain antibody polypeptide construct that antagonizes binding of human TNFα to the receptor and antagonizes binding of human VEGF to the receptor Wherein the single domain antibody polypeptide construct neutralizes human TNFα as measured in a standard L929 cell test, and the single domain antibody polypeptide construct has a Kd of less than 100 nM. Wherein the single domain antibody polypeptide construct inhibits the progression of rheumatoid arthritis.   Includes a single-domain antibody polypeptide construct that antagonizes binding of human TNFα to the receptor and antagonizes binding of human VEGF to the receptor to prevent an increase in arthritic score when administered to mice in a collagen-induced arthritis model of arthritis Wherein the single domain antibody polypeptide construct inhibits the progression of rheumatoid arthritis.   212. The composition of any one of claims 193 to 200, wherein the composition is administered with at least one additional therapeutic agent.   Said further therapeutic agents are corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDS), acetylsalicylic acid, pyrazolone, fenamic acid, diflunisal, acetic acid derivatives, propionic acid derivatives, oxicam, mefenamic acid, ponster, meclofenamic acid, meclomene, Phenylbutazone, Butazolidine, Diflunisal, Drobid, Diclofenac, Voltaren, Indomethacin, Indocin, Sulindac, Crinolyl, Etodolac, Rosin, Ketorolac, Tradol, Nabumetone, Leraphene, Tolmethine, Trectin, Ibuprofen, Motuline, Fenoprofen, Narphone, Flurbi Profen, Ante, Carprofen, Rimadil, Ketoprofen, Ordis, Naproxen, Anaprox, Naprosin, Piroxicam and Fe The composition of claim 201 which is selected from the group consisting den.

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