Dual variable region antibody-like binding proteins with swapped binding region orientations

Abstract

The present invention relates to a dual variable region antibody-like binding protein with an exchanged binding region orientation, and specifically provides an antibody-like binding protein comprising four polypeptide chains forming four antigen binding sites, wherein each pair of polypeptides forming the antibody-like binding protein has a dual variable domain with an exchanged orientation. The present invention also provides a method for preparing such an antigen-like binding protein.

Description

[0001] The present invention application is a divisional application based on the invention patent application filed on March 28, 2012, with application number 201280025960.2 (international application number PCT / US2012 / 030948) and entitled “Dual variable region antibody-like binding protein with exchange binding region orientation”. Field of the Invention

[0002] The present invention relates to an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein each pair of polypeptides forming the antibody-like binding protein has dual variable domains with an exchange orientation. The present invention also relates to a method for preparing such an antigen-like binding protein. Background of the Invention

[0003] Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies that have binding specificity for two different antigens can be produced using recombinant technology and are expected to have a wide range of clinical applications. As is well known, a complete IgG antibody molecule is a Y-shaped molecule consisting of four polypeptide chains: two heavy chains and two light chains. Each light chain consists of two domains, called variable or V domains. L The N-terminal domain of the domain (or region) and the constant (or C L The C-terminal domain of the heavy chain is called the variable (or V) domain. H ) domain (or region), which is followed by the first constant (or C H1 ) domain, the hinge region, and then the second and third constant (or C H2 and C H3 ) domain. In the assembled antibody, V L and V H The domains associate together to form the antigen binding site. L and C H1 The domains associate to keep one heavy chain associated with one light chain. The two heavy chain-light chain heterodimers are bound by the C H2 and C H3 The interactions of the domains are coupled to the interactions between the hinge regions of the two heavy chains.

[0004] It is known that proteolytic digestion of antibodies can lead to the production of antibody fragments (Fab and Fab2). Such fragments of intact antibodies can show antigen binding activity. Antibody fragments can also be produced recombinantly. Fv fragments can be obtained, which consist of the variable domains of the heavy and light chains associated with each other. These Fv fragments are monovalent for antigen binding. Smaller fragments, such as individual variable domains (domain antibodies or dABs; Ward et al., 1989, Nature 341(6242):544-46) and individual complementary determining regions or CDRs (Williams et al., 1989, Proc. Natl. Acad. Sci. USA 86(14):5537-41) have also been shown to retain the binding properties of the parent antibody, but most naturally occurring antibodies generally require V H and V L Both to maintain complete binding performance.

[0005] Single-chain variable fragment (scFv) constructs contain an antibody V contained in a single polypeptide chain. H and V L Domains, wherein the domains are separated by sufficiently long (greater than 12 amino acid residues) flexible linkers that force intramolecular interactions, allowing the two domains to self-assemble into a functional epitope binding site (Bird et al., 1988, Science 242(4877):423-26). These small proteins (MW about 25,000Da) generally retain specificity and affinity for their antigens in a single polypeptide and can provide suitable structural units (building blocks) for larger antigen-specific molecules.

[0006] The advantage of using antibody fragments rather than intact antibodies in diagnosis and treatment is their smaller size. They may be less immunogenic than intact antibodies and more able to penetrate tissues. A disadvantage associated with the use of such fragments is that they have only one antigen-binding site, resulting in reduced avidity. In addition, due to their small size, they are cleared from serum very quickly and therefore exhibit a short half-life.

[0007] What is of interest always is to produce bispecific antibodies (BsAb), which combines the antigen binding sites of two antibodies in a single molecule, therefore, can always combine two different antigens simultaneously.Except the application of diagnostic purposes, this class of molecules is also, for example, by redirecting effective effector systems to the diseased area (wherein cancer cells usually develop the mechanism of suppressing the normal immune response caused by monoclonal antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)) or by improving the neutralization or stimulating activity of antibodies, for new therapeutic applications to be prepared.This potential has been recognized in the early days, and multiple methods for obtaining this class of bispecific antibodies have been produced.The initial attempt to connect the binding specificity of two complete antibodies for different target antigens for therapeutic purposes has utilized the heterotypic conjugation (heteroconjugate) molecule (Staerz etc., 1985, Nature 314 (6012): 628-31) of chemical fusion.

[0008] Bispecific antibodies were initially prepared by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein et al., 1983, Nature 305(5934):537-40), but the complexity of the species produced in cell culture (up to 10 different species) makes purification difficult and expensive (George et al., 1997, The Antibodies 4:99-141 (Capra et al., eds., Harwood Academic Publishers). In this format, mouse IgG2a and rat IgG2b antibodies are produced simultaneously in the same cell (e.g., as a quadroma fusion of two hybridomas, or in engineered CHO cells). Since the light chain of each antibody preferentially associates with the heavy chain of its cognate type, three main types of antibodies are assembled: two parent antibodies and a heterodimer of the two antibodies comprising one heavy chain / light chain pair of each (associated via its Fc portion). The desired heterodimer can be purified from this mixture because its binding properties to Protein A differ from those of the parent antibody: rat IgG2b does not bind to Protein A, while mouse IgG2a does. Thus, the mouse-rat heterodimer binds to Protein A but elutes at a higher pH than the mouse IgG2a homodimer, which enables the selective purification of the bispecific heterodimer (Lindhofer et al., 1995, J. Immunol. 155(1):219-25). The resulting bispecific heterodimer is completely non-human and therefore highly immunogenic, which may have adverse side effects (e.g., "HAMA" or "HARA" reactions) and / or negate therapeutic properties. There is a need for engineered bispecifics with superior properties that can be readily produced in high yield from mammalian cell culture.

[0009] Despite promising results using heteroconjugates or bispecific antibodies produced by cell fusion as described above, several factors make them impractical for large-scale therapeutic applications, including the rapid clearance of heteroconjugate antibodies from the body, the intensive laboratory techniques required to produce any type of molecule, the need to purify heteroconjugates in large quantities from homoconjugates or monospecific antibodies, and the low yields typically achieved.

[0010] Genetic engineering has been used with increasing frequency to design, modify and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions. Various recombinant methods have been developed for the efficient production of BsAbs: both as antibody fragments (Carter et al., 1995, J. Hematother. 4(5):463-70; Pluckthun et al., 1997, Immunotechnology 3(2):83-105; Todorovska et al., 2001, J. Immunol. Methods 248(1-2):47-66) and full-length IgG forms (Carter, 2001, J. Immunol. Methods 248(1-2):7-15).

[0011] Combining two different scFvs results in a BsAb format with minimal molecular weight, termed sc-BsAb or Ta-scFv (Mack et al., 1995, Proc. Natl. Acad. Sci. USA 92(15):7021-25; Mallender et al., 1994, J. Biol. Chem. 269(1):199-206). BsAbs have been constructed by genetically fusing two scFvs to a dimerization functional group (e.g., a leucine zipper) (Kostelny et al., 1992, J. Immunol. 148(5):1547-53; de Kruif et al., 1996, J. Biol. Chem. 271(13):7630-34).

[0012] Diabodies are small bivalent and bispecific antibody fragments. L Connect V H, which uses a linker (less than 12 amino acid residues) that is too short to allow the two domains on the same chain to pair. The domains are forced to perform intermolecular pairing with the complementary domains of another chain and produce two antigen-binding sites. These dimeric antibody fragments, or "diabodies" are bivalent and bispecific (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90 (14): 6444-48). The size of diabodies is similar to that of Fab fragments. The V domains are connected by a linker between 3 and 12 amino acid residues. H and V L The polypeptide chains of the domains mainly form dimers (diabodies), while with a linker between 0 and 2 amino acid residues, trimers (tribodies) and tetrabodies (tetrabodies) are predominant. In addition to the linker length, the exact form of oligomerization also seems to depend on the composition and orientation of the variable domains (Hudson et al., 1999, J. Immunol. Methods 231 (1-2): 177-89). The predictability of the final structure of the diabody molecule is very poor.

[0013] Although sc-BsAb and diabody-based constructs show attractive clinical potential, it has been shown that such non-covalently associated molecules are not sufficiently stable under physiological conditions. The overall stability of scFv fragments depends on the V L and V H The intrinsic stability of the domain and the stability of the domain interface. H -V L Insufficient stability of the interface is often implicated as the primary cause of irreversible scFv inactivation, as transient opening of the interface (which is permitted by the peptide linker) exposes hydrophobic patches that promote aggregation, leading to instability and poor production yields ( et al., 2001, J. Mol. Biol. 305(5):989-1010).

[0014] Since V H and V L An alternative approach to preparing bispecific, bivalent antigen-binding proteins using a bicistronic vector encoding two polypeptide chains is described in U.S. Pat. No. 5,989,830. This type of double head and dual Fv configuration is achieved by expressing a bicistronic vector encoding two polypeptide chains. In a dual Fv configuration, the variable domains of two different antibodies are expressed in tandem on two separate chains (one heavy chain and one light chain), with one polypeptide chain having two tandem V domains separated by a peptide linker. H (V H1 -Connector-V H2 ), the other polypeptide chain consists of complementary VL Domain (V L1 -Connector-V L2 In the exchanged two-head configuration, the variable domains of two different antibodies are expressed in tandem on two separate polypeptide chains (one heavy chain and one light chain), with one polypeptide chain having two tandem V domains separated by a peptide linker. H (V H1 -Connector-V H2 ), the other polypeptide chain consists of complementary V L Domain (V L2 -Connector-V L1 ). Molecular modeling of the construct indicated that the linker size is long enough to span (15-20 amino acid residues).

[0015] Increasing the valency of an antibody is valuable because it increases the functional affinity of the antibody due to avidity. Polyvalent protein complexes (PPCs) with increased valency are described in U.S. Patent Application Publication No. US 2005 / 0003403 A1. A PPC comprises two polypeptide chains, typically arranged sideways to one another. Each polypeptide chain typically comprises three or four "v-regions," which comprise amino acid sequences capable of forming an antigen binding site when matched with corresponding v-regions on the opposing polypeptide chain. Up to about six "v-regions" may be used per polypeptide chain. The v-regions of each polypeptide chain may be linearly linked to one another and may be connected by interspersed linker regions. When arranged in the form of a PPC, the v-regions on each polypeptide chain form their own antigen binding site. The complex may contain one or more binding specificities.

[0016] Carter et al. (Ridgway et al., 1996, Protein Eng. 9(7):617-21; Carter, 2011, J. Immunol. Methods 248(1-2):7-15) proposed the use of Fc C H3 A strategy of generating Fc heterodimers by a set of “knob-into-hole” mutations in the domains. These mutations result in a structurally conserved hydrophobic core within the C H3 The altered residue packing complementarity between the domain interfaces favors the formation of heterodimers over homodimers, which enables good heterodimer expression from mammalian cell culture. Although this strategy results in higher heterodimer yields, homodimerization is not completely inhibited (Merchant et al., 1998, Nat. Biotechnol. 16(7):677-81).

[0017] Gunasekaran et al. studied the effect of C H3 Charge complementarity at the domain interface facilitates the formation of Fc heterodimers while maintaining the feasibility of the hydrophobic core intact (Gunasekaran et al., 2010, J. Biol. Chem. 285(25):19637-46). Taking advantage of the electrostatic manipulation mechanism, these constructs show that mutation of two pairs of peripherally located charged residues effectively promotes the formation of Fc heterodimers with minimal homodimer contamination. In contrast to the knob-into-hole design, homodimers are uniformly suppressed but not completely avoided due to the electrostatic repulsion mechanism.

[0018] Davis et al. described a novel method for the synthesis of human IgG and IgA C without introducing additional interchain disulfide bonds. H3 The β-chain segments of the Fc domains are staggered to convert Fc homodimers into heterodimers (Davis et al., 2010, Protein Eng. Des. Sel. 23(4):195-202). The SEEDbody (Sb) fusion protein is expressed in mammalian cells to produce Sb heterodimers in high yield, which can be easily purified to remove small amounts of byproducts.

[0019] US Patent Application Publication No. US 2010 / 331527 A1 describes a C-based H3 A bispecific antibody with a heterodimerization domain, in which a C H3 The mutations H95R and Y96F in the domain. These amino acid substitutions are derived from the C H3 domains and will heterodimerize with the IgG1 backbone. The common light chain that readily pairs with each heavy chain is based on the C H3 A prerequisite for all forms of domain heterodimerization. Thus, a total of 3 types of antibodies were produced: 50% with a pure IgG1 backbone, 1 / 3 with a pure H95R and Y96F mutant backbone, and 1 / 3 with two different heavy chains (bispecific). The desired heterodimer can be purified from this mixture because its binding properties to protein A are different from those of the parent antibody: IgG3-derived C H3 The domain does not bind to Protein A, but does bind to IgG1. Thus, the heterodimer binds to Protein A but elutes at a higher pH than the pure IgG1 homodimer, enabling selective purification of the bispecific heterodimer.

[0020] U.S. Patent No. 7,612,181 describes a dual variable domain IgG (DVD-IgG) bispecific antibody based on the dual Fv format described in U.S. Patent No. 5,989,830. Similar bispecific formats are also described in U.S. Patent Application Publication No. US 2010 / 0226923 A1. The constant domains are added to the corresponding chains of the dual Fv (C H1 -Fc added to the heavy chain, kappa or lambda constant domain added to the light chain) results in functional bispecific antibodies without any need for additional modifications (i.e., the obvious addition of constant domains to improve stability). Certain antibodies expressed in DVD-Ig / TBTI format show position effects on the second (or deepest) antigen binding site (Fv2). Depending on the sequence and nature of the antigen recognized by the Fv2 site, this antibody domain shows a reduced affinity for its antigen (i.e., loss of on-rate compared to the parent antibody). One possible explanation for this observation is that V L1 and V L2 The linker protrudes into the CDR region of Fv2, making Fv2 somewhat inaccessible to larger antigens.

[0021] The second configuration of bispecific antibody fragments described in U.S. Patent No. 5,989,830 is the cross-over double head (CODH), which has the following orientation of the variable domains expressed on both chains:

[0022] V L1 -Connector-V L2 , for the light chain, and

[0023] V H2 -Connector-V H1 , for heavy chain

[0024] The '830 patent discloses that a bispecific crossover two-headed antibody fragment (construct GOSA.E) retains higher binding activity than a dual Fv (see page 20, lines 20-50 of the '830 patent), and further discloses that this format is less affected by the linker used between the variable domains (see pages 20-21 of the '830 patent). SUMMARY OF THE INVENTION

[0025] The present invention provides an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have a structure represented by the following formula:

[0026] V L1 -L1-V L2 -L2-C L [I]

[0027] and two polypeptide chains having a structure represented by the following formula:

[0028] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0029] in:

[0030] V L1 is the first immunoglobulin light chain variable domain;

[0031] V L2 is a second immunoglobulin light chain variable domain;

[0032] V H1 is the first immunoglobulin heavy chain variable domain;

[0033] V H2 is a second immunoglobulin heavy chain variable domain;

[0034] C L is the immunoglobulin light chain constant domain;

[0035] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0036] Fc is the hinge region and C H2 、C H3 immunoglobulin heavy chain constant domain;

[0037] L1, L2, L3, and L4 are amino acid linkers;

[0038] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0039] The present invention also provides an antibody-like binding protein comprising two polypeptide chains forming two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the following formula:

[0040] V L1 -L1-V L2 -L2-C L [I]

[0041] and the second polypeptide chain has a structure represented by the following formula:

[0042] V H2 -L3-V H1 -L4-C H1 [II]

[0043] in:

[0044] V L1 is the first immunoglobulin light chain variable domain;

[0045] V L2 is a second immunoglobulin light chain variable domain;

[0046] V H1 is the first immunoglobulin heavy chain variable domain;

[0047] V H2 is a second immunoglobulin heavy chain variable domain;

[0048] C L is the immunoglobulin light chain constant domain;

[0049] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0050] L1, L2, L3, and L4 are amino acid linkers;

[0051] and wherein the first and second polypeptides form a crossover light chain-heavy chain pair.

[0052] The present invention also provides a method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, the method comprising identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, wherein each variable domain comprises a V L and V H ; specify light chain or heavy chain as template chain; specify V of first antibody variable domain or second antibody variable domain L As V L1 ; V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0053] V L1 -L1-V L2 -L2-C L [I]

[0054] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0055] Determining the maximum and minimum lengths of L1, L2, L3, and L4; generating polypeptide structures of Formula I and Formula II; selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0056] in:

[0057] V L1is the first immunoglobulin light chain variable domain;

[0058] V L2 is a second immunoglobulin light chain variable domain;

[0059] V H1 is the first immunoglobulin heavy chain variable domain;

[0060] V H2 is a second immunoglobulin heavy chain variable domain;

[0061] C L is the immunoglobulin light chain constant domain;

[0062] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0063] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and

[0064] L1, L2, L3, and L4 are amino acid linkers;

[0065] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0066] The present invention also provides a method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, the method comprising identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, wherein each variable domain comprises a V L and V H ; specify light chain or heavy chain as template chain; specify V of first antibody variable domain or second antibody variable domain L As V L1 ; V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0067] V L1 -L1-V L2 -L2-C L [I]

[0068] V H2 -L3-V H1 -L4-C H1 [II]

[0069] Determining the maximum and minimum lengths of L1, L2, L3, and L4; generating polypeptide structures of Formula I and Formula II; selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0070] in:

[0071] V L1 is the first immunoglobulin light chain variable domain;

[0072] V L2 is a second immunoglobulin light chain variable domain;

[0073] V H1 is the first immunoglobulin heavy chain variable domain;

[0074] V H2 is a second immunoglobulin heavy chain variable domain;

[0075] C L is the immunoglobulin light chain constant domain;

[0076] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0077] L1, L2, L3, and L4 are amino acid linkers;

[0078] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0079] Specific embodiments of the invention will be apparent from the following more detailed description of certain embodiments and the claims.

[0080] Specifically, the present invention relates to the following:

[0081] 1. An antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, wherein two of the polypeptide chains have a structure represented by the following formula:

[0082] V L1 -L1-V L2 -L2-C L [I]

[0083] And the two polypeptide chains have a structure represented by the following formula:

[0084] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0085] in:

[0086] VL1 is the first immunoglobulin light chain variable domain;

[0087] V L2 is a second immunoglobulin light chain variable domain;

[0088] V H1 is the first immunoglobulin heavy chain variable domain;

[0089] V H2 is a second immunoglobulin heavy chain variable domain;

[0090] C L is the immunoglobulin light chain constant domain;

[0091] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0092] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and L1, L2, L3, and L4 are amino acid linkers;

[0093] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0094] 2. The antibody-like binding protein of claim 1, wherein:

[0095] The length of L1 is 3–12 amino acid residues;

[0096] L2 is 3–14 amino acid residues in length;

[0097] L3 is 1-8 amino acid residues in length; and

[0098] The length of L4 is 1-3 amino acid residues.

[0099] 3. The antibody-like binding protein of item 1 or 2, wherein:

[0100] L1 is 5–10 amino acid residues in length;

[0101] L2 is 5–8 amino acid residues in length;

[0102] L3 is 1-5 amino acid residues in length; and

[0103] L4 is 1-2 amino acid residues in length.

[0104] 4. The antibody-like binding protein of any one of items 1 to 3, wherein:

[0105] L1 is 7 amino acid residues in length;

[0106] L2 is 5 amino acid residues in length;

[0107] L3 is 1 amino acid residue in length; and

[0108] L4 is 2 amino acid residues in length.

[0109] 5. The antibody-like binding protein of item 1, wherein:

[0110] L1 is 1–3 amino acid residues in length;

[0111] L2 is 1–4 amino acid residues in length;

[0112] L3 is 2-15 amino acid residues in length; and

[0113] The length of L4 is 2-15 amino acid residues.

[0114] 6. The antibody-like binding protein of item 1 or 5, wherein:

[0115] L1 is 1–2 amino acid residues in length;

[0116] L2 is 1–2 amino acid residues in length;

[0117] L3 is 4-12 amino acid residues in length; and

[0118] The length of L4 is 2-12 amino acid residues.

[0119] 7. The antibody-like binding protein of any one of items 1, 5 or 6, wherein:

[0120] L1 is 1 amino acid residue in length;

[0121] L2 is 2 amino acid residues in length;

[0122] L3 is 7 amino acid residues in length; and

[0123] L4 is 5 amino acid residues in length.

[0124] 8. The antibody-like binding protein of item 1, wherein the length of L1 is 0 amino acid residues and the length of L3 is 2 or more amino acid residues.

[0125] 9. The antibody-like binding protein of item 1, wherein the length of L3 is 0 amino acid residues and the length of L1 is 1 or more amino acid residues.

[0126] 10. The antibody-like binding protein of item 1, wherein the length of L4 is 0 amino acid residues and the length of L2 is 3 or more amino acid residues.

[0127] 11. The antibody-like binding protein of any one of items 1 to 10, wherein the binding protein is capable of specifically binding to one or more antigenic targets.

[0128] 12. The antibody-like binding protein of any one of items 1 to 11, wherein the one or more antigenic targets are selected from B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eosinophil chemoattractant), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2 / eosinophil chemoattractant-2), CCL25 (TECK), CCL26 (eosinophil chemoattractant-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2 ), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrins), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5 / CCXCR1), DNGR-1 (CLEC91), and HMGB1.

[0129] 13. The antibody-like binding protein of any one of items 1 to 12, wherein the binding protein is bispecific and is capable of binding to two different antigenic targets.

[0130] 14. The antibody-like binding protein of any one of items 1-13, wherein the two different antigen targets are selected from IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL1β, TNFα and IL-23 and IL17 and IL23.

[0131] 15. The antibody-like binding protein of any one of items 1 to 14, wherein the binding protein is capable of inhibiting one or more functions of the antigenic targets.

[0132] 16. The antibody-like binding protein of any one of items 1 to 15, wherein at least one of the linkers selected from L1, L2, L3 and L4 contains at least one cysteine ​​residue.

[0133] 17. An antibody-like binding protein comprising two polypeptide chains forming two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the following formula:

[0134] V L1 -L1-V L2 -L2-C L [I]

[0135] And the second polypeptide chain has a structure represented by the following formula:

[0136] V H2 -L3-V H1 -L4-C H1 [II]

[0137] in:

[0138] V L1 is the first immunoglobulin light chain variable domain;

[0139] V L2 is a second immunoglobulin light chain variable domain;

[0140] V H1 is the first immunoglobulin heavy chain variable domain;

[0141] V H2 is a second immunoglobulin heavy chain variable domain;

[0142] C L is the immunoglobulin light chain constant domain;

[0143] C H1 Immunoglobulin C H1a heavy chain constant domain; and

[0144] L1, L2, L3, and L4 are amino acid linkers;

[0145] and wherein the first and second polypeptides form a crossover light chain-heavy chain pair.

[0146] 18. The antibody-like binding protein of claim 17, wherein:

[0147] The length of L1 is 3–12 amino acid residues;

[0148] L2 is 3–14 amino acid residues in length;

[0149] L3 is 1-8 amino acid residues in length; and

[0150] The length of L4 is 1-3 amino acid residues.

[0151] 19. The antibody-like binding protein of item 17 or 18, wherein:

[0152] L1 is 5–10 amino acid residues in length;

[0153] L2 is 5–8 amino acid residues in length;

[0154] L3 is 1-5 amino acid residues in length; and

[0155] L4 is 1-2 amino acid residues in length.

[0156] 20. The antibody-like binding protein of any one of items 17 to 19, wherein:

[0157] L1 is 7 amino acid residues in length;

[0158] L2 is 5 amino acid residues in length;

[0159] L3 is 1 amino acid residue in length; and

[0160] L4 is 2 amino acid residues in length.

[0161] 21. The antibody-like binding protein of claim 17, wherein:

[0162] L1 is 1–3 amino acid residues in length;

[0163] L2 is 1–4 amino acid residues in length;

[0164] L3 is 2-15 amino acid residues in length; and

[0165] The length of L4 is 2-15 amino acid residues.

[0166] 22. The antibody-like binding protein of item 17 or 21, wherein:

[0167] L1 is 1–2 amino acid residues in length;

[0168] L2 is 1–2 amino acid residues in length;

[0169] L3 is 4-12 amino acid residues in length; and

[0170] The length of L4 is 2-12 amino acid residues.

[0171] 23. The antibody-like binding protein of any one of items 17, 21 or 22, wherein:

[0172] L1 is 1 amino acid residue in length;

[0173] L2 is 2 amino acid residues in length;

[0174] L3 is 7 amino acid residues in length; and

[0175] L4 is 5 amino acid residues in length.

[0176] 24. The antibody-like binding protein of item 17, wherein the length of L1 is 0 amino acid residues and the length of L3 is 2 or more amino acid residues.

[0177] 25. The antibody-like binding protein of item 17, wherein the length of L3 is 0 amino acid residues and the length of L1 is 1 or more amino acid residues.

[0178] 26. The antibody-like binding protein of item 17, wherein the length of L4 is 0 amino acid residues and the length of L2 is 3 or more amino acid residues.

[0179] 27. The antibody-like binding protein of any one of items 17-26, wherein the binding protein is capable of specifically binding to one or more antigenic targets.

[0180] 28. The antibody-like binding protein of any one of items 17-27, wherein the one or more antigenic targets are selected from B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eosinophil chemoattractant), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2 / eosinophil chemoattractant-2), CCL25 (TECK), CCL26 (eosinophil chemoattractant-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2 ), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrins), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5 / CCXCR1), DNGR-1 (CLEC91), and HMGB1.

[0181] 29. The antibody-like binding protein of any one of items 17-28, wherein the binding protein is bispecific and is capable of binding to two different antigenic targets.

[0182] 30. The antibody-like binding protein of any one of items 17-29, wherein the two different antigen targets are selected from IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL1β, TNFα and IL-23 and IL17 and IL23.

[0183] 31. The antibody-like binding protein of any one of items 17-30, wherein the binding protein is capable of inhibiting one or more functions of the antigen target.

[0184] 32. The antibody-like binding protein of any one of items 17-31, wherein at least one of the linkers selected from L1, L2, L3 and L4 contains at least one cysteine ​​residue.

[0185] 33. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody-like binding protein of any one of items 1-32.

[0186] 34. An expression vector comprising the nucleic acid molecule of item 33.

[0187] 35. An isolated host cell comprising the nucleic acid molecule of item 33 or the expression vector of item 34.

[0188] 36. The host cell of claim 35, wherein the host cell is a mammalian cell or an insect cell.

[0189] 37. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody-like binding protein of any one of items 1 to 32.

[0190] 38. A method for preparing the antibody-like binding protein of any one of items 1 to 16, the method comprising expressing one or more nucleic acid molecules encoding a polypeptide having a structure represented by the following formula [I] and formula [II] in a cell:

[0191] V L1 -L1-V L2 -L2-C L [I]

[0192] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0193] in:

[0194] V L1is the first immunoglobulin light chain variable domain;

[0195] V L2 is a second immunoglobulin light chain variable domain;

[0196] V H1 is the first immunoglobulin heavy chain variable domain;

[0197] V H2 is a second immunoglobulin heavy chain variable domain;

[0198] C L is the immunoglobulin light chain constant domain;

[0199] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0200] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and

[0201] L1, L2, L3, and L4 are amino acid linkers;

[0202] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0203] 39. A method for preparing the antibody-like binding protein of any one of items 17 to 32, the method comprising expressing one or more nucleic acid molecules encoding a polypeptide having a structure represented by the following formula [I] and formula [II] in a cell:

[0204] V L1 -L1-V L2 -L2-C L [I]

[0205] V H2 -L3-V H1 -L4-C H1 [II]

[0206] in:

[0207] V L1 is the first immunoglobulin light chain variable domain;

[0208] V L2 is a second immunoglobulin light chain variable domain;

[0209] V H1 is the first immunoglobulin heavy chain variable domain;

[0210] V H2is a second immunoglobulin heavy chain variable domain;

[0211] C L is the immunoglobulin light chain constant domain;

[0212] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0213] L1, L2, L3, and L4 are amino acid linkers;

[0214] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0215] 40. A method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, the method comprising:

[0216] (a) identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, wherein each variable domain comprises V L and V H ;

[0217] (b) designating the light chain or the heavy chain as the template chain;

[0218] (c) specifying the V domain of the first antibody variable domain or the second antibody variable domain L As V L1 ;

[0219] (d) V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0220] V L1 -L1-V L2 -L2-C L [I]

[0221] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0222] (e) Determine the maximum and minimum lengths of L1, L2, L3, and L4;

[0223] (f) generating polypeptide structures of Formula I and Formula II;

[0224] (g) selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0225] in:

[0226] V L1 is the first immunoglobulin light chain variable domain;

[0227] V L2 is a second immunoglobulin light chain variable domain;

[0228] V H1 is the first immunoglobulin heavy chain variable domain;

[0229] V H2 is a second immunoglobulin heavy chain variable domain;

[0230] C L is the immunoglobulin light chain constant domain;

[0231] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0232] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and

[0233] L1, L2, L3, and L4 are amino acid linkers;

[0234] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0235] 41. The method of claim 40, wherein the first antibody variable domain and the second antibody variable domain are the same.

[0236] 42. A method for preparing an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, the method comprising:

[0237] (a) identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, wherein each variable domain comprises V L and V H ;

[0238] (b) designating the light chain or the heavy chain as the template chain;

[0239] (c) specifying the V domain of the first antibody variable domain or the second antibody variable domain L As V L1 ;

[0240] (d) V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0241] V L1-L1-V L2 -L2-C L [I]

[0242] V H2 -L3-V H1 -L4-C H1 [II]

[0243] (e) Determine the maximum and minimum lengths of L1, L2, L3, and L4;

[0244] (f) generating polypeptide structures of Formula I and Formula II;

[0245] (g) selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0246] in:

[0247] V L1 is the first immunoglobulin light chain variable domain;

[0248] V L2 is a second immunoglobulin light chain variable domain;

[0249] V H1 is the first immunoglobulin heavy chain variable domain;

[0250] V H2 is a second immunoglobulin heavy chain variable domain;

[0251] C L is the immunoglobulin light chain constant domain;

[0252] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0253] L1, L2, L3, and L4 are amino acid linkers;

[0254] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0255] 43. The method of claim 42, wherein the first antibody variable domain and the second antibody variable domain are the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0256] Figure 1 Antigen binding domains Fv1 and Fv2 in a dual V region configuration and their respective peptide linkers L in the TBTI format L and L H Schematic diagram of the arrangement.

[0257] Figure 2Schematic representation of the arrangement of the antigen-binding domains Fv1 (anti-IL4) and Fv2 (anti-IL13) and their respective peptide linkers within the exchange dual variable (CODV) configuration (2D).

[0258] Figure 3 .Schematic diagram of Fv anti-IL4 and Fab anti-IL13, showing a possible spatial arrangement obtained by protein-protein docking of anti-IL4 Fv and anti-IL13 Fv.

[0259] Figure 4 In the BIACORE assay, the tetravalent bispecific binding capacity of CODV protein was evaluated by injecting two antigens sequentially or simultaneously into a DVD-Ig protein-coated chip. The maximum signal observed by sequential injection was obtained by co-injection of the two antigens, indicating that all binding sites were saturated.

[0260] Figure 5 .Antigen binding domains in CODV configuration and their respective peptide linkers L L (L1 and L2) and L H Schematic diagram of the arrangement of (L3 and L4) (2D). In panel A, the light chains remain in a "linear or template" arrangement, while the heavy chains are in an "exchange" configuration. In panel B, the heavy chains remain in a "linear or template" arrangement, while the light chains are in an "exchange" configuration.

[0261] Figure 6 Schematic diagram of CODV-Ig design depending on whether the light or heavy chain is used as the "template".

[0262] Figure 7 . Comparison of TBTI / DVD-Ig or CODV-Ig molecules incorporating anti-IL4 and anti-IL13 sequences.

[0263] Figure 8 Comparison of CODV-Fab and B-Fab formats in cytotoxicity assay using NALM-6 cells. Detailed Description of the Invention

[0264] The present invention provides antibody-like binding proteins comprising four polypeptide chains forming four antigen-binding sites, wherein each pair of polypeptides forming the antibody-like binding protein has dual variable domains with an exchange orientation. The present invention also provides methods for preparing such antigen-like binding proteins.

[0265] Computer modeling predicts that the exchange double-headed (CODH) design of U.S. Patent number 5,989,830 can produce such complex, wherein two binding sites face with relative orientation, without the constraint proposed to the dual Fv configuration of U.S. Patent number 7,612,181. Specifically, computer modeling shows that the length of amino acid linker between variable domains is not critical for CODH design, but is important for allowing sufficient access to two antigen binding sites in dual Fv design. As DVD-Ig / TBTI form, such antibody-like binding protein construct is prepared, wherein constant domain is connected with CODH configuration to form an antibody-like binding protein comprising 4 polypeptide chains forming 4 antigen binding sites, wherein every pair of polypeptides forming antibody-like binding protein has the dual variable domains (i.e. CODH-Ig) with exchange orientation. Expectation compares with CODH molecule, CODH-Ig molecule has significantly improved stability (because DVD-Ig / TBTI has the improved stability relative to dual Fv molecule).

[0266] To verify this hypothesis, CODH-Ig molecules were prepared using the anti-IL4 and anti-IL13 antibody sequences described in U.S. Patent Application Publication No. US 2010 / 0226923 A1. CODH-Ig molecules differ from the CODH molecules of US 2010 / 0226923 in terms of the length of the amino acid linker separating the variable domains on their respective polypeptide chains. CODH-Ig molecules were expressed in cells after transient transfection and then purified by protein A chromatography. Although their size exclusion chromatography (SEC) profiles showed 5-10% aggregation levels, none of the CODH-Ig molecules were functional, and therefore none of the CODH-Ig molecules were able to bind to all of their target antigens. The lack of antigen-binding activity may be due to disruption of the dimerization of the heavy and light chain Fv regions due to inappropriate linker lengths that impair correct paratope formation. Therefore, a scheme for determining a suitable amino acid linker was developed to be inserted between the two variable domains and the second variable domain and the constant domain on both the heavy and light chains of the antibody-like binding protein. The approach was based on protein-protein docking of homologous and experimental models of the FvIL4 and FvIL13 regions, incorporation of a model Fc1 domain, and construction of suitable linkers between the FvIL4 and FvIL13 regions and between the Fv and constant Fc1 regions, respectively.

[0267] Standard recombinant DNA methods are used to construct polynucleotides encoding polypeptides that form antibody-like binding proteins of the present invention, incorporate these polynucleotides into recombinant expression vectors, and import these vectors into host cells. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd edition). Enzymatic reactions and purification techniques can be carried out according to the manufacturer's instructions, as commonly implemented in the art, or as described herein. Unless specific definitions are provided, the terms used in conjunction with laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein are well-known and commonly used terms in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment.

[0268] 1. General Definition

[0269] As used in this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated: Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0270] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. In certain embodiments, the nucleotides comprising a polynucleotide can be a modified form of a ribonucleotide or a deoxyribonucleotide or any nucleotide type. The modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3'-dideoxyribose and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, selenophosphate (phosphoroselenoate), diselenphosphate, phosphoroanilothioate, phoshoraniladate and phosphoramidate (phosphoroamidate). The term "polynucleotide" clearly includes single-stranded and double-stranded forms of DNA.

[0271] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, by reason of its origin: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide naturally occurs, (2) is linked to a polynucleotide to which it is not naturally linked, or (3) does not naturally occur as part of a larger sequence.

[0272] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it is normally present, (2) is substantially free of other polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is naturally associated, (5) is not associated (by covalent or non-covalent interactions) with portions of polypeptides with which the "isolated polypeptide" is naturally associated, (6) is operatively associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) does not occur in nature. The isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants present in its natural environment that might interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0273] As used herein, the term "human antibody" includes antibodies whose variable and constant regions are substantially equivalent to those of human germline immunoglobulin sequences. In some embodiments, human antibodies are produced in non-human mammals, including but not limited to rodents (e.g., mice and rats) and lagomorphs (e.g., rabbits). In other embodiments, human antibodies are produced in hybridoma cells. In still other embodiments, human antibodies are produced recombinantly.

[0274] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100-110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines the constant domains responsible for effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide includes the variable domains (V H ) and three constant domains (C H1 、C H2 and C H3 ), where V H The domain is located at the amino terminus of the polypeptide, C H3 The variable domain is located at the carboxyl terminus, while the full-length light chain immunoglobulin polypeptide includes the variable domain (V L ) and constant domain (C L ), where V L The domain is located at the amino terminus of the polypeptide, C L The domain is located at the carboxyl terminus.

[0275] Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as μ, δ, γ, α, or ε, which define the isotype of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is also subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are generally connected by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair generally form an antigen binding site. The variable domains of naturally occurring antibodies typically display the same general structure of relatively conservative framework regions (FRs) connected by three hypervariable regions (also known as complementary determining regions or CDRs). The CDRs of each pair of two chains are typically aligned by the framework regions, which can enable binding to a specific epitope. From amino-terminal to carboxyl-terminal, the variable domains of both light and heavy chains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0276] The term "native Fc" as used herein refers to a molecule comprising a sequence of a non-antigen binding fragment obtained by antibody digestion or produced by other methods, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of natural Fc is preferably human, and may be any immunoglobulin, although IgG1 and IgG2 are preferred. Natural Fc molecules are composed of monomeric polypeptides that can be linked to form dimers or multimers by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomer subunits of natural Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of natural Fc is a disulfide-bonded dimer obtained from papain digestion of IgG. The term "native Fc" as used herein is a general term for monomeric, dimeric, and multimeric forms.

[0277] As used herein, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" may include a molecule or sequence that has been humanized from a non-human native Fc. Furthermore, a native Fc contains regions that can be removed because they provide structural features or biological activities that are not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues are modified, which affects or participates in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0278] As used herein, the term "Fc domain" includes native Fc and Fc variants and sequences defined above. Like Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from intact antibodies or produced by other methods.

[0279] As used herein, the term "antibody-like binding protein" refers to a non-naturally occurring (or recombinant) molecule that specifically binds to at least one target antigen and comprises four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have a structure represented by the following formula:

[0280] V L1 -L1-V L2 -L2-C L [I]

[0281] and two polypeptide chains having a structure represented by the following formula:

[0282] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0283] in:

[0284] V L1 is the first immunoglobulin light chain variable domain;

[0285] V L2 is a second immunoglobulin light chain variable domain;

[0286] V H1 is the first immunoglobulin heavy chain variable domain;

[0287] V H2 is a second immunoglobulin heavy chain variable domain;

[0288] C L is the immunoglobulin light chain constant domain;

[0289] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0290] Fc is the hinge region and C H2 、C H3 immunoglobulin heavy chain constant domain;

[0291] L1, L2, L3, and L4 are amino acid linkers;

[0292] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair. The term "antibody-like binding protein" as used herein also refers to a non-naturally occurring (or recombinant) molecule that specifically binds to at least one target antigen and comprises two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the following formula:

[0293] V L1 -L1-V L2 -L2-C L [I]

[0294] and the second polypeptide chain has a structure represented by the following formula:

[0295] V H2 -L3-V H1 -L4-C H1 [II]

[0296] in:

[0297] V L1 is the first immunoglobulin light chain variable domain;

[0298] V L2 is a second immunoglobulin light chain variable domain;

[0299] V H1 is the first immunoglobulin heavy chain variable domain;

[0300] V H2 is a second immunoglobulin heavy chain variable domain;

[0301] C L is the immunoglobulin light chain constant domain;

[0302] C H1 Immunoglobulin C H1heavy chain constant domain;

[0303] L1, L2, L3, and L4 are amino acid linkers;

[0304] and wherein the first and second polypeptides form a crossover light chain-heavy chain pair.A "recombinant" molecule is one that is prepared, expressed, produced or isolated by recombinant means.

[0305] One embodiment of the present invention provides an antibody-like binding protein with biological and immunological specificity for between 1 and 4 target antigens. Another embodiment of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide chain forming the antibody-like binding protein. Another embodiment of the present invention provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide chain forming the antibody-like binding protein. Yet another embodiment of the present invention provides a host cell expressing the antibody-like binding protein (i.e., comprising a nucleic acid molecule or vector encoding the polypeptide chain forming the antibody-like binding protein).

[0306] As used herein, the term "interchangeability" refers to the interchangeability of variable domains within the CODV format while maintaining folding and ultimately binding affinity. "Complete interchangeability" refers to exchanging the V domains in CODV-Ig (i.e., reversing the order) or CODV-Fab. H1 and V H2 The order of the two domains is thus swapped L1 and V L2 The ability to modify the order of the domains while maintaining the full functionality of the antibody-like binding protein, as demonstrated by retention of binding affinity. Furthermore, it should be noted that the identified V H and V L refers only to the position of the domain on a particular protein chain in its final form. For example, V H1 and V H2 Can be derived from V in the parent antibody L1 and V L2 domains and is placed in the V H1 and V H2 Position. Similarly, V L1 and V L2 Can be derived from V in the parent antibody H1 and V H2 domains and is placed in the V H1 and V H2 Therefore, V H and V L The labels refer to the present position, not the original position in the parent antibody. H and V LThe domains are thus "exchangeable".

[0307] An "isolated" antibody-like binding protein is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with the diagnostic or therapeutic use of the antibody-like binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody-like binding protein may be purified to: (1) greater than 95% by weight of the antibody and most preferably greater than 99% by weight as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody-like binding protein includes an antibody-like binding protein in situ within recombinant cells since at least one component of the natural environment of the antibody-like binding protein will not be present.

[0308] As used herein, the terms "substantially pure" or "substantially purified" refer to any compound or species that is the predominant species present (i.e., more abundant on a molar basis than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0309] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule that is capable of being bound by an antibody-like binding protein and that can be used in an animal to produce an antibody that binds to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes the target antigen. "Bivalent" antibody-like binding proteins, but not "multispecific" or "multifunctional" antibody-like binding proteins, are considered to contain antigen binding sites with the same antigenic specificity.

[0310] Bispecific or bifunctional antibodies are generally artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites or epitopes. Bispecific antibodies can be produced by various methods, including but not limited to fusion of hybridomas or linking of F(ab') fragments.

[0311] The F(ab) fragment usually contains a light chain and a heavy chain V H and CH1 domain, wherein the V H -C H1 The heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. The F(ab) fragment used herein may also include a light chain (containing two variable domains separated by an amino acid linker) and a heavy chain (containing two variable domains separated by an amino acid linker and a C H1 domain).

[0312] F(ab') fragments usually consist of a light chain and a more constant region (in C H1 with C H2 The F(ab')2 molecule is formed by binding to a portion of one heavy chain (between the two heavy chains) so that an interchain disulfide bond can form between the two heavy chains.

[0313] The phrases "biological property", "biological characteristic" and the term "activity" with respect to the antibody-like binding proteins of the present invention are used interchangeably herein and include, but are not limited to, epitope affinity and specificity, the ability to antagonize the activity of the antigenic target (or targeting polypeptide), the in vivo stability of the antibody-like binding protein and the immunogenic properties of the antibody-like binding protein. Other identifiable biological properties or characteristics of the antibody-like binding protein include, for example, cross-reactivity (i.e., generally with non-human homologs of the antigenic target or with other antigenic targets or tissues) and the ability to maintain high expression levels of the protein in mammalian cells. The above-mentioned properties or characteristics can be observed or measured using techniques recognized in the art, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis, in vitro and in vivo neutralization assays and immunohistochemistry using tissue sections from different sources (including humans, primates or any other sources that may be desired).

[0314] As used herein, the term "immunologically functional immunoglobulin fragment" refers to a polypeptide fragment that contains at least the CDRs of the immunoglobulin heavy chain or light chain from which the polypeptide fragment is derived. The immunologically functional immunoglobulin fragment is capable of binding to a target antigen.

[0315] As used herein, a "neutralizing" antibody-like binding protein refers to a molecule that blocks or substantially reduces the effector function of a target antigen to which it binds. "Substantially reduce" as used herein means that the effector function of the target antigen is reduced by at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, and most preferably at least about 90%.

[0316] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties. An epitope is a region of an antigen that is bound by an antibody or antibody-like binding protein. In certain embodiments, an antibody-like binding protein is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In preferred embodiments, the equilibrium dissociation constant is ≤10 -8 M, more preferably when the equilibrium dissociation constant ≤ 10 -9 M, most preferably when the dissociation constant ≤ 10 -10 When M, the antibody-like binding protein is said to bind specifically to the antigen.

[0317] The dissociation constant (K) of an antibody-like binding protein can be determined, for example, by surface plasmon resonance. D ). Generally, surface plasmon resonance analysis uses a BIAcore system (Pharmacia Biosensor; Piscataway, NJ) to measure the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (antibody-like binding protein in solution) by surface plasmon resonance (SPR). Surface plasmon resonance analysis can also be performed by immobilizing the analyte (antibody-like binding protein immobilized on a biosensor matrix) and providing the ligand (target antigen). As used herein, the term "K D ” refers to the dissociation constant of the interaction between a specific antibody-like binding protein and the target antigen.

[0318] As used herein, the term "specifically binds" refers to an antibody-like protein or antigen-binding fragment thereof that binds to an antigen with a specific binding affinity of at least about 1 x 10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 The ability of an antigen to bind to an antigen containing the epitope with a Kd of M or greater and / or to bind to the epitope with an affinity at least 2 times greater than its affinity for a nonspecific antigen.

[0319] The term "linker" as used herein refers to one or more amino acid residues inserted into the immunoglobulin domain to provide sufficient mobility for the domains of the light chain and heavy chain to fold into an exchange dual variable region immunoglobulin. At the sequence level, the linker is inserted at the transition between the variable domains or between the variable and constant domains, respectively. The transition between domains can be identified because the approximate size of the immunoglobulin domain is fully understood. The exact position of the domain transition can be determined by locating peptides that do not form secondary structural units such as β-sheets or α-helices, which can be confirmed by experimental data or can be assumed by modeling techniques or secondary structure predictions. The linker described herein is called L1, which is located at the N-terminal V L1 and V L2 On the interdomain light chain; L2, which is also located on the V L2 and C-end C L The heavy chain linker is called L3, which is located at the N-terminal V H2 and V H1 domains; and L4, which is located between the V H1 and C H1 - between Fc domains. Linkers L1, L2, L3 and L4 are independent, but they have the same sequence and / or length in some cases.

[0320] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term "vector" includes nucleic acid molecules capable of transporting another nucleic acid to which it has been connected. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which another DNA segment can be inserted. Another type of vector is a viral vector, in which another DNA segment can be inserted into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors having bacterial replication origins). Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are effectively connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, expression vectors used in recombinant DNA technology are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used vector form. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0321] As used herein, the term "operably linked" refers to an arrangement of flanking sequences, wherein the flanking sequences are configured or assembled so as to perform their normal functions. Thus, a flanking sequence that is operably linked to a coding sequence can be capable of effecting replication, transcription, and / or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of the coding sequence. A flanking sequence need not be contiguous with the coding sequence, as long as it functions correctly. Thus, for example, an intervening, untranslated, but transcribed sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0322] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell refers not only to a specific subject cell, but also to the progeny of such cells. Since certain modifications may occur in the offspring due to mutations or environmental influences, although such offspring may actually be different from the parent cell, such cells are still included within the scope of the term "host cell" as used herein. Various host cell expression systems can be used to express the antibody-like binding proteins of the present invention, including bacterial, yeast, baculovirus and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. In order to recombinantly express the antibody-like binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the antibody-like binding protein, so that the polypeptide chain is expressed in the host cell and preferably secreted into the culture medium in which the host cell is cultured, from which the antibody-like binding protein can be recovered.

[0323] As used herein, the term "transformation" refers to a change in the genetic properties of a cell. When a cell is modified to contain new DNA, the cell is transformed. For example, when it is genetically modified from its native state, the cell is transformed. After transformation, the transformed DNA can be recombined with the cell by naturally integrating into the cell chromosome, or can be instantaneously maintained as an additional element without replication, or can be independently replicated as a plasmid. When DNA replicates with cell division, the cell is considered to have been stably transformed. As used herein, the term "transfection" refers to the uptake of heterogeneous or exogenous DNA by the cell. When exogenous DNA is introduced into the cell membrane, the cell is "transfected." A variety of transfection techniques are well known in the art. Such techniques can be used to import one or more exogenous DNA molecules into suitable host cells.

[0324] As used herein and as applied to an object, the term "naturally occurring" refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans is naturally occurring. Similarly, "non-naturally occurring" as used herein refers to an object that does not exist in nature or has been structurally modified or synthesized by humans.

[0325] As used herein, the 20 commonly used amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 commonly used amino acids (e.g., d-amino acids); unnatural amino acids such as α-amino acids, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other uncommon amino acids may also be suitable components of the antibody-like binding protein polypeptide chains of the present invention. Examples of uncommon amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, in accordance with standard usage and convention, the left side is the amino terminal direction and the right side is the carboxyl terminal direction.

[0326] Naturally occurring residues can be classified according to the properties of their shared side chains:

[0327] (1) Hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0328] (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0329] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0330] (4) Aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

[0331] (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0332] (6) Acidic: Asp, Glu;

[0333] (7) Basic: His, Lys, Arg;

[0334] (8) Residues that affect chain orientation: Gly, Pro;

[0335] (9) Aromatic: His, Trp, Tyr, Phe; and

[0336] (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0337] Conservative amino acid substitutions can include exchanging a member of one of these classes with another member of the same class. Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid residues. Non-conservative substitutions can include exchanging a member of one of these classes for a member of another class.

[0338] Using well-known techniques, the skilled person will be able to determine suitable variants of the antibody-like binding protein polypeptide chains of the present invention. For example, by targeting regions that are not considered important for activity, one skilled in the art can identify suitable regions of the polypeptide chain that can be altered without destroying the activity. Alternatively, one skilled in the art can identify residues and portions of molecules that are conserved among similar polypeptides. Additionally, conservative amino acid substitutions can be made to regions that may be important for biological activity or structure without destroying the biological activity or adversely affecting the polypeptide structure.

[0339] As used herein, the term "patient" includes both human and animal subjects.

[0340] A "disorder" is any condition that may benefit from treatment using the antibody-like binding proteins of the invention."Disorder" and "condition" are used interchangeably herein and include chronic and acute disorders or diseases, including those pathological conditions that predispose a patient to the disorder.

[0341] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already with the disorder as well as those susceptible to the disorder or those in whom the disorder is to be prevented.

[0342] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to any compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0343] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or facilitating the delivery of an antibody-like binding protein.

[0344] The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more antibody-like binding proteins refer to an amount or dosage sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of an antibody-like binding protein that is sufficient to inhibit one or more clinically determined pathological processes associated with the condition to be treated for a certain period of time. The effective amount may vary with the specific antibody-like binding protein used, as well as various factors related to the patient being treated and the severity of the condition and disorder. For example, if the antibody-like binding protein is administered in vivo, factors such as the patient's age, weight and health status, as well as dose-response curves and toxicity data obtained in preclinical animal studies, will be among those considered. The determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of those skilled in the art.

[0345] One embodiment of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody-like binding protein.

[0346] 2. Antibody-like binding protein

[0347] In one embodiment of the present invention, the antibody-like binding protein comprises four polypeptide chains forming four antigen-binding sites, wherein two polypeptide chains have a structure represented by the following formula:

[0348] V L1 -L1-V L2 -L2-C L [I]

[0349] and two polypeptide chains having a structure represented by the following formula:

[0350] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0351] in:

[0352] V L1 is the first immunoglobulin light chain variable domain;

[0353] V L2 is a second immunoglobulin light chain variable domain;

[0354] V H1 is the first immunoglobulin heavy chain variable domain;

[0355] V H2 is a second immunoglobulin heavy chain variable domain;

[0356] C L is the immunoglobulin light chain constant domain;

[0357] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0358] Fc is the hinge region and C H2 、C H3 immunoglobulin heavy chain constant domain;

[0359] L1, L2, L3, and L4 are amino acid linkers;

[0360] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0361] In another embodiment of the present invention, the antibody-like binding protein comprises two polypeptide chains forming two antigen binding sites, wherein the first polypeptide chain has a structure represented by the following formula:

[0362] V L1 -L1-V L2 -L2-C L [I]

[0363] and the second polypeptide chain has a structure represented by the following formula:

[0364] V H2 -L3-V H1 -L4-C H1 [II]

[0365] in:

[0366] V L1 is the first immunoglobulin light chain variable domain;

[0367] V L2 is a second immunoglobulin light chain variable domain;

[0368] V H1 is the first immunoglobulin heavy chain variable domain;

[0369] V H2 is a second immunoglobulin heavy chain variable domain;

[0370] C L is the immunoglobulin light chain constant domain;

[0371] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0372] L1, L2, L3, and L4 are amino acid linkers;

[0373] and wherein the first and second polypeptides form a crossover light chain-heavy chain pair.

[0374] The antibody-like binding proteins of the present invention can be prepared from domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0375] In some antibody-like binding proteins of the invention, the length of L3 is at least twice the length of L1. In other antibody-like binding proteins of the invention, the length of L4 is at least twice the length of L2. In some antibody-like binding proteins of the invention, the length of L1 is at least twice the length of L3. In other antibody-like binding proteins of the invention, the length of L2 is at least twice the length of L4.

[0376] In some antibody-like binding proteins of the present invention, L1 is 3-12 amino acid residues in length, L2 is 3-14 amino acid residues in length, L3 is 1-8 amino acid residues in length, and L4 is 1-3 amino acid residues in length. In other antibody-like binding proteins, L1 is 5-10 amino acid residues in length, L2 is 5-8 amino acid residues in length, L3 is 1-5 amino acid residues in length, and L4 is 1-2 amino acid residues in length. In a preferred antibody-like binding protein, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length.

[0377] In some antibody-like binding proteins of the present invention, L1 is 1-3 amino acid residues in length, L2 is 1-4 amino acid residues in length, L3 is 2-15 amino acid residues in length, and L4 is 2-15 amino acid residues in length. In other antibody-like binding proteins, L1 is 1-2 amino acid residues in length, L2 is 1-2 amino acid residues in length, L3 is 4-12 amino acid residues in length, and L4 is 2-12 amino acid residues in length. In a preferred antibody-like binding protein, L1 is 1 amino acid residue in length, L2 is 2 amino acid residues in length, L3 is 7 amino acid residues in length, and L4 is 5 amino acid residues in length.

[0378] In some antibody-like binding proteins of the present invention, L1, L3, or L4 may be equal to zero. However, in antibody-like binding proteins in which L1, L3, or L4 are equal to zero, the corresponding transition linker between the variable region and the constant region or between the dual variable domains on the other chain cannot be zero. In some embodiments, L1 is equal to zero and L3 is 2 or more amino acid residues, L3 is equal to zero and L1 is equal to one or more amino acid residues, or L4 is equal to 0 and L2 is 3 or more amino acid residues.

[0379] In some antibody-like binding proteins of the invention, at least one linker selected from L1, L2, L3, and L4 contains at least one cysteine ​​residue.

[0380] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with 4 glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 25); a peptide with 5 glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 26); a peptide with 6 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 27); a peptide with 7 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 28); and a peptide with 8 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 29). Other combinations of amino acid residues can be used, for example, the peptide Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 30) and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31). Other suitable linkers include single Ser and Val residues; the dipeptides Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 52), Thr-Val-Ala-Ala-Pro (SEQ ID NO: 53), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 54), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 55), Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 48), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 49), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 50), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 51). The above examples are not intended to limit the scope of the invention in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable for the antibody-like binding proteins of the invention (see Example 12).

[0381] In the joint, the characteristic and sequence of amino acid residues can change with the type of secondary structure element that must be realized in the joint.For example, glycine, serine and alanine are most suitable for joints with maximum flexibility.If a more rigid and extended joint is needed, some combination of glycine, proline, threonine and serine is useful.When needed, any amino acid residue can be considered to be combined with other amino acid residues to build a joint of a larger peptide joint, which depends on the required properties.

[0382] In some antibody-like binding proteins of the present invention, V L1 comprising the amino acid sequence of SEQ ID NO: 1; V L2 comprising the amino acid sequence of SEQ ID NO: 3; V H1 comprising the amino acid sequence of SEQ ID NO: 2; V H2 Comprising the amino acid sequence of SEQ ID NO:4.

[0383] In some embodiments of the present invention, the antibody-like binding protein is capable of specifically binding to one or more antigen targets. In a preferred embodiment of the present invention, the antibody-like binding protein is capable of specifically binding to at least one antigen target selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eosinophil chemoattractant), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2 / eosinophil chemoattractant-2), CCL25 (TECK), CCL26 (eosinophil chemoattractant-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp -2), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrins), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNF-a, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5 / CCXCR1), DNGR-1 (CLEC91) and HMGB1. In other embodiments of the present invention, the antibody-like binding protein is capable of inhibiting the function of one or more antigenic targets.

[0384] In some embodiments of the present invention, the antibody-like binding protein is bispecific and is capable of binding to two different antigenic targets or epitopes. In a preferred embodiment of the present invention, the antibody-like binding protein is bispecific and each light chain-heavy chain pair is capable of binding to two different antigenic targets or epitopes. In a more preferred embodiment of the present invention, the antibody-like binding protein is capable of binding to two different antigenic targets selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL-1β, TNFα and IL-23, and IL17 and IL23. In an even more preferred embodiment, the antibody-like binding protein is capable of binding to the antigenic targets IL4 and IL13.

[0385] In some embodiments of the invention, the antibody-like binding protein specifically binds to IL4 with an association rate of 2.97E+07 and a dissociation rate of 3.30E-04, and specifically binds to IL13 with an association rate of 1.39E+06 and a dissociation rate of 1.63E-04. In other embodiments of the invention, the antibody-like binding protein specifically binds to IL4 with an association rate of 3.16E+07 and a dissociation rate of 2.89E-04, and specifically binds to IL13 with an association rate of 1.20E+06 and a dissociation rate of 1.12E-04.

[0386] In one embodiment of the invention, an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites is prepared by identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, each of the variable domains comprising a V L and V H ; specify light chain or heavy chain as template chain; specify V of first antibody variable domain or second antibody variable domain L As V L1 ; V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0387] V L1 -L1-V L2 -L2-C L [I]

[0388] V H2 -L3-V H1 -L4-C H1 -Fc [II]

[0389] Determining the maximum and minimum lengths of L1, L2, L3, and L4; generating polypeptide structures of Formula I and Formula II; selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0390] in:

[0391] V L1 is the first immunoglobulin light chain variable domain;

[0392] V L2 is a second immunoglobulin light chain variable domain;

[0393] V H1 is the first immunoglobulin heavy chain variable domain;

[0394] V H2 is a second immunoglobulin heavy chain variable domain;

[0395] C L is the immunoglobulin light chain constant domain;

[0396] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0397] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and

[0398] L1, L2, L3, and L4 are amino acid linkers;

[0399] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0400] In another embodiment of the invention, an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites is prepared by identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, wherein each variable domain comprises a V L and V H ; specify light chain or heavy chain as template chain; specify V of first antibody variable domain or second antibody variable domain L As V L1 ; V is specified according to the following formula [I] and formula [II] L2 、V H1 and V H2 :

[0401] V L1 -L1-V L2 -L2-CL [I]

[0402] V H2 -L3-V H1 -L4-C H1 [II]

[0403] Determining the maximum and minimum lengths of L1, L2, L3, and L4; generating polypeptide structures of Formula I and Formula II; selecting polypeptide structures of Formula I and Formula II that, when combined to form an antibody-like binding protein, bind to a first target antigen and a second target antigen;

[0404] in:

[0405] V L1 is the first immunoglobulin light chain variable domain;

[0406] V L2 is a second immunoglobulin light chain variable domain;

[0407] V H1 is the first immunoglobulin heavy chain variable domain;

[0408] V H2 is a second immunoglobulin heavy chain variable domain;

[0409] C L is the immunoglobulin light chain constant domain;

[0410] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0411] L1, L2, L3, and L4 are amino acid linkers;

[0412] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0413] In other embodiments of the invention, antibody-like binding proteins are prepared in which the first antibody variable domain and the second antibody variable domain are identical.

[0414] One embodiment of the present invention provides a method for preparing an antibody-like binding protein, the method comprising expressing in a cell one or more nucleic acid molecules encoding a polypeptide having a structure represented by the following formula [I] and formula [II]:

[0415] V L1 -L1-V L2 -L2-C L [I]

[0416] V H2 -L3-V H1 -L4-CH1 -Fc [II]

[0417] in:

[0418] V L1 is the first immunoglobulin light chain variable domain;

[0419] V L2 is a second immunoglobulin light chain variable domain;

[0420] V H1 is the first immunoglobulin heavy chain variable domain;

[0421] V H2 is a second immunoglobulin heavy chain variable domain;

[0422] C L is the immunoglobulin light chain constant domain;

[0423] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0424] Fc is the hinge region and C H2 、C H3 an immunoglobulin heavy chain constant domain; and

[0425] L1, L2, L3, and L4 are amino acid linkers;

[0426] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0427] Another embodiment of the present invention provides a method for preparing an antibody-like binding protein, the method comprising expressing in a cell one or more nucleic acid molecules encoding a polypeptide having a structure represented by the following formula [I] and formula [II]:

[0428] V L1 -L1-V L2 -L2-C L [I]

[0429] V H2 -L3-V H1 -L4-C H1 [II]

[0430] in:

[0431] V L1 is the first immunoglobulin light chain variable domain;

[0432] V L2 is a second immunoglobulin light chain variable domain;

[0433] V H1 is the first immunoglobulin heavy chain variable domain;

[0434] V H2 is a second immunoglobulin heavy chain variable domain;

[0435] C L is the immunoglobulin light chain constant domain;

[0436] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0437] L1, L2, L3, and L4 are amino acid linkers;

[0438] And wherein the polypeptide of Formula I and the polypeptide of Formula II form an exchange light chain-heavy chain pair.

[0439] 3. Uses of antibody-like binding proteins

[0440] The antibody-like binding proteins of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for detection and quantification of one or more target antigens. The antibody-like binding proteins will bind to one or more target antigens with an affinity suitable for the assay method being employed.

[0441] For diagnostic applications, in certain embodiments, the antibody-like binding protein can be labeled with a detectable moiety. The detectable moiety can be any moiety that is capable of directly or indirectly producing a detectable signal. For example, the detectable moiety can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S. 125 I. 99 Tc, 111 In or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0442] The antibody-like binding proteins of the present invention can also be used for in vivo imaging. The antibody-like binding proteins labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host can be determined. The antibody-like binding proteins can be labeled with any moiety detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection methods known in the art.

[0443] The present invention also relates to a kit comprising an antibody-like binding protein and other reagents that can be used to detect the level of a target antigen in a biological sample. The reagents may include a detectable label, blocking serum, positive and negative control samples, and a detection reagent.

[0444] 4. Antibody-like binding protein therapeutic compositions and their administration

[0445] Therapeutic or pharmaceutical compositions comprising antibody-like binding proteins are within the scope of the present invention. The therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of an antibody-like binding protein or an antibody-like binding protein-drug conjugate mixed with a pharmaceutically or physiologically acceptable formulation agent selected to be compatible with the mode of administration.

[0446] Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

[0447] Pharmaceutical compositions may contain formulation materials for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants, or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 or polysorbate 80, triton, tromethamine, lecithin, cholesterol, or tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides (preferably sodium chloride or potassium chloride) or mannitol, sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical excipients (see, e.g., Remington's Pharmaceutical Sciences (18th ed., AR Gennaro, ed., Mack Publishing Company 1990) and subsequent editions of said document, which are incorporated herein by reference for any purpose).

[0448] The skilled artisan can determine the most suitable pharmaceutical composition based on, for example, the intended route of administration, delivery form and desired dosage. The composition can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the antibody-like binding protein.

[0449] In the pharmaceutical composition, main solvent or carrier can be aqueous or non-aqueous in nature. For example, suitable injection solvent or carrier can be water, physiological saline solution or artificial cerebrospinal fluid, may have supplemented other common substances in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary solvents. Other exemplary pharmaceutical compositions comprise the Tris buffer of about pH 7.0-8.5, or the acetate buffer of about pH 4.0-5.5, which may also include sorbitol or suitable substitute. In one embodiment of the invention, the antibody-like conjugated protein composition in the form of lyophilized cake or aqueous solution can be prepared by mixing the selected composition with required purity with optional modulator. In addition, suitable excipient (for example sucrose) can be used, and the antibody-like conjugated protein is made into lyophilized material.

[0450] The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0451] The components of the formulation are present in concentrations acceptable to the site of administration. For example, a buffering agent is used to maintain the composition at physiological pH or at a slightly lower pH, typically within the pH range of about 5 to about 8.

[0452] In one embodiment, the present invention provides the pharmaceutical composition of the present invention.When considering parenteral administration, can be pyrogen-free, parenteral acceptable aqueous solution form, it is included in the required antibody-like conjugated protein in the pharmaceutically acceptable solvent.The especially suitable solvent for parenteral injection is sterile distilled water, and wherein the antibody-like conjugated protein is made into the sterile isotonic solution of suitable antiseptic.Another prepared product can comprise the preparation of required molecule and agent, described agent is for example injectable microsphere, biological erodible particle, polymer (for example polylactic acid or polyglycolic acid), bead or liposome, and it provides controlled release or the sustained release of product, and then product is sent by long-acting injection.Also can use hyaluronic acid, this can have the effect of prolonging the persistent period in circulation.Other suitable methods for introducing required molecule comprise implantable drug delivery device.

[0453] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the antibody-like binding protein can be formulated as a dry powder for inhalation. The antibody-like binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In another embodiment, the solution can be atomized.

[0454] It is also contemplated that certain formulations can be administered orally. In one embodiment of the invention, the antibody-like binding protein administered in this manner may be formulated with or without those carriers commonly used in solid dosage forms (e.g., tablets and capsules). For example, a capsule may be designed to release the active portion of the formulation at a certain point in the gastrointestinal tract, where bioavailability is maximized and degradation prior to internal absorption is minimized. Other agents may be included to promote the absorption of the antibody-like binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and adhesives may also be used.

[0455] Another pharmaceutical composition may include an effective amount of an antibody-like binding protein admixed with a non-toxic excipient suitable for tablet manufacture. A solution in unit dosage form may be prepared by dissolving the tablet in sterile water or another suitable solvent. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0456] Other pharmaceutical compositions of the present invention will be apparent to those skilled in the art, including preparations comprising antibody-like binding proteins in a sustained-release or controlled-release formulation. Technologies for preparing various other sustained-release or controlled-release methods, such as liposome carriers, bioerodible microparticles or porous beads, and long-acting injections, are also known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of molded articles, such as membranes or microcapsules. Sustained-release matrices can include copolymers of polyesters, hydrogels, polylactides, L-glutamic acid and gamma ethyl-L-glutamic acid, poly-(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0457] Pharmaceutical compositions of the present invention for in vivo administration generally must be sterile. This can be achieved by filtration through a sterile filter membrane. When the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally packaged in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be penetrated by a hypodermic needle.

[0458] Once the pharmaceutical composition is prepared, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilization) before administration.

[0459] The present invention also includes kits for producing single-dose administration units. The kits can each contain a first container with a dry protein and a second container with an aqueous formulation. Kits containing single-chamber and multi-chamber preloaded syringes (e.g., liquid syringes and lyosyringes) are also within the scope of the present invention.

[0460] The effective amount of the antibody-like binding protein pharmaceutical composition to be used therapeutically will depend on, for example, treatment background and purpose. Those skilled in the art will appreciate that the appropriate dosage level for the treatment of will therefore partly vary with the molecule delivered, the indication targeted by the antibody-like binding protein, route of administration and patient size (body weight, body surface or organ size) and general condition (age and general health). Therefore, clinicians can determine (titer) dosage and modify route of administration to obtain optimal therapeutic effect. The scope of typical dose can be approximately 0.1 μ g / kg to as much as approximately 100 mg / kg or higher, depending on the above factors. In other embodiments, the dosage range may be 0.1 μg / kg up to about 100 mg / kg; or 1 μg / kg up to 100 mg / kg; or 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg up to about 100 mg / kg.

[0461] The frequency of administration will depend on the pharmacokinetic parameters of the antibody-like binding protein in the preparation to be used. Typically, the clinician will give the composition until the dosage is reached to obtain the desired effect. The composition can therefore be given as follows: as a single dose, over time as two or more doses (which may or may not contain the same amount of desired molecule) or as a continuous infusion by an implant or catheter. Further improvements in appropriate dosages are routinely performed by those of ordinary skill in the art and fall within the scope of the tasks they routinely perform. Suitable dosage can be determined by utilizing suitable dose-response data.

[0462] The route of administration of the pharmaceutical composition follows known methods, such as oral administration; by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system or by implant device. When necessary, the composition is administered by bolus injection or by continuous infusion or by implant device.

[0463] The composition can also be administered locally by implanting a membrane, sponge or other suitable material into which the desired molecule has been adsorbed or encapsulated. When an implant device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, timed release bolus, or continuous administration.

[0464] 5. Examples

[0465] The following examples are illustrative of specific embodiments of the present invention and its various uses. They are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0466] Example 1. Design and Engineering of Bispecific Exchange Dual Variable Region Antibody-Like Binding Proteins

[0467] The exchanged dual variable region Fv format is described in U.S. Patent No. 5,989,830 and is referred to as the exchanged dual head (CODH) configuration. Molecular modeling predicted that the exchanged dual head (CODH) design results in a complex with two binding sites facing in opposite directions, without the constraints suggested by the dual Fv configuration. The CODH Fv format was examined to determine whether it could be constructed by replacing the C L The domain is added to the light chain and the Fc region is added to the heavy chain to be converted into a complete antibody-like molecule. For the corresponding dual variable domains (DVD-Ig) and TBTI, similar conversions are successful, as described in U.S. Patent No. 7,612,181 and International Publication No. WO2009 / 052081. The arrangement of the variable regions in the CODH format is shown in the following structure, which indicates the amino to carboxyl orientation of the peptide chain:

[0468] (a) Light chain: NH2-V L1 -Connector-V L2 -COOH

[0469] (b) Heavy chain: NH2-V H2 -Connector-V H1 -COOH

[0470] The amino to carboxyl terminus arrangement of the variable regions in (a) and (b) above can be distinguished from the arrangements in the dual Fv configurations shown in (c) and (d) below:

[0471] (c) Light chain: NH2-V L1 -Connector-V L2 -COOH

[0472] (d) Heavy chain: NH2-V H1 -Connector-V H2 -COOH

[0473] The main difference to note is the different placement of the corresponding light and heavy chain variable regions relative to each other in the two dual variable region configurations (V H1 / V L1 and V H2 / V L2 ). In the dual variable region configuration, the corresponding V L1 and V H1 Both domains are located at the N-terminus of the light and heavy chains. In contrast, in the exchange configuration, the halves of a pair of antibody variable regions are spatially separated within the protein chains of the exchange configuration. In the exchange configuration, the V L1 The domain can be located at the N-terminus of the protein light chain, but the paired V H1 The domain is located at the C-terminus of the heavy chain in the exchange configuration. L1 and V H1 The spatial relationship between them is the arrangement that exists in natural antibodies.

[0474] A potential disadvantage of the dual Fv configuration is the linker L that separates the two variable domains. L The antigen binding site of the Fv2 domain (see Figure 1 ). This protrusion may interfere with antigen binding and cause the accessibility of antigen 2 to Fv2 to be disturbed. This disturbed accessibility or interference may prevent antigen binding. In addition, when antigen 2 is larger in size, this interference may be more significant. In fact, it has been demonstrated in U.S. Patent No. 7,612,181 that the binding affinity and neutralizing ability of DVD-Ig molecules depend on which antigen specificity is present at the N-terminus or the C-terminus. See U.S. Patent No. 7,612,181, Example 2.

[0475] Therefore, in order to generate antibody-like binding proteins that are more stable than the parent antibody and do not suffer from a loss of antigen affinity, a C-terminal domain on the light chain was designed and constructed. L The polypeptides that form these antibody-like proteins have the following structures, where the amino-to-carboxyl-terminal orientation of the polypeptide chains is indicated:

[0476] (e) Light chain: NH2-V L1 -Connector-V L2 -C L -COOH

[0477] (f) Heavy chain: NH2-V H2 -Connector-V H1 -C H1 -Fc-COOH

[0478] To evaluate whether this bispecific antibody-like protein design could bind to two different antigens, two previously generated humanized variable regions from antibodies specific for IL4 (parent humanized anti-IL4) and IL13 (parent humanized anti-IL13) were used to construct the bispecific antibody-like molecules shown in Table 1. The sequencing and humanization methods for mouse antibodies are described in International Publication No. WO 2009 / 052081 (TBTI). Briefly, the amino acid sequences of the variable heavy and light chains of the mouse anti-IL13 clone B-B13 and the mouse anti-IL4 clone 8D4-8 were determined by amino acid sequencing. The mouse sequences were humanized and then reverse translated into the nucleotide sequences described in Example 5 of International Publication No. WO 2009 / 052081, which is incorporated herein by reference in its entirety. The parent humanized anti-IL4 V H and V L and parental humanized anti-IL13V H and V L Sequence combinations and permutations. The abbreviated codes in the first column of Table 1 were created to simplify the description of these antibody-like binding proteins. As shown in Table 1, the antibody-like binding proteins differ in the size of the linker inserted between the two variable regions. DNA molecules encoding the polypeptides shown in Table 1 were generated from reverse-translated parental anti-IL4 and anti-IL13 antibodies. H1 、C L The and Fc domains were obtained from IGHG1 (GenBank Accession No. 569F4) and IGKC (GenBank Accession No. Q502W4).

[0479] Table 1. Exchangeable dual-headed immunoglobulins

[0480]

[0481] The protein combinations shown in Table 2 were expressed by transient transfection and purified by Protein A chromatography. Size exclusion chromatography revealed less than 12% aggregation in each case, with most exhibiting less than 7% aggregation; however, the exchanged two-headed immunoglobulins were not found to exhibit any binding to IL4 or IL13. However, functional antibody-like binding could not be detected, and the reason for this lack of activity could not be determined. It was previously predicted that this arrangement would exhibit superior stability, surpassing that of the dual variable region domain antibodies described in U.S. Patent No. 7,612,181 and International Publication No. WO 2009 / 052081.

[0482] Table 2. Binding of CODH-Ig to IL4 and IL13

[0483]

[0484] ＊ ND means not detected

[0485] Example 2. Design of CODV-Ig protein by molecular modeling

[0486] In order to utilize the modified Fc and C L1 To obtain fully functional antibody-like proteins by swapping the domains in a dual-head configuration, a molecular modeling approach was developed to incorporate and evaluate different linkers between the constant and variable domains on both the heavy and light chains and between the dual variable domains. The question was whether the inclusion of unique linkers between each constant / variable domain interface and between the two variable / variable domain interfaces on both the heavy and light chains would allow the proper protein folding to occur in the swapped dual variable region configuration and generate functional antibody-like molecules (see Figure 2 In other words, a total of 4 independent and unique linkers were evaluated (see Figure 2 ). The molecular modeling scheme is based on Fv IL4 and Fv IL13 Protein-protein docking of homology models and experimental models in the Fv IL4 and Fv IL13 Suitable linker combinations between regions and between the Fv and constant or Fc regions.

[0487] Unique names for the individual linkers are assigned as follows: L1 refers to the N-terminal V on the light chain. L and C-end V L The linker between the two; L2 refers to the C-terminal V L and C L L3 refers to the N-terminal V H and C-end V H The linker between the heavy chain and the C-terminal V H and C H1 (and Fc). Note that the V H and V L refers only to the position of the domain on a particular protein chain in its final form. For example, V H1 and V H2 Can be derived from the parent antibody V L1 and V L2 domain, and placed in the V H1 and V H2 Position. Similarly, V L1 and V L2 Can be derived from the parent antibody V H1 and V H2 domain, and placed in the V H1 and V H2 Therefore, V H and V LThe labels refer to the present position, not the original position in the parent antibody.

[0488] In more detail, Fv IL4 The homology model of Fv was constructed based on PDB entries 1YLD (light chain) and 1IQW (heavy chain). IL4 Dimerization in IL13 / anti-IL13 Fab IL13 The internal crystal structure of the complex was recomposed and optimized. IL4 Estimation of the volume required for IL4 binding to dock the crystal structure of IL4 (1RCB.pdb) to Fv IL4 Next, 22 putative models of the complex were generated that warranted further consideration.

[0489] In parallel, Fv IL4 The homology model of IL13 / Fab was docked to IL13 The internal crystal structure of the complex selected Fv IL13 A good solution was found that allows the construction of relatively short linkers while not showing steric interference with the antigen binding and arrangement of the constant domains as is the case in dual variable region immunoglobulins (see Figure 3 ). In this arrangement, Fv IL4 (V L1 ) is placed at the N-terminus of the light chain, followed by the Fv IL13 (V L2 ) and Fc(C L1 ). On the heavy chain, Fv IL13 (V H2 ) is placed at the N-terminus, followed by Fv IL4 (V H1 ) and constant region (C H1 -C H2 -C H3 ).

[0490] As shown in Table 3, the model proposed for the light chain V L1 and V L2 Linkers L1 and V between domains L2 and C L1 The linker L2 between the domains should be between 1-3 and 0-2 glycine residues long, respectively. H2 and V H1 The linkers L3 and V between the domains H1 and C H1 The linker L4 between the domains should be between 2-6 and 2-7 glycine residues long, respectively (see Tables 3 and Figure 2In this example, glycine was used as the prototype linker amino acid, but other amino acid residues can also be used as linkers. The structural stability of the proposed model was verified by optimization of the linker conformation, minimization, and molecular dynamics calculations. Systematic combination of 4 light chain and 6 heavy chain constructs resulted in 24 possible exchanged dual variable region bispecific anti-IL4 and anti-IL13 antibody-like binding proteins (see Table 4).

[0491] Table 3. Proposed connector lengths

[0492]

[0493] Table 4. CODV-Ig used for expression

[0494]

[0495] ＊ Abbreviated codes were designed to represent related structures. Codes beginning with HC represent adjacent heavy chains, and codes beginning with LC represent adjacent light chains.

[0496] In Table 4, the prefix "anti" is not included, but is intended to mean that IL13 refers to anti-IL13 and IL4 refers to anti-IL4.

[0497] Example 3. Generation of CODV-Ig expression plasmid

[0498] Nucleic acid molecules encoding the variable heavy and light chains of the six heavy and four light chains described in Table 4 were generated by gene synthesis at Geneart (Regensburg, Germany). The variable light chain domains were fused to the constant light chain (IGKC, GenBank Accession No. Q502W4) by digestion with the restriction endonucleases ApaLI and BsiWI followed by ligation into the ApaLI / BsiWI sites of the episomal expression vector pFF (an analog of the pTT vector described by Durocher et al. (2002, Nucl. Acids Res. 30(2):E9)) to generate mammalian expression plasmids for light chain expression.

[0499] The variable heavy chain domain was fused to the "Ted" variant of human constant heavy chain (IGHG1, GenBank accession number 569F4), or to a 6x His-tagged C-terminal fragment from human constant IGHG1. H1 The domains were fused to generate bispecific Fab. H The domain was digested with restriction endonucleases ApaLI and ApaI and then ligated into the ApaLI / ApaI sites of the episomal expression vector pFF to express IGHG1 or His-tagged C H1The domains were fused to generate mammalian expression plasmids for heavy chain expression (IgG1 or Fab, respectively).

[0500] Example 4. Expression of CODV-Ig

[0501] The expression plasmids encoding the heavy and light chains of the corresponding constructs were propagated in E. coli DH5a cells. Plasmids for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.

[0502] HEK 293-FS cells grown in Freestyle medium (Invitrogen) were transfected with the indicated LC and HC plasmids encoding the heavy and light chains shown in Table 4 using 293fectin (Invitrogen) transfection reagent as described by the manufacturer. After 7 days, the cells were removed by centrifugation and the supernatant was passed through a 0.22 μm filter to remove particulates.

[0503] The CODV-IgG1 construct was purified by affinity chromatography on protein A column (HiTrap Protein A HP column, GE Life Sciences). After eluting from the column with 100mM acetate buffer and 100mM NaCl (pH 3.5), the CODV-IgG1 construct was desalted using HiPrep 26 / 10 desalting columns, prepared in PBS at a concentration of 1mg / mL, and filtered using 0.22 μm membranes.

[0504] Bispecific CODV Fab construct is passed through IMAC purification on HiTrap IMAC HP post (GE Life Sciences).After eluting from post with linear gradient (elution buffer: 20mM sodium phosphate, 0.5M NaCl, 50-500mM imidazoles, pH 7.4), merge the flow point containing protein, and use HiPrep 26 / 10 desalting column desalination, prepare in PBS with the concentration of 1mg / mL, and use 0.22 μm membrane filtration.

[0505] Protein concentration was determined by measuring absorbance at 280 nm. Each batch was analyzed by SDS-PAGE under reducing and non-reducing conditions to determine the purity and molecular weight of each subunit and monomer.

[0506] Nunc F96-MaxiSorp-Immuno plates were coated with goat anti-human IgG (Fc specific) [NatuTec A80-104A]. The antibody was diluted to 10 μg / ml in carbonate coating buffer (50 mM sodium carbonate, pH 9.6) and aliquoted at 50 μL / well. The plates were sealed with tape and stored at 4°C overnight. The plates were washed three times with wash buffer (PBS, pH 7.4 and 0.1% Tween20). 150 μL of blocking solution (1% BSA / PBS) was aliquoted into each well to cover the plate. After 1 hour at room temperature, the plates were washed three times with wash buffer. 100 μL of sample or standard (range 1500 ng / ml-120 ng / ml) was added and allowed to stand at room temperature for 1 hour. The plates were washed three times with wash buffer. Using incubation solution (0.1% BSA, PBS, pH 7.4 and 0.05% Tween20), 100 μL of goat anti-human IgG-FC-HRP conjugate [NatuTec A80-104P-60] diluted 1:10.000 was added. After incubation at room temperature for 1 hour, the plate was washed 3 times with wash buffer. 100 μL of ABTS substrate (0.1 M Na2HPO4, 0.05 M citric acid solution (pH 5.0) containing 10 mg of ABTS tablets (Pierce 34026) was added. Before use, 10 μL of 30% H2O2 / 10 ml of substrate buffer was added and dispensed into each well to allow color development. After color development (approximately 10-15 minutes), 50 μL of 1% SDS solution was added to terminate the reaction. 405 Read the board.

[0507] Example 5. Characterization of CODV-Ig variants

[0508] To determine whether the heavy and light chains of the CODV-Ig antibody-like protein are correctly paired and folded, the aggregation level was measured by analytical size exclusion chromatography (SEC). A TSKgel G3000SWXL column (7.8 mm x 30 cm) and a TSKgel SWXL guard column (Tosoh Bioscience) were used. Analytical SEC was performed on each pair of the assembled samples using a detector 10 (GE Healthcare). The analysis was run at 1 ml / min using 250 mM NaCl, 100 mM sodium phosphate (pH 6.7) and detected at 280 nm. 30 μL of protein sample (at 0.5-1 mg / ml) was applied to the column. To estimate molecular size, the column was calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using UNICORN software v5.11.

[0509] Table 5 shows the results of the first set of 24 different CODV-Ig molecules using the anti-IL4 and anti-IL13 variable region combinations described in Table 4. The codes specified in Table 4 represent the adjacent structures shown in Table 4. For the light chain and heavy chain pairs in which proteins were generated, the aggregation level was measured using SEC. The results are shown in Table 5, where LC4 (L1=1; L2=2) was the most successful pairing with all 6 heavy chains. LC4 corresponds to the structure IL4 V with a linker L1 equal to 1. L -(Gly)-IL13 V L -(Gly2)-C L1 , in which a single amino acid residue separates the two V L In addition, LC4 has an L2 of 2, which contains a V L and C-end C H1 A Gly-Gly dipeptide linker is present between the two.

[0510] Table 5. Aggregation levels in heavy and light chain pairs

[0511]

[0512] *ND indicates no protein production

[0513] Wherein CODV-Ig molecules were generated, single concentration BIACORE experiments were performed at intermediate IL13 and IL4 concentrations to verify binding to the target antigen. CODV-Ig antibody-like molecules corresponding to the LC4:HC4 and LC4:HC6 combinations described in Table 4 were selected for evaluation of the complete kinetic analysis using surface plasmon resonance.

[0514] As shown in Table 5, the majority of CODV-Ig molecules were not produced at all or only as aggregates (up to 90%). The heavy chain / light chain combinations that produced acceptable aggregation levels (5-10%) after one chromatography step were the heavy chain / light chain IL4V L -(Gly)-IL13V L -(Gly2)-C L1 The light chain is the most demanding chain within these CODV-Ig variants and was used as a platform to accept different heavy chain and different linker compositions.

[0515] 1. Kinetic analysis

[0516] Two heavy chain and light chain pairs were selected for complete kinetic analysis. Recombinant human IL13 and IL4 were purchased from Chemicon (USA). Kinetic characterization of the purified antibodies was performed on a BIACORE 3000 (GE Healthcare) using surface plasmon resonance technology. A capture assay was used that used species-specific antibodies (e.g., human-Fc specific MAB1302, Chemicon) to capture and localize the antibodies under study. Standard methods were used to immobilize the capture antibodies on a research-grade CM5 chip (GE Life Sciences) via primary amine groups (11,000 RU). The antibodies analyzed were captured at a flow rate of 10 μL / min at an adjusted RU value that resulted in a maximum analyte binding of 30 RU. Binding kinetics were measured for recombinant human IL4 and IL13 in HBS EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) over a concentration range of 0-25 nM at a flow rate of 30 μl / min. The chip surface was regenerated with 10 mM glycine (pH 2.5). Kinetic parameters were analyzed and calculated using the BIA Evaluation package v4.1, using a flow cell without capture antibody as a reference.

[0517] Table 6 below shows the kinetic comparison of the corresponding domain (table 4, code LC4:HC4 and LC4:HC6) in parental generation BB13 (anti-IL13) and 8D4 (anti-IL4) antibody (expressed as IgG) and CODV-Ig form.As shown in Table 6, when compared with parental generation anti-IL13 and anti-IL4 antibody, CODV-Ig construct does not show that the binding properties for the corresponding antigen are reduced.Using identical Fv sequence, the binding rate loss observed in DVD-Ig / TBTI form does not occur with CODV-Ig configuration.Relatively facing the binding site will allow to combine large antigen or the different cells of bridging with bispecific antibody sample configuration, and is also suitable for wider parental antibody selection.Other advantages of CODV-Ig are that no linker residue protrudes into the antigen binding site and reduces the accessibility of antigen.

[0518] Table 6. Kinetic analysis of LC4:HC4 and LC4:HC6

[0519]

[0520] 2. Co-injection of IL4 and IL13 to confirm additive antigen binding by CODV-Ig

[0521] To investigate the additive binding of the two antigens, a wizard-driven co-injection method was applied, in which one antigen was injected followed by the other antigen (IL4 then IL13 and vice versa) after a delay time. The resulting binding levels were compared to those achieved with a 1:1 mixture of the two antigens at the same concentrations. To demonstrate additive binding of both IL4 and IL13 antigens by CODV-Ig molecules, BIACORE experiments were performed with the CODV-Ig combination [HC4:LC4] by co-injection of the two antigens in three separate analysis cycles (see Figure 4 ). Co-injection was performed with 3.125nM IL4 / 25nM IL13 (and vice versa) and with a 1:1 mixture of 3.125nM IL4 and 25nM IL13. Co-injection of HBS-EP buffer was performed as a reference. At the time point of 800 seconds, no matter how the co-injection order was, after the antigen mixture was injected or the antigens were co-injected, the same binding level of 63RU was reached. When the CODV-Ig protein was saturated with the first antigen (IL4), the second antigen (IL13) was injected and a second binding signal was observed. When the order of antigen injection was reversed, this observation was reproduced. This shows that the binding of the two antigens due to CODV-Ig is additive, not inhibitory. Therefore, the CODV-Ig construct can bind to the two antigens simultaneously (i.e., show bispecificity), saturating all binding sites (i.e., showing tetravalence).

[0522] Example 6. Linker length tolerance for CODV-Ig

[0523] The tolerance of various linker lengths was evaluated by constructing CODV-Ig molecules with different combinations of linker lengths for L1, L2 on the light chain and L3 and L4 on the heavy chain. CODV-Ig constructs with heavy chain linkers L3 and L4 were generated, varying between 1-8 residues for L3 and 0 or 1 residue for L4. The heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the C-terminal binding domain, followed by the C H1 -Fc. The light chain linkers L1 and L2 vary from 3 to 12 residues for L1 and from 3 to 14 residues for L2. The light chain contains an anti-IL13 binding domain as the N-terminal and an anti-IL4 binding domain as the C-terminal, followed by a C L1 .

[0524] 1. Characterization of CODV-Ig variants

[0525] Aggregation levels were determined by size exclusion chromatography (SEC) using a TSKgel G3000 SWXL column (7.8 mm x 30 cm) and a TSKgel SWXL guard column (Tosoh Bioscience). Analytical SEC was performed using a detector 10 (GE Healthcare). The analysis was run at 1 ml / min using 250 mM NaCl, 100 mM sodium phosphate (pH 6.7) and detected at 280 nm. 30 μL of protein sample (at 0.5-1 mg / ml) was applied to the column. To estimate molecular size, the column was calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using UNICORN software v5.11.

[0526] Recombinant human IL13 and IL4 were purchased from Chemicon (USA). Recombinant human TNF-α was purchased from Sigma Aldrich (H8916-10 μg), and recombinant human IL-1β (201-LB / CF), recombinant human IL-23 (1290-IL / CF), recombinant human EGFR (344ER), and recombinant human HER2 (1129-ER-50) were purchased from R&D Systems.

[0527] Carry out kinetic binding analysis as follows by Biacore.Adopt the surface plasmon resonance technology on Biacore 3000 (GE Healthcare) for the detailed kinetic characterization of purified antibodies.Adopt capture assay, this method uses species-specific antibody (for example people-Fc specific MAB 1302, Chemicon) for the capture and positioning of studied antibody.In order to measure IL4 and IL13 binding kinetics, use anti-human Fab capture kit (GE Healthcare), capture the corresponding CODV Fab as in embodiment 10, table 12.Adopt standard method, make capture antibody be fixed on research grade CM5 chip (GE Life Sciences) by primary amine group (11000RU).With the RU value of the adjustment that can cause the maximum analyte of 30RU to be combined, the antibody analyzed is captured by the flow velocity capture of 10 μ L / minute. Binding kinetics were measured for recombinant human IL4 and IL13 in HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) over a concentration range of 0-25 nM at a flow rate of 30 μl / min. The chip surface was regenerated with 10 mM glycine (pH 2.5). Kinetic parameters were analyzed and calculated using the BIA Evaluation package v4.1, using a flow cell without capture antibody as a reference.

[0528] Use Proteon XPR36 protein interaction array system (Biorad), measure the binding affinity of CODV-Ig, CODV-Fab and TBTI for EGFR and HER2.Antigen is fixed on the GLC sensor chip (Biorad) by amine reactive coupling.With the single trigger kinetics mode (one-shot kinetics mode) with dual reference, the dilution series of bispecific antibody variant in PBSET buffer (Biorad) is analyzed in parallel.Application has the Langmuir 1:1 model with mass transfer (masstransfer) or the Proteon Manager software v3.0 (Biorad) of bivalent analyte model, data are analyzed.

[0529] Table 7 summarizes the results of yield, aggregation (measured by size exclusion chromatography) and binding affinity of CODV-Ig with different size combinations of linkers. The results show that CODV-Ig molecules in which L2 is zero are generally not produced, or if the protein is produced, there is a high level of aggregation (see batch identification numbers 101, 102, 106-111 and 132-137 in Table 7). Therefore, unlike the molecular modeling predictions of Example 2 (in which L2 is zero is within the acceptable range), these results indicate that V L2 -C L The switch (or L2) requires a linker of at least one residue (see Table 7).

[0530] Table 7. Optimization of linker size of CODV-Ig

[0531]

[0532] ＊ The arrangement on the heavy chain must be IL13V H -L3-IL4V H -L4-C H1 -Fc

[0533] 1 np means the construct was not producible.

[0534] In addition, it was found that increasing the length of the CODV-Ig linker by 1 amino acid residue was more sensitive than increasing it by 2 amino acid residues. For example, although batch IDs 103 and 104 differed by 1 amino acid residue in L2, batch ID 103 showed more than 6 times the aggregation, while batch ID 104 showed less aggregation and twice the yield. In contrast, batch IDs 104 and 105, which differed by 2 residues in L2, showed similar characteristics in terms of yield, aggregation, and binding.

[0535] Example 7. Heavy chain as template chain for CODV-Ig

[0536] In Examples 1-5, the optimal short linker size on the light chain suggests that the light chain serves as a template by maintaining a linear arrangement, and a larger linker is required on the heavy chain to allow the heavy chain to fold correctly into an exchange conformation to coincide with the template light chain (see Figure 5 , Figure A). We next evaluated whether a short linker specifically placed on the heavy chain to maintain linear alignment on the heavy chain conferred a "template" heavy chain, and whether this pattern itself repeated and whether a larger linker was required to allow the non-template chain to fold correctly and accommodate the existing template heavy chain (see Figure 5 , Figure B).

[0537] Figure 6 The design principles of these CODV-Igs are described based on either the light chain or the heavy chain as a "template". To evaluate the general properties of this concept, CODV-Ig constructs were generated with heavy chain linkers L3 and L4 varying between 1-8 residues for L3 and 0 or 1 residue for L4. The heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the C-terminal binding domain, followed by the C H1 -Fc. The light chain linkers L1 and L2 vary from 3 to 12 residues for L1 and from 3 to 14 residues for L2. The light chain contains an anti-IL13 binding domain as the N-terminal and an anti-IL4 binding domain as the C-terminal, followed by a C L1 .

[0538] Table 8 summarizes the output, aggregation (by size exclusion chromatography measurement) and the result of binding affinity of the CODV-Ig with the joint of different size combination, and in described CODV-Ig, heavy chain remains as linear arrangement as template chain and allows light chain to fold with exchange configuration.Result shows, generally can not produce the CODV-Ig molecule that wherein L4 is zero, or if protein produces, then there is high-caliber aggregation (being similar to the molecule that wherein L2 equals zero) (referring to the batch identification number 207-209,211-212,219-224,231-236,243-252 and 263-266 in Table 8).An exception is batch identification number 210, and wherein L1 is 7, and L2 is 5, and L3 is 2, and L4 is zero.This arrangement produces enough protein, and has the aggregation and combination of acceptable level, and this just shows, can exist in some cases the zero-length joint on some combinations compensation L4 of joint size.

[0539] Table 8. Optimization of linker size using heavy chain as template

[0540]

[0541]

[0542] ＊ The arrangement on the light chain must be IL13V L -L1-IL4V L -L2-C L1

[0543] The results of Tables 7 and 8 clearly show that a linker is required between the variable and constant domains for optimal folding. Only in rare arrangements is a linker equal to zero allowed (see lot IDs 103-105, where L1 (LC) is zero, and lot ID 210, where L4 is zero). However, in each case, the corresponding transition linker between the variable and constant regions on the other chain cannot be zero.

[0544] The above results indicate that the combination L1=7, L2=5, L3=1 and L4=2 is a good starting point for optimizing new CODV-Ig in which the heavy chain is the template. The ranges in Table 9 indicate reasonable ranges for successfully engineering new CODV-Ig from two parent antibodies.

[0545] Table 9. Linker size ranges for either LC or HC as templates

[0546]

[0547] Example 8. Universal utility of CODV-Ig formats

[0548] To evaluate the suitability of the CODV-Ig format for engineering new antibody-like binding proteins, variable regions from several existing human and humanized antibodies specific for insulin-like growth factor 1 receptor (IGF1R(1)), a secondary antibody to insulin-like growth factor 1 receptor (IGF1R(2)), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), tumor necrosis factor-α (TNFα), interleukins 12 and 23 (IL-12 / 23), and interleukin 1β (IL-1β) were incorporated into the CODV-Ig format (see Table 10).

[0549] Table 10. Descriptive codes for the heavy and light chains of bispecific CODV-Ig

[0550]

[0551] * The abbreviation codes are designed to represent related structures. Codes starting with HC represent adjacent heavy chains, and codes starting with LC represent adjacent light chains.

[0552] In designing bispecific antibody-like binding proteins, the universal utility of the CODV-Ig format was tested using antibody variable regions from known human and humanized antibodies. Furthermore, the potential for positional effects regarding the position of certain antibody variable regions at the N-terminus or C-terminus of the heavy or light chain was investigated. Based on the design of a CODV-Ig molecule with a linker composition of L1=7, L2=5, L3=1, and L4=2, different antibody sequences were introduced into the CODV-Ig format.

[0553] The activity of bispecific antibodies or derivatives against IL1β and TNFα was determined using commercially available HEK-Blue TNFα / IL1β reporter cells (InvivoGen). To determine antibody activity against TNFα and IL1β, the cytokines were preincubated with varying concentrations of antibody for 1 hour and added to 50,000 HEK Blue TNFα / IL1β cells. Cytokine-mediated SEAP induction was measured in culture supernatants after 24 hours using the QU Anti-Blue assay (InvivoGen).

[0554] As shown in Table 11, all constructs showed good to excellent protein yields and acceptable aggregation levels (specifically referring to batch identification numbers 301 and 302 in Table 11). The affinities measured for each antibody variable domain were within published and expected affinities. In the case of evaluating affinity, no position effect was detected. In general, as shown in the table below, no position effect was found in any of the antibody variable domains used or when these domains were used on any antibody chain.

[0555] Table 11. Common applications of CODV-Ig formats of bispecific antibody-like binding proteins

[0556]

[0557] Example 9. Preservation of parent antibody affinity in CODV-Ig format

[0558] The same antibody sequences for anti-IL4 and anti-IL13 were incorporated into TBTI / DVD-Ig or CODV-Ig formats for direct comparison of these configurations, linker positions, and the affinities of the resulting molecules. Figure 7 As shown, the parental affinity of each antibody was maintained in the CODV format. Figure 7 As shown in the figure above, when the variable region is placed in the TBTI / DVD-Ig format, the affinity of the IL4 antibody located at the internal Fv2 position is reduced, which is manifested by a decrease in the binding rate of the antibody to the antigen. In contrast, the affinity of the CODV-Ig format is not lost compared to the parent antibody (see Figure 7 (see the figure below).

[0559] Example 10. Adaptation of CODV-Ig to Fab format

[0560] Next, the ability of the CODV-Ig format to provide fragments (e.g., Fab fragments) was evaluated. Two different variable heavy chains were fused to each other via joint L3 and extended at the C-terminus via joint L4. H The complex with IGHG1 (GenBank accession number Q569F4) C H1 The IGHG1 domain fusion carries the C-terminal five amino acid sequence DKTHT (SEQ ID NO: 60) from the hinge region followed by six histidine residues. Two different variable light chains are fused to each other via linker L1 in an exchange configuration with the corresponding heavy chains, and are C-terminally extended by linker L2 before being fused to a constant kappa chain (IGKC, GenBank Accession No. Q502W4).

[0561] Fab fragments were expressed by transient transfection as described above. Seven days after transfection, cells were removed by centrifugation, 10% vol / vol 1M Tris-HCl, pH 8.0, was added, and the supernatant was passed through a 0.22 μm filter to remove particulates. Fab protein was captured using a HisTrap HighPerformance column (GE Healthcare) and eluted by an imidazole gradient. Protein-containing fractions were combined and desalted using PD-10 or Sephadex columns. The concentrated and sterile-filtered (0.22 μm) protein solution was adjusted to 1 mg / ml and kept at 4°C until use.

[0562] The Fab sample molecule in the CODV orientation shows no aggregation tendency and keeps the avidity of parental antibody and observes direct advantage (referring to Table 12).Directly compare with the antibody-like protein from the associated protein construct of batch identification number 401-421, in described antibody-like protein, antibody variable region is by following arranging: using heavy chain as 4 kinds of domain antibody-like molecules (404,409,414 and 419) of CODV-Ig molecule (401,402,406 and 407), CODV Fab sample fragment (402,408,413,418 and 421), TBTI / DVD-Ig form and the CODV-Ig (405,410,415 and 420) without joint.As shown in Table 12, this comparative result shows, when variable region mixed in TBTI or DVD-Ig form, compared with parental antibody, avidity more may lose. In contrast, both CODV-Ig and CODV-Ig Fab-like formats were better able to retain parental affinity.The results also demonstrated that CODV-Ig molecules require linkers between the variable regions and between the variable regions and constant domains (see Table 12).

[0563] Table 12. Application of CODV-Ig formats for Fab-like fragments

[0564]

[0565] Example 11. Substitution of variable domains in CODV-Ig and CODV-Fab

[0566] In order to characterize CODV form in T cell engagement method (T-cell engaging approach), produce the bispecific CODV Fab sample binding protein (CODV-Fab) with TCR binding site (CD3ε) and CD19 binding site, and compared with the bispecific Fab (B-Fab) derived from TBTI / DVD-Ig form.In order to study the importance of the orientation of binding site (TCR x CD19 is relative to CD19x TCR), two kinds of orientations are evaluated for each associated protein.

[0567] The binding proteins were characterized in a cytotoxicity assay using NALM-6 (CD19 expressing) cells as target cells and primary human T cells as effector cells. CD3 positive cells were isolated from freshly prepared human PBMCs. Effector and target cells were mixed at a ratio of 10:1 and incubated with the indicated concentrations of the bispecific binding proteins for 20 hours (see Table 1). Figure 8 ). Apoptotic target cells were determined in a FACS-based assay using 7-aminoactinomycin staining.

[0568] The B-Fab form of the CD3-CD19 configuration (1060) was shown to be active in inducing T cell-mediated cytotoxicity against NALM-6 cells with an EC50 of 3.7 ng / ml. Similar high activity was observed for the CD19-CD3CODV-Fab (1109) with an EC50 of 3.2 ng / ml (see Figure 8 ).

[0569] The exchange of the conformation of the B-Fab molecule (Fab in TBTI / DVD-Ig format) with the CD19-CD3 orientation resulted in a significant loss of activity (see Figure 8). The exchanged B-Fab molecules showed no activity at concentrations that were maximal for both orientations of CODV-Ig Fab and the other orientation of B-Fab. Maximum responses were observed for CODV-Ig Fab and one orientation of B-Fab (ranging between 1 and 100 ng / ml). For the CD19-CD3 orientation of B-Fab, even at the maximum concentration (30 μg / ml), the optimal toxic response was not reached. In sharp contrast, the change in domain orientation in CODV-Fab towards CD3-CD19 (1108) produced a molecule with significant activity in this assay (see Figure 8 ). Although the domain exchange in CODV-Fab also reduces the induction of T cell-mediated cytotoxicity (EC50 increases by about 100 times), this effect is far less significant than that observed in the B-Fab format, and the molecule is able to induce cytotoxicity until the maximum level. Data are representative and obtained from 3 independent experiments.

[0570] Example 12. Effect of amino acid sequence characteristics on CODV-Ig linker

[0571] The optimized construct corresponding to batch ID 204 (see Example 7 and Table 8) was selected to study the effect of linker composition on linkers L1-L4. The linker lengths of L1, L2, L3, and L4 were set to 7, 5, 1, and 2 residues, respectively (see Table 13). The test sequence was derived from a natural antibody V H and C H1 between the domains of the antibody Fv and C L The naturally occurring linker at the transition between domains. The candidate sequence is ASTKGPS (SEQ ID NO: 48) (which is derived from V H and C H1 domain switching), RTVAAPS (SEQ ID NO: 49) and GQPKAAP (SEQ ID NO: 50) (derived from the Fv and C of the kappa and lambda light chains, respectively). L Domain switching). In addition, a construct was generated with an arbitrary linker composition to demonstrate that any sequence can be used for linkers L1-L4. This linker composition was obtained by randomly distributing the following amino acids at 15 positions across the four linkers: valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline. The aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as the amino acids methionine and cysteine, were intentionally excluded to avoid potential increases in aggregation.

[0572] The construct three-dimensional model of batch identification number 204 is generated to ensure suitability or to improve the selection of joint composition. Therefore, for joint L3, serine is selected as positively charged, and negatively charged residues are observed near this three-dimensional model. The residue in the joint L4 is selected to be compatible with the solvent exposure of these positions suggested by the model. Similarly, for the joint composition of L1 and L2, no expectation or prediction is problematic. For the joint composition, the three-dimensional model of selected suggestion has been constructed.

[0573] As shown in Table 12, the joint composition can have a great influence on the yield. The sequence derived from the lambda chain on L1 (comparing batch identification numbers 505-507 with batch identification numbers 501-503) is a more prolific protein generator (increased up to 8 times). In fact, the joint based on random generation also produces good yields, as shown in Table 13 batch identification number 508. Therefore, the joint composition should be a parameter considered during CODV-Ig optimization.

[0574] Table 13. Effect of linker composition on CODV-Ig

[0575]

[0576] In commercially available HEK-Blue IL-4 / IL-13 reporter cells (InvivoGen), the activity of bispecific antibodies or derivatives for cytokines IL4 and IL13 was determined. HEK-Blue IL-4 / IL-13 cells are designed to monitor the activation of the STAT6 pathway caused by IL-4 or IL13. Cells are stimulated with any cytokine to produce a reporter gene secreting embryonic alkaline phosphatase (SEAP), which can be measured in the culture supernatant using the QUANTI-Blue assay. In order to test the activity of antibodies for IL4 or IL13, cytokines are pre-incubated with antibodies of varying concentrations for 1 hour and added to 50,000 HEK-Blue IL-4 / IL-13 cells. Using the QUANTI-Blue assay (InvivoGen), after incubation for 24 hours in the cell culture supernatant, cytokine-mediated SEAP induction is measured.

[0577] Example 13. Introduction of Cysteine ​​into the CODV-Ig Linker

[0578] Published data indicate that the stability of antibodies and antibody-derived proteins can be improved by the introduction of non-native disulfide bridges (see Wozniak-Knopp et al., 2012, "Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds," PLoS ONE 7(1):e30083). To examine whether the introduction of inter- and intra-chain disulfide bridges stabilizes the equivalent Fc fragment derived from a human IgG1 antibody engineered into a CODV-Ig molecule, the equivalent Fc position of CODV-Ig construct batch ID 204 (from Example 7) was mutated to a cysteine ​​residue, and the mutant protein was overproduced, purified, and characterized (see Table 14).

[0579] As shown in Table 14, each of the mutated CODV-Ig molecules containing an additional cysteine ​​residue had the same melting temperature as that of CODV-Ig construct lot ID 204.

[0580] In addition, two simultaneous cysteines were introduced at the Kabat positions in each variable domain (position 100 in the light chain and position 44 in the heavy chain) as described by Brinkmann et al., 1993, Proc. Natl. Acad. Sci. USA 90:7538-42. These positions have been shown to be structurally conserved within the antibody fold and thus can tolerate cysteine ​​substitutions without disturbing the integrity of the respective domains.

[0581] As shown in Table 15, CODV and CODV-Ig constructs in which a cysteine ​​residue was introduced at the Kabat position of each variable domain (position 100 on the light chain and position 44 on the heavy chain) had higher melting temperatures than CODV and CODV-Ig constructs in which no cysteine ​​residue was introduced at these positions (see, e.g., Lot IDs 704 and 706 and Lot IDs 713 and 714).

[0582] 1. Thermal stability measurements of CODV and TBTI variants

[0583] Using differential scanning fluorimetry (DSF), the melting temperature (Tm) of CODV and TBTI variants was determined. The sample was diluted to a final concentration of 0.2 μg / μl in D-PBS buffer (Invitrogen) and added to a 40x concentrated solution of D-PBS containing SYPRO-Orange dye (Invitrogen) in 2 μl of a white semi-skirt 96-well plate. All measurements were performed in duplicate using a MyiQ2 real-time PCR instrument (Biorad). The Tm value was calculated using iQ5 software v2.1, derived from the negative first derivative of the melting curve.

[0584] Next, checked the impact that cysteine ​​residues are directly introduced into joint or variable region.In this embodiment, batch identification number 204 (from embodiment 7 and table 8) is used as pattern CODV-Ig conjugated protein, makes cysteine ​​replace the glycine in variable region, L1 or L3 on the three-dimensional model basis.As shown in the following table 16, the result shows how to introduce cysteine ​​to can affect output and gathering.With the all modeling of expected sudden change to determine the suitable formation disulfide bond and to the joint in the model and environment thereof, keep correct geometry.But batch identification number 808 shows good output and seldom gathers, therefore shows and may form suitable cysteine ​​bridge.

[0585] Although the present invention has been described in terms of various embodiments, it will be understood that variations and modifications will occur to those skilled in the art. It is therefore intended that the appended claims encompass all such equivalent variations within the scope of the claimed invention. Furthermore, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0586] Each embodiment described herein may be combined with any one or more other embodiments, unless expressly stated otherwise. Specifically, any feature or embodiment indicated as preferred or advantageous may be combined with any one or more other features or embodiments indicated as preferred or advantageous, unless expressly stated otherwise.

[0587] All references cited in this application are expressly incorporated herein by reference.

[0588] Table 14. Effect of disulfide bridge stability on CODV-Ig

[0589]

[0590] Table 15. Effect of disulfide bridge stability on CODV-Ig

[0591]

[0592] Table 16A. Effect of disulfide bridge stabilization on CODV-Ig

[0593]

[0594] Table 16B. Effect of disulfide bridge stabilization on CODV-Ig

[0595] Sequence Listing <110> E. Rao, Ercole V. Mikol (Vincent) C. Corvey, Carsten C. Beil, Christian C. Lange (Christian Lange) A. Steinmetz, Anke N. Pauling (Baurin, Nicolas) D. Li (Li, Danxi) <120> Dual variable region antibody-like binding proteins with swapped binding region orientations <130> 11-414-WO-CN DIV.2 <140> PCT / US2012 / 030948 <141> 2012-03-28 <150> 61 / 468276 <151> 2011-03-28 <150> FR1160311 <151> 2011-11-14 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Humanized antibody light chain (VL) from anti-IL4 <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 2 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Humanized heavy chain V region (VH) from anti-IL4 antibody <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 <210> 3 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized light chain V region (VL) from anti-IL13 antibody <400> 3 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala 85 90 95 Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 4 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Humanized heavy chain V region (VH) from anti-IL-13 antibody <400> 4 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 5 <211> 575 <212> PRT <213> Artificial sequence <220> <223> Exchange the heavy chain of the dual-head construct IL13(G4S)IL4CHFc <400> 5 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln 115 120 125 Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser 130 135 140 Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile 145 150 155 160 Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro 165 170 175 Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr 180 185 190 Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser 195 200 205 Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu 210 215 220 Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 245 250 255 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 260 265 270 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 275 280 285 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 290 295 300 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 305 310 315 320 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 325 330 335 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 340 345 350 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 355 360 365 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 370 375 380 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 385 390 395 400 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 405 410 415 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 420 425 430 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 435 440 445 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 450 455 460 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 465 470 475 480 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 485 490 495 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 500 505 510 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 515 520 525 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 530 535 540 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 545 550 555 560 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 575 <210> 6 <211> 580 <212> PRT <213> Artificial sequence <220> <223> Exchange the heavy chain of the dual-head construct IL13(G4S2)IL4CHFc <400> 6 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 130 135 140 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 145 150 155 160 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 165 170 175 Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 180 185 190 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 195 200 205 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 225 230 235 240 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 245 250 255 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 260 265 270 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 275 280 285 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 290 295 300 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 305 310 315 320 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 325 330 335 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 340 345 350 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 355 360 365 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 370 375 380 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 385 390 395 400 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 405 410 415 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 420 425 430 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 435 440 445 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 450 455 460 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 465 470 475 480 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 485 490 495 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 500 505 510 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 515 520 525 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 530 535 540 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 545 550 555 560 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 565 570 575 Leu Ser Pro Gly 580 <210> 7 <211> 698 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Swap Bifunctional Construct IL4(G4S)IL13CHFc <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 130 135 140 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 145 150 155 160 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 165 170 175 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 180 185 190 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 195 200 205 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 210 215 220 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu 245 250 255 Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 260 265 270 Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys Gln Arg Pro Gly 275 280 285 Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Gly Glu Thr 290 295 300 Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu Thr Val Asp Glu 305 310 315 320 Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro Thr Ser Glu Asp 325 330 335 Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp 340 345 350 Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu Val Thr Val Ser 355 360 365 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 370 375 380 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 385 390 395 400 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 405 410 415 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 420 425 430 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 435 440 445 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 450 455 460 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 465 470 475 480 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 485 490 495 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 500 505 510 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 515 520 525 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 530 535 540 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 545 550 555 560 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 565 570 575 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 580 585 590 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 595 600 605 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 610 615 620 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 625 630 635 640 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 645 650 655 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 660 665 670 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 675 680 685 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 <210> 8 <211> 703 <212> PRT <213> Artificial sequence <220> <223> Heavy chain exchange dual-head construct IL4(G4S2)IL13CHFc <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu 130 135 140 Val Ala Pro Gly Gly Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe 145 150 155 160 Ser Leu Thr Asp Ser Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys 165 170 175 Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr 180 185 190 Ala Asp Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys 195 200 205 Ser Gln Val Phe Leu Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala 210 215 220 Thr Tyr Tyr Cys Ala Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe 225 230 235 240 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gln Val Gln Leu Gln 245 250 255 Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser 260 265 270 Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile 275 280 285 Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro 290 295 300 Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr 305 310 315 320 Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser 325 330 335 Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu 340 345 350 Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr 355 360 365 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 370 375 380 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 385 390 395 400 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 405 410 415 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 420 425 430 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 435 440 445 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 450 455 460 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 465 470 475 480 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 485 490 495 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 500 505 510 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 515 520 525 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 530 535 540 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 545 550 555 560 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 565 570 575 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 580 585 590 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 595 600 605 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 610 615 620 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 625 630 635 640 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 645 650 655 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 660 665 670 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 675 680 685 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 <210> 9 <211> 330 <212> PRT <213> Artificial sequence <220> <223> Light chain exchange dual-head construct IL13(G4S)IL4CL <400> 9 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala 85 90 95 Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 115 120 125 Val Ser Val Gly Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn 130 135 140 Ile Asp Val Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro 145 150 155 160 Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser 165 170 175 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser 180 185 190 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His 195 200 205 Ser Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 210 215 220 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 225 230 235 240 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 245 250 255 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 260 265 270 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 275 280 285 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 290 295 300 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 305 310 315 320 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 10 <211> 335 <212> PRT <213> Artificial sequence <220> <223> Light chain exchange dual-head construct IL13(G4S2)IL4CL <400> 10 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala 85 90 95 Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 115 120 125 Pro Ala Ser Leu Ser Val Ser Val Gly Asp Thr Ile Thr Leu Thr Cys 130 135 140 His Ala Ser Gln Asn Ile Asp Val Trp Leu Ser Trp Phe Gln Gln Lys 145 150 155 160 Pro Gly Asn Ile Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His 165 170 175 Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe 180 185 190 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr 195 200 205 Cys Gln Gln Ala His Ser Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys 210 215 220 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 245 250 255 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 260 265 270 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 275 280 285 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 290 295 300 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 305 310 315 320 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 335 <210> 11 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Light chain-swapped bivalent construct IL4(G4S)IL13CL <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 115 120 125 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 130 135 140 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 145 150 155 160 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 165 170 175 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 180 185 190 Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala 195 200 205 Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 210 215 220 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 225 230 235 240 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 245 250 255 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 260 265 270 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 275 280 285 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 290 295 300 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 305 310 315 320 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 12 <211> 335 <212> PRT <213> Artificial sequence <220> <223> Light chain exchange dual-head construct IL4(G4S2)IL13CL <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu 115 120 125 Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu 130 135 140 Ser Val Asp Ser Tyr Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys 145 150 155 160 Ala Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu 165 170 175 Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe 180 185 190 Thr Leu Thr Ile Asp Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr 195 200 205 Cys Gln Gln Asn Ala Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys 210 215 220 Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 245 250 255 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 260 265 270 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 275 280 285 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 290 295 300 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 305 310 315 320 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 335 <210> 13 <211> 583 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC1 <400> 13 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gln Val Gln Leu 115 120 125 Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile 130 135 140 Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp 145 150 155 160 Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp 165 170 175 Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala 180 185 190 Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg 195 200 205 Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys 210 215 220 Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala Ser 245 250 255 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 260 265 270 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 275 280 285 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 290 295 300 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 305 310 315 320 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 325 330 335 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 340 345 350 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 355 360 365 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 370 375 380 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 385 390 395 400 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 405 410 415 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 420 425 430 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 435 440 445 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 450 455 460 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 465 470 475 480 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 485 490 495 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 500 505 510 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 515 520 525 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 530 535 540 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 545 550 555 560 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 565 570 575 Ser Leu Ser Leu Ser Pro Gly 580 <210> 14 <211> 580 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC2 <400> 14 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gln Val Gln Leu 115 120 125 Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile 130 135 140 Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp 145 150 155 160 Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp 165 170 175 Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala 180 185 190 Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg 195 200 205 Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys 210 215 220 Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ala Ser Thr Lys Gly 245 250 255 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 260 265 270 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 275 280 285 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 290 295 300 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 305 310 315 320 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 325 330 335 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 340 345 350 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 355 360 365 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 370 375 380 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 385 390 395 400 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 405 410 415 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 420 425 430 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 435 440 445 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 450 455 460 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 465 470 475 480 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 485 490 495 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 500 505 510 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 515 520 525 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 530 535 540 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 545 550 555 560 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 565 570 575 Leu Ser Pro Gly 580 <210> 15 <211> 579 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC3 <400> 15 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gln Val Gln Leu Gln Gln Ser Gly 115 120 125 Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 130 135 140 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys Gln Arg 145 150 155 160 Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Gly 165 170 175 Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu Thr Val 180 185 190 Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro Thr Ser 195 200 205 Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr Gly Asn 210 215 220 Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala Ser Thr Lys Gly Pro 245 250 255 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 260 265 270 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 275 280 285 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 290 295 300 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 305 310 315 320 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 325 330 335 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 340 345 350 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 355 360 365 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 370 375 380 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 385 390 395 400 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 405 410 415 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 420 425 430 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 435 440 445 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 450 455 460 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 465 470 475 480 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 485 490 495 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 500 505 510 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 515 520 525 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 530 535 540 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 545 550 555 560 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 565 570 575 Ser Pro Gly <210> 16 <211> 576 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC4 <400> 16 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gln Val Gln Leu Gln Gln Ser Gly 115 120 125 Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 130 135 140 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys Gln Arg 145 150 155 160 Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Gly 165 170 175 Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu Thr Val 180 185 190 Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro Thr Ser 195 200 205 Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr Gly Asn 210 215 220 Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser Gly Gly Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe 245 250 255 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 260 265 270 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 275 280 285 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 290 295 300 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 305 310 315 320 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 325 330 335 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 340 345 350 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 355 360 365 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 370 375 380 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 385 390 395 400 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 405 410 415 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 420 425 430 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 435 440 445 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 450 455 460 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 465 470 475 480 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 485 490 495 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 500 505 510 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 515 520 525 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 530 535 540 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 545 550 555 560 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 575 <210> 17 <211> 581 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC5 <400> 17 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser 165 170 175 Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu 180 185 190 Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr 210 215 220 Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala Ser Thr Lys 245 250 255 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 260 265 270 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 275 280 285 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 290 295 300 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 305 310 315 320 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 325 330 335 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 340 345 350 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 485 490 495 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 530 535 540 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 545 550 555 560 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 565 570 575 Ser Leu Ser Pro Gly 580 <210> 18 <211> 578 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of the exchanged double V-Ig with code HC6 <400> 18 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser 165 170 175 Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe Gln Gly Arg Ala Thr Leu 180 185 190 Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser Pro 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu Tyr 210 215 220 Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val Trp Gly Ala Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Gly Gly Gly Gly Ala Ser Thr Lys Gly Pro Ser 245 250 255 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 260 265 270 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 275 280 285 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 290 295 300 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 305 310 315 320 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 325 330 335 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 340 345 350 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 355 360 365 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 370 375 380 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 385 390 395 400 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 405 410 415 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 420 425 430 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 435 440 445 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 450 455 460 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 465 470 475 480 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 485 490 495 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 500 505 510 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 515 520 525 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 530 535 540 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 545 550 555 560 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 565 570 575 Pro Gly <210> 19 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC1 <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Asp Ile 100 105 110 Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg 115 120 125 Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln 130 135 140 Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu 145 150 155 160 Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe 165 170 175 Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val 180 185 190 Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp 195 200 205 Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 210 215 220 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 225 230 235 240 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 245 250 255 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 260 265 270 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 275 280 285 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys [[ID=??]]290 295 300 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 305 310 315 320 Lys Ser Phe Asn Arg Gly Glu Cys 325 <210> 20 <211> 326 <212> PRT <213> Artificial Sequence <220> It seems there is a mistake in the original text where the line break for "290 295 300" is marked as "??" instead of "28". The above translation has been adjusted accordingly.<223> Light chain sequence of the exchanged double V-Ig with code LC2 <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Asp Ile Val Leu 100 105 110 Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr 115 120 125 Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser Tyr 130 135 140 Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile 145 150 155 160 Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly 165 170 175 Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala 180 185 190 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser Arg 195 200 205 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 210 215 220 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 225 230 235 240 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 245 250 255 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 260 265 270 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 275 280 285 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 290 295 300 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 305 310 315 320 Phe Asn Arg Gly Glu Cys 325 <210> twenty one <211> 330 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC3 <400> twenty one Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Asp Ile 100 105 110 Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg 115 120 125 Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln 130 135 140 Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu 145 150 155 160 Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe 165 170 175 Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val 180 185 190 Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp 195 200 205 Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Arg 210 215 220 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 225 230 235 240 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 245 250 255 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 260 265 270 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 275 280 285 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 290 295 300 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 305 310 315 320 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> twenty two <211> 328 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC4 <400> twenty two Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Asp Ile Val Leu 100 105 110 Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr 115 120 125 Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln Ser Tyr 130 135 140 Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys Leu Leu Ile 145 150 155 160 Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly 165 170 175 Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Gln Ala 180 185 190 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala Glu Asp Ser Arg 195 200 205 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Arg Thr Val 210 215 220 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 225 230 235 240 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 245 250 255 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 260 265 270 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 275 280 285 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 290 295 300 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 305 310 315 320 Lys Ser Phe Asn Arg Gly Glu Cys 325 <210> 23 <211> 107 <212> PRT <213> Homo sapiens <400> 23 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 24 <211> 329 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 25 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Peptide linker with 4 glycine residues <400> 25 Gly Gly Gly Gly 1 <210> 26 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker with 5 glycine amino acid residues <400> 26 Gly Gly Gly Gly Gly 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Peptide linker with 6 glycine amino acid residues <400> 27 Gly Gly Gly Gly Gly Gly 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Peptide linker with 7 glycine amino acid residues <400> 28 Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Peptide linker with 8 glycine amino acid residues <400> 29 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 30 Gly Gly Gly Gly Ser 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 31 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 32 <211> 582 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC10 <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr 100 105 110 Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 145 150 155 160 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ala Ser Thr 245 250 255 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 260 265 270 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 275 280 285 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 290 295 300 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 305 310 315 320 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 325 330 335 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 340 345 350 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 355 360 365 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 370 375 380 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 385 390 395 400 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 405 410 415 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 420 425 430 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 435 440 445 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 450 455 460 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 465 470 475 480 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 485 490 495 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 500 505 510 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 515 520 525 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 530 535 540 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 545 550 555 560 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 565 570 575 Leu Ser Leu Ser Pro Gly 580 <210> 33 <211> 582 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC11 <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Glu Val Gln Leu Val Gln Ser 115 120 125 Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys 130 135 140 Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe 165 170 175 Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr 180 185 190 Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 195 200 205 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Pro Leu Arg Phe 210 215 220 Leu Glu Trp Ser Thr Gln Asp His Tyr Tyr Tyr Tyr Tyr Met Asp Val 225 230 235 240 Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Ala Ser Thr 245 250 255 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 260 265 270 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 275 280 285 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 290 295 300 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 305 310 315 320 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 325 330 335 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 340 345 350 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 355 360 365 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 370 375 380 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 385 390 395 400 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 405 410 415 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 420 425 430 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 435 440 445 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 450 455 460 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 465 470 475 480 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 485 490 495 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 500 505 510 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 515 520 525 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 530 535 540 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 545 550 555 560 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 565 570 575 Leu Ser Leu Ser Pro Gly 580 <210> 34 <211> 571 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain sequence of a swapped dual V-Ig with code HC12 <400> 34 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Val Ile Asp Thr Arg Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Asn Phe Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gln Val Gln Leu Gln Glu Ser Gly Pro 115 120 125 Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser 130 135 140 Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser Trp Ile Arg Gln 145 150 155 160 Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly 165 170 175 Ser Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val 180 185 190 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Val Asn Ser Val Thr Ala 195 200 205 Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Ser Ile Phe Gly Val 210 215 220 Gly Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 245 250 255 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 260 265 270 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 275 280 285 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 290 295 300 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 305 310 315 320 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 325 330 335 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 340 345 350 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 355 360 365 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 370 375 380 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 385 390 395 400 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 405 410 415 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 420 425 430 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 435 440 445 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 450 455 460 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 465 470 475 480 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 485 490 495 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 500 505 510 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 515 520 525 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 530 535 540 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 545 550 555 560 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 35 <211> 571 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC13 <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Val Gln Leu Val Gln 115 120 125 Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Ala Met His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser Val Ile Asp Thr Arg 165 170 175 Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 180 185 190 Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg 195 200 205 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Asn Phe Tyr 210 215 220 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 225 230 235 240 Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 245 250 255 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 260 265 270 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 275 280 285 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 290 295 300 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 305 310 315 320 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 325 330 335 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 340 345 350 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 355 360 365 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 370 375 380 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 385 390 395 400 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 405 410 415 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 420 425 430 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 435 440 445 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 450 455 460 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 465 470 475 480 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 485 490 495 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 500 505 510 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 515 520 525 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 530 535 540 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 545 550 555 560 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 36 <211> 572 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC14 <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys 130 135 140 Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr Trp Leu Gly Trp Val Arg 145 150 155 160 Gln Met Pro Gly Lys Gly Leu Asp Trp Ile Gly Ile Met Ser Pro Val 165 170 175 Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Met 180 185 190 Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr Leu Gln Trp Asn Ser Leu 195 200 205 Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Arg Pro Gly 210 215 220 Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ser Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 245 250 255 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 260 265 270 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 275 280 285 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 290 295 300 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 305 310 315 320 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 325 330 335 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 340 345 350 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 355 360 365 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 370 375 380 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 385 390 395 400 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 405 410 415 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 420 425 430 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 435 440 445 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 450 455 460 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 465 470 475 480 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 485 490 495 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 500 505 510 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 515 520 525 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 530 535 540 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 545 550 555 560 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 37 <211> 572 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC15 <400> 37 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile 35 40 45 Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Glu Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala 145 150 155 160 Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly 165 170 175 His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Tyr Leu Ser Thr 210 215 220 Ala Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ser Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 245 250 255 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 260 265 270 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 275 280 285 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 290 295 300 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 305 310 315 320 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 325 330 335 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 340 345 350 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 355 360 365 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 370 375 380 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 385 390 395 400 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 405 410 415 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 420 425 430 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 435 440 445 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 450 455 460 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 465 470 475 480 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 485 490 495 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 500 505 510 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 515 520 525 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 530 535 540 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 545 550 555 560 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 38 <211> 571 <212> PRT <213> Artificial sequence <220> <223> Heavy chain sequence of the exchanged double V-Ig with code HC16 <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gln Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr Gly Met Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ile Ile Trp Tyr Asp 165 170 175 Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Gly Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu Arg Thr 210 215 220 Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 245 250 255 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 260 265 270 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 275 280 285 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 290 295 300 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 305 310 315 320 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 325 330 335 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 340 345 350 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 355 360 365 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 370 375 380 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 385 390 395 400 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 405 410 415 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 420 425 430 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 435 440 445 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 450 455 460 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 465 470 475 480 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 485 490 495 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 500 505 510 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 515 520 525 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 530 535 540 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 545 550 555 560 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 39 <211> 571 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain sequence of the swapped dual V-Ig with code HC17 <400> 39 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly 115 120 125 Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser 130 135 140 Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro 145 150 155 160 Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly His 165 170 175 Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp 180 185 190 Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 195 200 205 Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Tyr Leu Ser Thr Ala 210 215 220 Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 Gly Gly Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 245 250 255 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 260 265 270 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 275 280 285 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 290 295 300 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 305 310 315 320 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 325 330 335 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 340 345 350 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 355 360 365 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 370 375 380 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 385 390 395 400 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 405 410 415 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 420 425 430 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 435 440 445 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 450 455 460 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 465 470 475 480 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 485 490 495 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 500 505 510 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 515 520 525 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 530 535 540 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 545 550 555 560 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 <210> 40 <211> 334 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC10 <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu 115 120 125 Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr 130 135 140 Tyr Ala Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val 145 150 155 160 Ile Tyr Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 165 170 175 Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln 180 185 190 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly 195 200 205 Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 210 215 220 Gly Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 245 250 255 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 260 265 270 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 275 280 285 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 290 295 300 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 305 310 315 320 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 41 <211> 334 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC11 <400> 41 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45 Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly 100 105 110 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 115 120 125 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 130 135 140 Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 145 150 155 160 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg 165 170 175 Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 180 185 190 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr 195 200 205 Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly 210 215 220 Gly Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 245 250 255 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 260 265 270 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 275 280 285 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 290 295 300 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 305 310 315 320 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 42 <211> 333 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC12 <400> 42 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Ala Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser 115 120 125 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly 130 135 140 Ser Ser Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 145 150 155 160 Leu Ile Lys Tyr Ala Ser Gln Ser Leu Ser Gly Ile Pro Asp Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 180 185 190 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ser Ser Arg Leu 195 200 205 Pro His Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 43 <211> 333 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC13 <400> 43 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Leu Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ser Ser Arg Leu Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 115 120 125 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 130 135 140 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 145 150 155 160 Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 180 185 190 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Thr 195 200 205 Pro Leu Thr Phe Gly Gly Gly Thr Lys Ala Glu Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 44 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> It has the code LC14 of exchange double chain V-Ig sequence <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 115 120 125 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg 130 135 140 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 145 150 155 160 Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 180 185 190 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala 195 200 205 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 45 <211> 333 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC15 <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 115 120 125 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser 130 135 140 Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser 145 150 155 160 Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 180 185 190 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr 195 200 205 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 46 <211> 333 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC16 <400> 46 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 115 120 125 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg 130 135 140 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 145 150 155 160 Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 180 185 190 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala 195 200 205 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 47 <211> 333 <212> PRT <213> Artificial sequence <220> <223> Light chain sequence of the exchanged double V-Ig with code LC17 <400> 47 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly 100 105 110 Gly Gly Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr 115 120 125 Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly 130 135 140 Ser Ser Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu 145 150 155 160 Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe 165 170 175 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu 180 185 190 Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu 195 200 205 Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Gly Gly Gly 210 215 220 Gly Gly Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 225 230 235 240 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 245 250 255 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 260 265 270 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 275 280 285 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 290 295 300 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 305 310 315 320 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325 330 <210> 48 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 48 Ala Ser Thr Lys Gly Pro Ser 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 49 Arg Thr Val Ala Ala Pro Ser 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 50 Gly Gln Pro Lys Ala Ala Pro 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 51 His Ile Asp Ser Pro Asn Lys 1 5 <210> 52 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 52 Thr Lys Gly Pro Ser 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 53 Thr Val Ala Ala Pro 1 5 <210> 54 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 54 Gln Pro Lys Ala Ala 1 5 <210> 55 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 55 Gln Arg Ile Glu Gly 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 56 Gly Gly Cys Gly Gly Gly Gly 1 5 <210> 57 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 57 Gly Gly Gly Cys Gly Gly Gly 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 58 Gly Gly Gly Gly Cys Gly Gly 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial peptide linker <400> 59 Gly Gly Gly Gly Gly Cys Gly 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence from the IGHG1 hinge region <400> 60 Asp Lys Thr His Thr 1 5

Claims

1. An antibody-like binding protein comprising two polypeptide chains forming two antigen binding sites, wherein the first polypeptide chain comprises structure V L1 -L1-V L2 -L2-C L , and the second polypeptide chain comprises structure V H2 -L3-V H1 -L4-C H1 ; in: (a)(i) V L1 V is the variable domain of the first immunoglobulin heavy chain H , V L2 is the second immunoglobulin light chain variable domain, V H2 is a second immunoglobulin heavy chain variable domain, and V H1 V is the first immunoglobulin light chain variable domain L ;or (ii) V L1 is the first immunoglobulin light chain variable domain, V L2 V is the second immunoglobulin heavy chain variable domain H , V H2 V is the second immunoglobulin light chain variable domain L , and V H1 is a first immunoglobulin heavy chain variable domain; or (iii) V L1 V is the variable domain of the first immunoglobulin heavy chain H , V L2 V is the second immunoglobulin heavy chain variable domain H , V H2 V is the second immunoglobulin light chain variable domain L , and V H1 V is the first immunoglobulin light chain variable domain L ; Where V L1 and V H1 associates to form a first antigen binding site, and V L2 and V H2 associate to form a second antigen binding site; (b) C L is the immunoglobulin light chain constant domain; (c) C H1 Immunoglobulin C H1 a heavy chain constant domain; and (d) L1, L2, L3 and L4 are amino acid linkers having amino acid lengths according to the following combination: L1=1, L2=1, L3=2, L4=4; L1=1, L2=2, L3=1, L4=4; L1=1, L2=2, L3=12, L4=12; L1=1, L2=2, L3=15, L4=15; L1=1, L2=2, L3=2, L4=2; L1=1, L2=2, L3=2, L4=4; L1=1, L2=2, L3=2, L4=7; L1=1, L2=2, L3=4, L4=4; L1=1, L2=2, L3=4, L4=7; L1=1, L2=2, L3=6, L4=4; L1=1, L2=2, L3=6, L4=7; L1=1, L2=2, L3=9, L4=12; L1=1, L2=3, L3=2, L4=4; L1=1, L2=4, L3=2, L4=4; L1=2, L2=2, L3=2, L4=4; L1=5, L2=5, L3=1, L4=2; L1=7, L2=3, L3=1, L4=2; L1=7, L2=5, L3=1, L4=2; L1=10, L2=3, L3=1, L4=2; L1=10, L2=5, L3=1, L4=2; L1=5, L2=5, L3=2, L4=2; L1=7, L2=5, L3=2, L4=2; L1=10, L2=3, L3=2, L4=2; L1=10, L2=5, L3=2, L4=2; or L1=12, L2=6, L3=8, L4=2; And wherein the first and second polypeptides form an exchange light chain-heavy chain pair.

2. The antibody-like binding protein according to claim 1, wherein the binding protein is capable of specifically binding to one or more antigenic targets.

3. The antibody-like binding protein according to claim 2, wherein the one or more antigen targets are selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11, CCL15, CCL17, CCL19, CCL2, CCL20, CCL21, SLC, CCL24, CCL25, CCL26, CCL3, CCL4, CCL5, CCL7, CCL8, CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1, chitin, , CSF1, CSF2, CSF3, CTLA4, CX3CL1, CXCL12, CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, ITGB4, LEP, MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4, TNFSF5, Toll-like receptor, TREM1, TSLP, TWEAK, XCR1, DNGR-1, and HMGB1. The antibody-like binding protein according to claim 1 , wherein the binding protein is bispecific and is capable of binding to two different antigenic targets.

5. The antibody-like binding protein according to claim 4, wherein the two different antigen targets are selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL1β, TNFα and IL-23, and IL17 and IL23. The antibody-like binding protein according to claim 1 , wherein the binding protein is capable of inhibiting the function of one or more of the antigen targets.

7. The antibody-like binding protein according to claim 1, wherein at least one of the linkers selected from L1, L2, L3 and L4 contains at least one cysteine ​​residue.

8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antibody-like binding protein of claim 1.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody-like binding protein of any one of claims 1-7.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. An isolated host cell comprising the nucleic acid molecule of claim 9 or an expression vector containing the nucleic acid molecule of claim 9.

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