RU2823693C2 - Antibody-like binding proteins with double variable regions having cross-cross orientation of binding regions - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to an antibody-like binding protein construct. This construct binds a first antigen and a second antigen and has interchangeable variable domains containing two polypeptide chains that form two antigen-binding sites. The invention also relates to an isolated nucleic acid molecule encoding the said antibody-like binding protein construct, as well as an expression vector and an isolated host cell containing it.

EFFECT: invention makes it possible to effectively obtain a bispecific antibody-like binding protein that binds a pair of antigens.

26 cl, 8 dwg, 16 tbl, 13 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to antibody-like binding proteins containing four polypeptide chains that form four antigen-binding sites, wherein each pair of polypeptides forming the antibody-like binding protein has double variable domains having a "cross-over" orientation. The invention also relates to methods for producing such antigen-like binding proteins.

LEVEL OF TECHNOLOGY

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies, which have binding specificities for two different antigens, can be produced using recombinant techniques and may be intended for widespread clinical use. It is well known that complete IgG antibody molecules are Y-shaped molecules containing four polypeptide chains: two heavy chains and two light chains. Each light chain contains two domains, with the N-terminal domain called the variable or VL domain (or region), and the C-terminal domain called the constant (or CL) domain (constant kappa (Cκ) or constant lambda (Cλ) domain). Each heavy chain consists of four or five domains, depending on the class of antibody. The N-terminal domain is called the variable (or VH) domain (or region), followed by the first constant (or CH1) domain, the hinge region, and then the second and third constant (or CH2 and CH3) domains. In the assembled antibody, the VL and VH domains associate together to form the antigen-binding site. Also, the CL and CH1 domains associate together, holding one heavy chain in association with one light chain. The two heavy-light chain heterodimers are associated together by interactions between the CH2 and CH3 domains and interactions between the hinge regions of the two heavy chains.

It is known that proteolytic cleavage of an antibody can lead to the formation of antibody fragments (Fab and Fab2). Such fragments of whole antibodies can exhibit antigen-binding activity. Antibody fragments can also be produced recombinantly. Fv fragments can be produced consisting only of the variable domains of the heavy and light chains associated with each other. Such Fv fragments are monovalent with respect to antigen binding. Smaller fragments such as individual variable domains (domain antibodies or dABs; Ward et al., 1989, Nature 341(6242): 544–46) and individual complementarity-determining regions (CDRs) (Williams et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86(14): 5537–41) have also been shown to retain the binding characteristics of the parent antibody, although most naturally occurring antibodies require both VH and VL to fully retain binding efficiency.

Single-chain variable fragment (scFv) constructs contain the VH and VL domains of an antibody in a single polypeptide chain, with the domains separated by a flexible linker of sufficient length (>12 amino acid residues) to facilitate forced intramolecular interactions that allow self-assembly of the two domains into a functional epitope-binding site (Bird et al., 1988, Science 242(4877): 423–26). Such small proteins (Mw ~25,000 Da) typically retain specificity and affinity for their antigen in a single polypeptide and can serve as a convenient building block for larger antigen-specific molecules.

The advantage of using antibody fragments rather than whole antibodies in diagnostics and therapy is their small size. They are apparently less immunogenic than whole antibodies and have a greater ability to penetrate tissues. A disadvantage associated with the use of such fragments is that they have only one antigen-binding site, which results in reduced avidity. In addition, due to their small size, they are cleared very quickly from the serum and, therefore, have a short half-life.

Of interest has been the production of bispecific antibodies (BsAbs), which combine the antigen-binding sites of two antibodies in a single molecule and may therefore be able to bind two different antigens simultaneously. In addition to diagnostic applications, such molecules open the way to new therapeutic applications, for example by redirecting potent effector systems to disease sites (in which malignant cells often develop mechanisms to suppress normal monoclonal antibody-triggered immune responses such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)), or by enhancing the neutralizing or stimulatory activity of antibodies. This ability has been known for some time, leading to the development of a number of methods for producing such bispecific antibodies. Initial attempts to combine the binding specificities of two intact antibodies against different target antigens for therapeutic purposes used chemically fused heteroconjugate molecules (Staerz et al., 1985, Nature 314(6012): 628-31).

Bispecific antibodies were originally produced by fusing two hybridomas that produce different immunoglobulins (Milstein et al., 1983, Nature 305(5934): 537–40), but the complexity of the species composition (up to ten different species) produced in cell culture makes purification difficult and expensive (George et al., 1997, The antibodies 4: 99–141 (Capra et al., ed., Harwood Academic Publishers)). Using this format, a mouse IgG2a antibody and a rat IgG2b antibody have been produced in the same cell (e.g., either as a quadratic fusion of two hybridomas or in engineered CHO cells). Because the light chains of each antibody bind preferentially to the heavy chains of their cognate species, three main antibody species are assembled: the two parent antibodies and a heterodimer of the two antibodies, containing one heavy/light chain pair from each antibody associated with their Fc portions. The desired heterodimer can be purified from this mixture because its protein A binding properties differ from those of the parent antibodies: rat IgG2b does not bind protein A, whereas mouse IgG2a does. The mouse-rat heterodimer therefore binds protein A but elutes at a higher pH than the mouse IgG2a homodimer, allowing 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 cause deleterious side effects (e.g., "HAMA" or "HARA" reactions) and/or neutralize the therapeutic agent. There remains a need for engineered bispecific agents with superior properties that can be readily produced in high yields from mammalian cell culture.

Despite the promising results obtained using heteroconjugates or bispecific antibodies derived from cell fusions as described above, several factors make them unsuitable for large-scale therapeutic applications. These factors include: rapid clearance of heteroconjugates in vivo, labor-intensive laboratory techniques required to generate either type of molecule, the need for careful purification of heteroconjugates from homoconjugates or monospecific antibodies, and the generally low yields obtained.

Genetic engineering has been increasingly used to design, modify, and produce antibodies or antibody derivatives with the desired combination of binding properties and effector functions. Several recombination-based methods have been developed for the efficient production of BsAbs, both in the form of 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 in the form of full-length IgG (Carter, 2001, J. Immunol. Methods 248(1–2): 7–15).

The fusion of two different scFvs produces forms of BsAb with minimal molecular weight, termed sc-BsAb or Ta-scFv (Mack et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92(15): 7021–25; Mallender et al., 1994, J. Biol. Chem. 269(1): 199–206). BsAbs have been constructed using genetic fusion of two scFvs using a functional group such as a leucine zipper for dimerization (Kostelny et al., 1992, J. Immunol. 148(5): 1547–53; de Kruif et al., 1996, J. Biol. Chem. 271(13): 7630–34).

Diabodies are small bivalent and bispecific antibody fragments. The fragments contain VH linked to VL on the same polypeptide chain using a linker that is too short (less than 12 amino acid residues) to allow pairing between the two domains on the same chain. The domains are forced to pair intermolecularly with complementary domains on the other chain and create two antigen-binding sites. Such dimeric antibody fragments or "diabodies" are bivalent and bispecific (Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48). Diabodies are similar in size to a Fab fragment. The polypeptide chains of the VH and VL domains are linked by a linker of 3 to 12 amino acid residues in length, predominantly forming dimers (diabodies), whereas in the case of linkage by a linker of 0 to 2 amino acid residues in length, trimers (triantibodies) and tetramers (tetraantibodies) predominate. In addition to the dependence on the linker length, the exact pattern of oligomerization apparently depends on the composition as well as the orientation of the variable domains (Hudson et al., 1999, J. Immunol. Methods 231(1-2): 177-89). The probability of predicting the final structure of diabody molecules is very low.

Although sc-BsAb and diabody based constructs show interesting clinical potential, it has been shown that such non-covalently associated molecules are not sufficiently stable under physiological conditions. The overall stability of the scFv fragment depends on the intrinsic stability of the VL and VH domains as well as on the stability of the interface of the domains. Often, the lack of stability of the VH-VL interface of scFv fragments has been considered the main reason for the irreversible inactivation of scFv, since the transient opening of the interface, which can be allowed by the peptide linker, exposes hydrophobic regions that promote aggregation and hence instability and low product yield (Worn et al., 2001, J. Mol. Biol. 305(5): 989-1010).

An alternative method for producing bispecific bivalent antigen-binding proteins from VH and VL domains is described in U.S. Patent No. 5,989,830. Such dual-head, dual-Fv configurations are produced by expression of a bicistronic vector that encodes two polypeptide chains. In the dual-Fv configuration, the variable domains of two different antibodies are expressed in tandem orientation on two different chains (one heavy chain and one light chain), with one polypeptide chain having two VHs arranged sequentially and separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consisting of complementary VL domains joined sequentially by a peptide linker (VL1-linker-VL2). In the dual-headed criss-cross configuration, the variable domains of two different antibodies are expressed in tandem orientation in two separate polypeptide chains (one heavy chain and one light chain), with one polypeptide chain having two VHs arranged sequentially and separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consisting of complementary VL domains joined sequentially by a peptide linker in the opposite orientation (VL2-linker-VL1). Molecular modeling of the constructs suggests that the linker size should be long enough to extend over 30-40 (15-20 amino acid residues).

Increasing the valency of an antibody is of interest because it enhances the functional affinity of the antibody due to an avidity effect. Multivalent protein complexes (PPCs) of increased valency are described in U.S. Patent Application Publication No. US 2005/0003403 A1. PPCs contain two polypeptide chains, typically arranged laterally to each other. Each polypeptide chain typically contains three or four "v regions" that contain amino acid sequences capable of forming an antigen-binding site when paired with a corresponding v region on the opposite polypeptide chain. Up to about six "v regions" on each polypeptide chain can be used. The v regions of each polypeptide chain are linked to each other in a linear fashion and may be linked by intervening binding regions. When arranged in the form of a PPC, the v regions on each polypeptide chain form antigen-binding sites. The complex may have one or more binding specificities.

Carter and colleagues (Ridgway et al., 1996, Protein Eng. 9(7): 617–21; Carter, 2011, J. Immunol. Methods 248(1–2): 7–15) proposed a method for generating an Fc heterodimer using a set of knob-into-hole mutations in the CH3 domain of Fc. These mutations alter the complementarity of the packing of residues between the CH3 domain interface and the structurally conserved hydrophobic core so that heterodimer formation is preferentially favored over homodimers, resulting in good expression of the heterodimers in mammalian cell culture. Although the method resulted in higher yields of heterodimers, homodimer formation was not completely suppressed (Merchant et al., 1998, Nat. Biotechnol. 16(7): 677–81).

Gunasekaran et al. investigated the possibility of preserving the integrity of the hydrophobic core while driving Fc heterodimer formation by altering the charge complementarity at the interface of the CH3 domain (Gunasekaran et al., 2010, J. Biol. Chem. 285(25): 19637-46). Taking advantage of the electrostatic field-guided orientation mechanism, such constructs effectively promote Fc heterodimer formation with minimal homodimer contamination by mutating two pairs of peripherally located charged residues. In contrast to the knob-into-hole construct, homodimer formation is equally suppressed due to the nature of the electrostatic repulsion mechanism, but it is not completely avoided.

Davis et al. describe a method for engineering antibodies to convert Fc homodimers into heterodimers by intertwining the β-lactamase regions of the CH3 domains of human IgG and IgA without introducing additional interchain disulfide bonds (Davis et al., 2010, Protein Eng. Des. Sel. 23(4): 195–202). Expression of SEEDbody (Sb) fusion proteins in mammalian cells produces Sb heterodimers in high yields that are easily purified without minor byproducts.

US Patent Application Publication No. US 2010/331527 A1 describes a bispecific antibody based on heterodimerization of the CH3 domain, introducing into one heavy chain the mutations H95R and Y96F in the CH3 domain. Such amino acid substitutions are derived from the CH3 domain of the IgG3 subtype, and will heterodimerize with the IgG1 backbone. A typical light chain prone to pairing with any heavy chain is a prerequisite for all forms based on heterodimerization via the CH3 domain. Therefore, a total of three types of antibodies are obtained: 50% having a pure IgG1 backbone, one third having a pure backbone with the mutations H95R and Y96F, and one third having two different heavy chains (bispecific). The desired heterodimer can be purified from such a mixture because its binding properties to protein A differ from those of the parent antibodies: IgG3-derived CH3 domains do not bind to protein A, whereas IgG1 does. Therefore, the heterodimer binds to protein A but elutes at a higher pH than the pure IgG1 homodimer, allowing selective purification of the bispecific heterodimer.

U.S. Patent No. 7,612,181 describes bispecific IgG dual variable domain (DVD-IgG) antibodies that are based on the dual Fv format described in U.S. Patent No. 5,989,830. A similar bispecific form is also described in U.S. Patent Application Publication No. US 2010/0226923 A1. The addition of constant domains to the respective chains of the dual Fv (CH1-Fc to the heavy chain and a kappa or lambda constant domain to the light chain) results in the formation of functional bispecific antibodies without any need for further modifications (i.e., the apparent addition of constant domains to improve stability). For some of the antibodies expressed in the DVD-Ig/TBTI form, a position effect with respect to the second (or deeper inside) antigen-binding position (Fv2) is observed. Depending on the sequence and nature of the antigen recognized by the Fv2 position, such an antibody domain exhibits reduced affinity for its antigen (i.e., decreased rate of association compared to the parent antibody). One possible explanation for this observation is that the linker between VL1 and VL2 projects into the CDR region of Fv2, rendering Fv2 somewhat inaccessible to larger antigens.

The second configuration of the bispecific antibody fragment described in U.S. Patent No. 5,989,830 is a cross-headed dual (CODH) configuration having the following orientation of the variable domains expressed in two chains:

VL1-linker-VL2 in case of light chain and

VH2-linker-VH1 in case of heavy chain.

The '830 patent describes the fact that a bispecific antibody fragment with two cross-shaped heads (the GOSA.E construct) retains higher binding activity than a dual Fv (see page 20, lines 20-50 of the '830 patent), and further discloses the fact that such a form is less susceptible to interference from linkers used between variable domains (see pages 20-21 of the '830 patent).

ESSENCE OF THE INVENTION

The invention relates to an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and two polypeptide chains have a structure represented by the formula:

VH2-L3-VH1- _L4 -C _H1 -Fc [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3;

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

The invention also relates to an antibody-like binding protein comprising two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and the second polypeptide chain has a structure represented by the formula:

VH2-L3-VH1-L4-CH1 [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

L1, L2, L3 and L4 represent amino acid linkers;

and the first and second polypeptides form a light chain-heavy chain pair in a crosswise orientation.

The invention further relates to a method for producing an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, comprising identifying a first variable domain of the antibody that binds a first target antigen and a second variable domain of the antibody that binds a second target antigen, each comprising a VL and a VH; determining either the light chain or the heavy chain as a template chain; determining the VL of the first variable domain of the antibody or the second variable domain of the antibody as VL1; determining VL2, VH1 and VH2 according to formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1-Fc [II]

determining the maximum and minimum lengths of L1, L2, L3, and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that bind a first target antigen and a second target antigen when combined to form an antibody-like binding protein;

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

The invention further relates to a method for producing an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites, comprising identifying a first variable domain of the antibody that binds a first target antigen and a second variable domain of the antibody that binds a second target antigen, each comprising a VL and a VH; determining either the light chain or the heavy chain as a template chain; determining the VL of the first variable domain of the antibody or the second variable domain of the antibody as VL1; determining VL2, VH1 and VH2 according to formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1 [II]

determining the maximum and minimum lengths of L1, L2, L3, and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that bind a first target antigen and a second target antigen when combined to form an antibody-like binding protein;

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 denotes the constant domain of the immunoglobulin heavy chain C _H1 ; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

Specific embodiments of the invention will become apparent from the following detailed description of certain embodiments and the claims.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Schematic representation of the antigen-binding domains of Fv1 and Fv2 in the double V-region configuration and the arrangement of their corresponding peptide linkers LL and LH in the TBTI form.

Figure 2. Schematic diagram (2D) of the antigen-binding domains of Fv1 (anti-IL4) and Fv2 (anti-IL13) in a cross-over dual variable region (CODV) configuration and the arrangement of their corresponding peptide linkers.

Figure 3. Schematic representation of anti-IL4 Fv and anti-IL13 Fab showing one possible spatial arrangement resulting from protein-protein docking of anti-IL4 Fv and anti-IL13 Fv.

Figure 4. Evaluation of the tetravalent and bispecific binding capacity of CODV protein in the BIACORE assay by injecting two antigens sequentially or simultaneously onto a chip coated with DVD-Ig protein. The maximum signal observed with sequential injection can be obtained by co-injection of two antigens, indicating saturation of all binding sites.

Figure 5. Schematic diagram (2D) of the antigen-binding domains in the CODV configuration and the arrangement of their corresponding LL (L1 and L2) and LH (L3 and L4) peptide linkers. In panel A, the light chain is maintained in a “linear or matrix” arrangement while the heavy chain is in a “criss-cross” arrangement. In panel B, the heavy chain is maintained in a “linear or matrix” arrangement while the light chain is in a “criss-cross” arrangement.

Figure 6. Schematic representation of the CODV-Ig design based on whether the light chain or heavy chain is used as the "template".

Figure 7. Comparison of TBTI/DVD-Ig or CODV-Ig molecules containing anti-IL4 and anti-IL13 sequences.

Figure 8. Comparison of CODV-Fab and B-Fab forms in a cytotoxicity assay using NALM-6 cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to antibody-like binding proteins containing four polypeptide chains that form four antigen-binding sites, wherein each pair of polypeptides that form the antibody-like binding protein has double variable domains that have a crosswise orientation. The invention also relates to methods for producing such antigen-like binding proteins.

Using computer modeling, it was predicted that the cross-head dual (CODH) design described in U.S. Patent No. 5,989,830 could yield a complex in which both binding sites are face-to-face in the opposite direction without the limitations implied by the dual-Fv configuration described in U.S. Patent No. 7,612,181. In particular, computer modeling showed that the length of the amino acid linkers between the variable domains is not critical in the CODH design, but is important to ensure full access to both antigen-binding sites in the dual-Fv design. As with the DVD-Ig/TBTI form, antibody-like binding proteins were constructed in which the constant domains were linked in a CODH configuration to form antibody-like binding proteins containing four polypeptide chains that form four antigen-binding sites, with each pair of polypeptides forming the antibody-like binding protein having dual variable domains in a cross-like orientation (i.e., CODH-Ig). CODH-Ig molecules are expected to have significantly increased stability compared to CODH molecules (since DVD-Ig/TBTI had increased stability compared to dual Fv molecules).

To test the above hypotheses, a CODH-Ig molecule was generated using the anti-IL4 and anti-IL13 antibody sequences described in U.S. Patent Application Publication No. US 2010/0226923 A1. The CODH-Ig molecule differed from the CODH molecule described in US 2010/0226923 with respect to the lengths of the amino acid linkers separating the variable domains on the respective polypeptide chains. The CODH-Ig molecules were expressed in transiently transfected cells and then purified by protein A chromatography. Although they exhibited 5-10% aggregation levels in size exclusion chromatography (SEC) profiles, none of the CODH-Ig molecules were functional and, therefore, none of the CODH-Ig molecules were able to bind any of their target antigens. The lack of antigen-binding activity could be due to impaired dimerization of the Fv regions of the heavy and light chains due to inappropriate linker lengths preventing formation of the correct paratope. As a result, a protocol was developed to identify suitable amino acid linkers for insertion between the two variable domains and the second variable domain and constant domain on both the heavy and light polypeptide chains of the antibody-like binding protein. Such a protocol was based on protein-protein homology docking and experimental models of the FvIL4 and FvIL13 regions, respectively, incorporation of the Fc1 domain into the model and construction of suitable linkers between the FvIL4 and FvIL13 regions and between Fv and the Fc1 constant regions.

Standard recombinant DNA technology is used to construct polynucleotides that encode polypeptides that form the antibody-like binding proteins of the invention, incorporate such polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification methods can be carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. Unless otherwise specified, the nomenclature used in connection with the analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry laboratory methods and techniques described herein is that well known and commonly used in the art. Similarly, conventional techniques for chemical synthesis, chemical analysis, preparation, formulation, delivery of pharmaceuticals, and treatment of patients can be employed.

1. General definitions

As used in this description, the following terms, unless otherwise specified, shall be understood to have the following meanings. Unless the context otherwise requires, terms used in the singular shall include the plural forms, and terms used in the plural shall include the singular forms.

The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In some embodiments, the nucleotides comprising a polynucleotide may be ribonucleotides or deoxyribonucleotides, or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothiate, phosphoraniladate, and phosphoramidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

The term "isolated polynucleotide" means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, wherein by virtue of its origin the isolated polynucleotide: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide naturally occurs, (2) is associated with a polynucleotide with which it is not naturally associated, or (3) is not naturally occurring as part of a larger sequence.

An "isolated polypeptide" is a polypeptide that: (1) is free of at least some other polypeptides with which it may be found in nature, (2) is substantially free of other polypeptides from the same source, such as the same species, (3) is expressed by a cell of another species, (4) has been separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature, (5) is not associated (through covalent or non-covalent interactions) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is functionally associated (through covalent or non-covalent interactions) with a polypeptide with which it is associated in nature, or (7) is not found in nature. Such an isolated polypeptide may be encoded in 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 impurities that occur in its natural environment that may interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

The term "human antibody" as used herein includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences. In some embodiments, human antibodies are produced in non-human mammals, including, but not limited to, rodents such as mice and rats, and lagomorphs such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In further embodiments, human antibodies are produced recombinantly.

Naturally occurring antibodies typically comprise a tetramer. Each such tetramer typically consists 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). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids in length, which is typically responsive to antigen recognition. The carboxyl-terminal portion of each chain typically defines the boundaries of a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length immunoglobulin heavy chain polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2, and CH3), with the VH domain at the amino terminus of the polypeptide and the CH3 domain at the carboxyl terminus, and a full-length immunoglobulin light chain polypeptide comprises a variable domain (VL) and a constant domain (CL), with the VL domain at the amino terminus of the polypeptide and the CL domain at the carboxyl terminus.

Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype 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 similarly divided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains are typically linked by a "J" region of approximately 12 or more amino acids, with the heavy chain also including a "D" region of approximately another 10 amino acids. See, e.g., 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 typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically have the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically lined up in a row with framework regions that can mediate binding to a particular epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The term "native Fc" as used herein refers to a molecule comprising a non-antigen-binding fragment sequence obtained by cleavage of an antibody or produced by other means, either in monomeric or multimeric form, and may comprise a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc molecules are composed of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomeric subunits of native 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 IgA2). One example of a native Fc is the disulfide-linked dimer produced by papain cleavage of IgG. The term "native Fc" as used herein is a generic term for monomeric, dimeric, and multimeric forms.

The term "Fc variant" as used herein refers to a molecule or sequence that is modified compared to a native Fc but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Examples of Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" may include a molecule or sequence that is humanized compared to a native Fc of a non-human animal. In addition, a native Fc contains regions that may be deleted because they provide structural features or biological activity that is not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" includes a molecule or sequence that lacks one or more regions or residues of a native Fc, or that has modified one or more regions or residues of an Fc that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity when expressed in the 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).

The term "Fc domain" as used herein includes native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, either cleaved from a whole antibody or produced by other means.

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

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and two polypeptide chains have a structure represented by the formula:

VH2-L3-VH1- _L4 -C _H1 -Fc [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3;

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation. 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 that comprises two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and two polypeptide chains have a structure represented by the formula:

VH2-L3-VH1- _L4 -C _H1 [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the first and second polypeptides form a light chain-heavy chain pair in a criss-cross orientation. A "recombinant" molecule is one that has been produced, expressed, created, or isolated by recombinant means.

One embodiment of the invention relates to antibody-like binding proteins having biological and immunological specificity for one to four target antigens. Another embodiment of the invention relates to nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such antibody-like binding proteins. Another embodiment of the invention relates to expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such antibody-like binding proteins. Yet another embodiment of the invention relates to host cells that express such antibody-like binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such antibody-like binding proteins).

The term "swappability" as used herein refers to the interchangeability of the variable domains in a CODV form with preservation of folding and final binding affinity. "Fully swappable" refers to the ability to change the order of the VH1 and VH2 domains, and hence the order of the VL1 and VL2 domains in a CODV-Ig (i.e., reverse the order) or a CODV-Fab, while maintaining full functionality of the antibody-like binding protein, as evidenced by preservation of binding affinity. It should also be noted that the designation of VH and VL in a particular CODV-Ig or CODV-Fab refers only to the location of the domain in a particular protein chain in the final form. For example, VH1 and VH2 may be derived from the VL1 and VL2 domains in the parent antibodies and placed at the VH1 and VH2 positions in the antibody-like binding protein. Similarly, VL1 and VL2 can be derived from the VH1 and VH2 domains in the parent antibodies and placed at the VH1 and VH2 positions in the antibody-like binding protein. Thus, the VH and VL designations refer to the present localization, not the original localization in the parent antibody. Therefore, the VH and VL domains "can be swapped."

An "isolated" antibody-like binding protein is a protein that has been identified and separated and/or recovered from a mixture with a component of its natural environment. Contaminating components from its natural environment are substances that may interfere with diagnostic or therapeutic uses of the antibody-like binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous soluble substances. In preferred embodiments, the antibody-like binding protein may be purified: (1) to greater than 95% by weight of antibody, as determined by the Lowry method, and most preferably greater than 99% by weight, (2) sufficiently to obtain at least 15 residues of the N-terminal or internal amino acid sequence, using a spinning cup sequencer, or (3) to homogeneity on SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue staining, or, preferably, silver staining. Isolated antibody-like binding proteins include antibody-like binding protein in situ in recombinant cells, since in this case at least one component of the natural environment of the antibody-like binding protein is absent.

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

The term "antigen" or "target antigen" as used herein refers to a molecule or portion of a molecule that can be bound by an antibody-like binding protein and can further be used in an animal to produce antibodies capable of binding to an epitope of such an antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by the antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes that target antigen. It should be understood that a "bivalent" antibody-like binding protein, other than a "polyspecific" or "polyfunctional" antibody-like binding protein, contains antigen-binding sites that have identical antigen specificities.

A bispecific or bifunctional antibody is typically an artificial hybrid antibody having two different heavy chain/light chain pairs and two different binding sites or epitopes. Bispecific antibodies can be produced by a variety of methods, including but not limited to hybridoma fusion or F(ab') fusion.

An F(ab) fragment typically comprises one light chain and the VH and CH1 domains of one heavy chain, wherein the VH-CH1 portion of the heavy chain of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, an F(ab) fragment may also include one light chain comprising two variable domains separated by an amino acid linker and one heavy chain comprising two variable domains separated by an amino acid linker and a CH1 domain.

The F(ab') fragment typically comprises one light chain and a portion of one heavy chain that contains an additional constant region (between the CH1 and CH2 domains) so that an interchain disulfide bond can be formed between the two heavy chains to form an F(ab')2 molecule.

The phrases "biological property," "biological characteristic," and the term "activity" with respect to the antibody-like binding protein of the invention are used interchangeably herein and include, without limitation, affinity and specificity for an epitope, the ability to antagonize the activity of a target antigen (or target polypeptide), the stability of the antibody-like binding protein in vivo, 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., with non-human homologs of the target antigen, or with other target antigens or tissues in general) and the ability to maintain high levels of protein expression in mammalian cells. The above properties or characteristics can be observed and measured using methods known in the art, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance assay, in vitro and in vivo neutralization assays, and immunohistochemistry using tissue sections from various sources, including human, primate, or any other source that may be necessary.

The term "immunologically functional fragment of an immunoglobulin" as used herein refers to a polypeptide fragment that comprises at least the CDRs of the heavy and light chains of the immunoglobulin from which the polypeptide fragment is derived. An immunologically functional fragment of an immunoglobulin is capable of binding to a target antigen.

A "neutralizing" antibody-like binding protein as used herein refers to a molecule that is capable of blocking or significantly reducing the effector function of a target antigen to which it binds. As used herein, "significantly reducing" means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, most preferably at least about 90% reduction in the effector function of the target antigen.

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

The dissociation constant (KD) of an antibody-like binding protein can be determined, for example, using surface plasmon resonance. In general, a real-time surface plasmon resonance assay measures the binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (antibody-like binding protein in solution) based on surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed using immobilization of the analyte (antibody-like binding protein on a biosensor matrix) and presentation of the ligand (target antigen). The term "KD" as used herein refers to the dissociation constant for the interaction between a particular antibody-like binding protein and a target antigen.

The term "specifically binds" as used herein refers to the ability of an antibody-like protein or antigen-binding fragment thereof to bind to an antigen containing an epitope with a Kd of at least about 1×10-6 M, 1×10-7 M, 1×10-8 M, 1×10-9 M, 1×10-10 M, 1×10-11 M, 1×10-12 M or greater, and/or to bind to the epitope with an affinity that is at least two to three times higher than its affinity for a non-specific antigen.

The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient flexibility for the light and heavy chain domains to fold into dual variable region immunoglobulins in a crosswise fashion. The linker is inserted into the transition region between the variable domains or between the variable and constant domains, respectively, at the sequence level. The transition region between the domains can be identified because the approximate size of the immunoglobulin domains is well known. The exact position of the transition region between the domains can be determined by the location of peptide regions that do not form secondary structure elements such as beta sheets or alpha helices, which are shown by experimental data or which can be inferred using secondary structure modeling or prediction methods. The linkers described herein are referred to as: L1, which is located in the light chain between the N-terminal VL1 domain and the VL2 domain; L2, which is also found in the light chain and is located between the VL2 domain and the C-terminal CL domain. The heavy chain linkers are named: L3, which is located between the N-terminal VH2 and VH1 domains; and L4, which is located between the VH1 and CH1-Fc domains. The linkers L1, L2, L3, and L4 are independent, but in some cases they may have the same sequence and/or length.

The term "vector" as used herein refers to any molecule (e.g., a nucleic acid, plasmid, or virus) that is used to transfer coding information into a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular, double-stranded DNA molecule into which additional sections of DNA can be inserted. Another type of vector is a viral vector, in which case additional fragments of DNA can be inserted into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the host cell's genome upon introduction into the host cell and are thereby replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in DNA recombination techniques are often in the form of plasmids. In the present specification, the terms "plasmid" and "vector" may be used interchangeably, since the plasmid is the most widely used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The term "operably linked" is used herein to refer to an arrangement of flanking sequences, wherein the flanking sequences so described are configured or assembled such that they perform their normal function. Thus, a flanking sequence operably linked to a coding sequence may be capable of influencing the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter if the promoter is capable of directing the transcription of the coding sequence. The flanking sequence need not be adjacent to the coding sequence, so long as it functions properly. Thus, for example, intervening untranslated but still transcribed sequences may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to a particular cell of a subject, but also to the progeny of such a cell. Since some modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the original cell, but will still be within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the antibody-like binding proteins of the 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. To express an antibody-like binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the antibody-like binding protein, such that the polypeptide chains are expressed in the host cell and are preferably secreted into a medium in which the host cells are cultured, and from such medium the antibody-like binding protein can be recovered.

The term "transformation" as used herein refers to a change in the genetic characteristics of a cell, and a cell is transformed when it is modified to contain new DNA. For example, a cell is transformed when it is genetically modified compared to its native state. Following transformation, the transforming DNA may recombine with the cell's DNA by physical integration into the cell's chromosome, or may be maintained as an episomal element without replication, or may replicate independently as a plasmid. A cell is considered stably transformed when the DNA replicates with cell division. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA has been introduced through the cell membrane. Several transfection methods are well known in the art. Such methods can be used to introduce one or more exogenous DNA molecules into suitable host cells.

The term "naturally occurring" as used herein and applied to a particular object refers to the fact that the object can be found in nature and is not produced by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses), that can be isolated from a source in nature and that has not been intentionally modified by man is naturally occurring. Similarly, "non-naturally occurring" as used herein refers to an object that does not occur in nature or that has been structurally modified or synthesized by man.

As used herein, the names of the twenty common amino acids and their abbreviations are consistent with conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty common amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unusual amino acids may also be suitable components of the polypeptide chains of the antibody-like binding proteins of the invention. Examples of unusual 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 designation used herein, the leftward direction is the amino-terminal direction and the rightward direction is the carboxyl-terminal direction in accordance with standard usage and conventions.

Naturally occurring residues can be divided into classes based on common side chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

(2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

(3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

(4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(6) acidic: Asp, Glu;

(7) basic: His, Lys, Arg;

(8) residues that influence chain orientation: Gly, Pro;

(9) aromatic: His, Trp, Tyr, Phe; and

(10) aromatic hydrophobic: Phe, Trp, Tyr.

Conservative amino acid substitutions may involve replacing a member of one such class with another member of the same class. Conservative amino acid substitutions may involve non-naturally occurring amino acid residues that are typically incorporated in chemical peptide synthesis but that are not incorporated in biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid residues. Non-conservative substitutions may involve replacing a member of one such class with a member of another class.

A person skilled in the art can determine suitable variants of the polypeptide chains of the antibody-like binding proteins of the invention using well-known techniques. For example, a person skilled in the art can identify suitable regions of the polypeptide chain that can be altered without impairing activity by directly targeting regions that are not believed to be important for activity. Alternatively, a person skilled in the art can identify residues and portions of molecules that are conserved in similar polypeptides. In addition, even regions that may be important for biological activity or for structure can be subjected to conservative amino acid substitutions without impairing biological activity or without adversely affecting the structure of the polypeptide.

The term "patient" as used herein includes humans and animals.

"Disorder" is any condition that may benefit from treatment using the antibody-like binding proteins of the invention. The terms "disorder" and "condition" are used interchangeably herein, and the terms encompass chronic and acute disorders or diseases, including those pathological conditions that predispose the patient to the disorder in question.

The terms "treatment" or "treat" as used herein refer to both therapeutic treatment and prophylactic or preventive measures. Subjects in need of treatment include subjects already having a disorder, as well as subjects who are prone to developing a disorder or subjects in whom it is necessary to prevent a disorder.

The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more substances in a formulation suitable for effecting or enhancing delivery of an antibody-like binding protein.

The terms "effective amount" and "therapeutically effective amount" when used in relation to a pharmaceutical composition comprising one or more antibody-like binding proteins refer to an amount or dose sufficient to obtain the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of antibody-like binding protein sufficient to inhibit, for a specified period of time, one or more clinically defined pathological processes associated with the condition being treated. An effective amount may vary depending on the particular antibody-like binding protein used, and also depends on a variety of factors and conditions associated with the patient being treated and the severity of the disorder. For example, if the antibody-like binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient, as well as dose-response curves and toxicity data, may be among the factors to be considered. Those skilled in the art can determine an effective amount or therapeutically effective amount of a given pharmaceutical composition.

One embodiment of the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody-like binding protein.

2. Antibody-like binding proteins

In one embodiment of the invention, the antibody-like binding proteins comprise four polypeptide chains that form four antigen-binding sites, wherein two polypeptide chains have a structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and two polypeptide chains have a structure represented by the formula:

VH2-L3-VH1- _L4 -C _H1 -Fc [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3;

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

In another embodiment of the invention, the antibody-like binding proteins comprise two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has a structure represented by the formula:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I]

and the second polypeptide chain has a structure represented by the formula:

VH2-L3-VH1-L4-CH1 [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

L1, L2, L3 and L4 represent amino acid linkers;

and the first and second polypeptides form a light chain-heavy chain pair in a crosswise orientation.

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

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.

In some antibody-like binding proteins of the invention, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In other antibody-like binding proteins, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 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.

In some antibody-like binding proteins of the invention, L1 is from 1 to 3 amino acid residues in length, L2 is from 1 to 4 amino acid residues in length, L3 is from 2 to 15 amino acid residues in length, and L4 is from 2 to 15 amino acid residues in length. In other antibody-like binding proteins, L1 is from 1 to 2 amino acid residues in length, L2 is from 1 to 2 amino acid residues in length, L3 is from 4 to 12 amino acid residues in length, and L4 is from 2 to 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.

In some antibody-like binding proteins of the invention, L1, L3 or L4 may be zero. However, in antibody-like binding proteins in which L1, L3 or L4 is zero, the corresponding transition linker between the variable region and the constant region or between two variable domains on another chain may not be zero. In some embodiments, L1 is zero and L3 consists of 2 or more amino acid residues, L3 is zero and L1 consists of 1 or more amino acid residues, or L4 is 0 and L2 consists of 3 or more amino acid residues.

In some antibody-like binding proteins of the invention, at least one of the linkers selected from the group consisting of L1, L2, L3 and L4 comprises at least one cysteine residue.

Examples of suitable linkers include a single glycine residue (Gly); a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 25); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 26); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 27); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 28); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 29). Other combinations of amino acid residues may also be used, such as the Gly-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 30) and the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 31). Other suitable linkers include one Ser residue and a Val residue; a dipeptide of 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 amino acids randomly selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine and proline have been shown to be suitable for the antibody-like binding proteins of the invention (see Example 12).

The individual composition and sequence of amino acid residues in the linker may vary depending on the type of secondary structure element that is desired in the linker. For example, glycine, serine, and alanine are best for linkers that have maximum flexibility. A certain combination of glycine, proline, threonine, and serine is applicable when a more rigid and elongated linker is needed. Any amino acid residue can be considered as a linker in combination with other amino acid residues to construct larger peptide linkers if necessary, depending on the desired properties.

In some antibody-like binding proteins of the invention, VL1 comprises the amino acid sequence of SEQ ID NO: 1; VL2 comprises the amino acid sequence of SEQ ID NO: 3; VH1 comprises the amino acid sequence of SEQ ID NO: 2; and VH2 comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments of the invention, the antibody-like binding protein is capable of specifically binding one or more target antigens. In preferred embodiments, the antibody-like binding protein is capable of specifically binding at least one target antigen selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-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, 5, IL17, IL18, ILIA, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (integrin b4), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNF-α, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5/CCXCR1), DNGR-1(CLEC91), and HMGB1. In other embodiments, the antibody-like binding protein is capable of inhibiting the function of one or more target antigens.

In some embodiments, the antibody-like binding protein is bispecific and is capable of binding two different target antigens or epitopes. In a preferred embodiment, the antibody-like binding protein is bispecific and each light chain-heavy chain pair is capable of binding two different target antigens or epitopes. In a more preferred embodiment, the antibody-like binding protein is capable of binding two different target antigens 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 the target antigens IL4 and IL13.

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

In one embodiment of the invention, an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites is obtained by identifying a first variable domain of an antibody that binds a first target antigen and a second variable domain of an antibody that binds a second target antigen, each comprising a VL and a VH; defining either a light chain or a heavy chain as a template chain; defining the VL of the first variable domain of the antibody or the second variable domain of the antibody as VL1; defining VL2, VH1 and VH2 according to formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1-Fc [II],

determining the maximum and minimum lengths of L1, L2, L3, and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that bind a first target antigen and a second target antigen when combined to form an antibody-like binding protein;

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

In another embodiment of the invention, an antibody-like binding protein comprising four polypeptide chains that form four antigen-binding sites is obtained by identifying a first variable domain of an antibody that binds a first target antigen and a second variable domain of an antibody that binds a second target antigen, each comprising a VL and a VH; defining either a light chain or a heavy chain as a template chain; defining the VL of the first variable domain of the antibody or the second variable domain of the antibody as VL1; defining VL2, VH1 and VH2 according to formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1 [II]

determining the maximum and minimum lengths of L1, L2, L3, and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that bind a first target antigen and a second target antigen when combined to form an antibody-like binding protein;

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 denotes the constant domain of the immunoglobulin heavy chain C _H1 ; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

In other embodiments of the invention, an antibody-like binding protein is obtained in which the first variable domain of the antibody and the second variable domain of the antibody are the same.

One embodiment of the invention relates to a method for producing an antibody-like binding protein comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1-Fc [II],

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 stands for immunoglobulin heavy chain constant domain CH1;

Fc means the immunoglobulin hinge region and the immunoglobulin heavy chain constant domains CH2, CH3; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptides of formula I and the polypeptides of formula II form a light chain-heavy chain pair in a crosswise orientation.

Another embodiment of the invention relates to a method for producing an antibody-like binding protein, comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by formulas [I] and [II] below:

V _L1 -L ₁ -V _L2 -L ₂ -C _L [I] VH2-L3-VH1-L4-CH1 [II]

Where:

VL1 stands for the first variable domain of the immunoglobulin light chain;

VL2 stands for immunoglobulin light chain variable domain two;

VH1 stands for the first variable domain of the immunoglobulin heavy chain;

VH2 stands for the second variable domain of the immunoglobulin heavy chain;

CL stands for immunoglobulin light chain constant domain;

CH1 denotes the constant domain of the immunoglobulin heavy chain C _H1 ; and

L1, L2, L3 and L4 represent amino acid linkers;

and wherein the polypeptide of formula I and the polypeptide of formula II form a light chain-heavy chain pair in a crosswise orientation.

3. Applications of antibody-like binding proteins

The antibody-like binding proteins of the invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, to detect and quantify one or more target antigens. The antibody-like binding proteins will bind one or more target antigens with an affinity that is appropriate for the assay method used.

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

The antibody-like binding proteins of the invention are also useful for in vivo imaging. An antibody-like binding protein labeled with a detectable label can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host organism can be analyzed. The antibody-like binding protein can be labeled with any residue that can be detected in the animal using either nuclear magnetic resonance, radiology, or other detection methods known in the art.

The invention also relates to a kit containing an antibody-like binding protein and other reagents useful for detecting levels of a target antigen in biological samples. Such reagents may include a detectable label, a blocking serum, positive and negative control samples, and detection reagents.

4. Compositions of antibody-like binding proteins and their administration

Therapeutic or pharmaceutical compositions containing antibody-like binding proteins are within the scope of the invention. Such therapeutic or pharmaceutical compositions may contain a therapeutically effective amount of an antibody-like binding protein or an antibody-like binding protein-drug conjugate in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for its suitability for the route of administration.

Suitable substances for the preparation of the drug are preferably non-toxic to recipients at the doses and concentrations used.

The pharmaceutical composition may contain substances for the preparation of the composition for the purpose of modifying, maintaining or preserving, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. Suitable substances for preparing the composition include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelators (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrins), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavoring agents and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerol, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or humectants (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - preferably sodium or potassium chloride, or mannitol, sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions, incorporated herein by reference for any purpose).

The optimal pharmaceutical composition can be determined by a person skilled in the art, for example, depending on the intended route of administration, the form of delivery and the required dose. Such compositions can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the antibody-like binding protein.

The main vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection may be water, physiological saline solution or artificial cerebrospinal fluid, optionally with the addition of other substances customary in the case of compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional examples of vehicles. Other examples of pharmaceutical compositions contain a tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the invention, the antibody-like binding protein compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional means for preparing the compositions in the form of a lyophilized residue or an aqueous solution. In addition, the antibody-like binding protein can be prepared as a lyophilisate using suitable excipients such as sucrose.

The pharmaceutical compositions of the invention may be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery via the digestive tract, such as oral delivery. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The components of the preparation are present in concentrations that are acceptable for the site of administration. For example, buffers are used to maintain the pH of the composition at physiological pH or at a slightly lower pH, typically in the range of about pH 5 to about pH 8.

If parenteral administration is contemplated, the therapeutic compositions for use in the present invention may be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired antibody-like binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the antibody-like binding protein is formulated as a sterile isotonic solution with appropriate preservatives. Another method of preparation involves formulating the desired molecule with an agent, for example, in the form of microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes that provide controlled or sustained release of the product, which can then be delivered by injection of a depot agent. Hyaluronic acid can also be used and may have the effect of increasing the duration of circulation. Other suitable methods of administering the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition may be prepared for inhalation. For example, the antibody-like binding protein may be prepared as a dry powder for inhalation. Solutions of antibody-like binding proteins may also be prepared for inhalation using a propellant for aerosol delivery. In another embodiment, the solutions may be nebulized.

It is also contemplated that some preparations may be administered orally. In one embodiment, antibody-like binding proteins that are administered in this manner may be formulated with or without carriers commonly used in the preparation of compositions in solid dosage forms such as tablets and capsules. For example, a capsule may be formulated to release the active portion of the preparation at a specific point in the gastrointestinal tract when bioavailability is maximized and presystemic degradation is minimized. Additional agents may be included to facilitate absorption of the antibody-like binding protein. Diluents, flavors, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

Another pharmaceutical composition may include an effective amount of antibody-like binding proteins in admixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, solutions in unit dosage form may be obtained. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions of the invention will be apparent to those skilled in the art, including preparations containing antibody-like binding proteins in the form of sustained or controlled release preparations. Methods for preparing a variety of other sustained or controlled delivery vehicles, such as liposome carriers, biodegradable microparticles or porous beads, and injectable depots are also known to those skilled in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained release matrices may comprise polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

The pharmaceutical compositions of the invention, which are to be used for in vivo administration, generally must be sterile. This can be accomplished by filtration through sterile filtration membranes. In the case where the composition is lyophilized, sterilization using such a method can be carried out before or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are typically placed in a container having a sterile entry port, for example, a bag for an intravenous solution or a vial having a stopper pierced by a hypodermic needle.

Once the pharmaceutical composition has been prepared, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such preparations may be stored either in a form ready for use or in a form (e.g., lyophilized) requiring reconstitution before administration.

The invention also covers kits for producing unit doses for administration. Each of the kits may comprise a first container containing a dried protein and a second container containing an aqueous preparation. Also included within the scope of the present invention are kits containing single-chamber and multi-chamber pre-fillable syringes (e.g., syringes with liquid and syringes with lyophilisate).

The effective amount of the antibody-like binding protein pharmaceutical composition to be used therapeutically will depend, for example, on the therapeutic context and objectives. One skilled in the art will appreciate that appropriate dosage levels for treatment will therefore vary, in part, depending on the molecule being delivered, the indication for which the antibody-like binding protein is used, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, the treating physician can titrate the dose and modify the route of administration to obtain the optimal therapeutic effect. A typical dose can be in the range of about 0.1 mcg/kg to about 100 mg/kg or more, depending on the factors noted above. In other embodiments, the dose can be in the range of 0.1 mcg/kg to about 100 mg/kg; or 1 mcg/kg to about 100 mg/kg; or 5 mcg/kg, 10 mcg/kg, 15 mcg/kg, 20 mcg/kg, 25 mcg/kg, 30 mcg/kg, 35 mcg/kg, 40 mcg/kg, 45 mcg/kg, 50 mcg/kg, 55 mcg/kg, 60 mcg/kg, 65 mcg/kg, 70 mcg/kg, 75 mcg/kg, up to approximately 100 mg/kg.

The frequency of dosing will depend on the pharmacokinetic parameters of the antibody-like binding protein in the preparation used. Typically, the treating physician will administer the composition until a dose is reached that results in the desired effect. Therefore, the composition may be administered as a single dose, as two or more doses (which may contain the same or different amounts of the desired molecule) over a period of time, or as a continuous infusion using an implantable device or a catheter. Further refinement of the appropriate dose is routinely performed by those skilled in the art, and such refinement is within the scope of their usual tasks. Appropriate doses can be established using appropriate dose-response study data.

The route of administration of the pharmaceutical composition corresponds to known methods, for example, oral administration; by injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; using sustained release systems or using implantable devices. If necessary, the compositions can be administered by bolus injection or continuously by infusion or using an implantable device.

The composition can also be administered locally by implanting a membrane, sponge or other suitable material on which the desired molecule has been absorbed or in which it has been encapsulated. In the case of using an implantable device, such a device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule can be carried out by diffusion, a timed bolus or continuous administration.

5. Examples

The examples that follow are illustrative examples of specific embodiments of the invention and its various applications. The examples are provided for illustrative purposes only and should in no way be interpreted as limiting the scope of the invention.

Example 1. Design and construction of bispecific antibody-like binding proteins with dual variable regions in a criss-cross orientation

A dual variable region in a criss-cross orientation in the Fv form is described in U.S. Patent No. 5,989,830 and has been termed the cross-cross dual head (CODH) configuration. Using molecular modeling, the cross-cross dual head (CODH) design was predicted to result in a complex in which both binding sites face each other in the opposite direction without the constraints expected in the dual Fv configuration. The Fv form of CODH was examined to determine whether it could be converted into complete antibody-like molecules by adding a CL domain to the light chain and an Fc region to the heavy chain. A similar conversion was successful in the case of the corresponding dual variable domains (DVD-Ig) and TBTI, which are described in U.S. Patent No. 7,612,181 and International Publication No. WO 2009/052081. The arrangement of the variable regions in the CODH form is shown in the structures below, where the orientation of the peptide chains is indicated from the amino terminus to the carboxyl terminus:

a) light chain: NH2-VL1-linker-VL2-COOH

(b) heavy chain: NH2-VH2-linker-VH1-COOH

The arrangement of the variable regions from amino terminus to carboxyl terminus in (a) and (b) can be distinguished from the arrangement in the case of the dual Fv configuration shown in (c) and (d) below:

(c) light chain: NH2-VL1-linker-VL2-COOH

(d) heavy chain: NH2-VH1-linker-VH2-COOH

The main difference to note is the different arrangement of the corresponding variable regions of the light chain and heavy chain (VH1/VL1 and VH2/VL2) with respect to each other in the two dual variable region configurations. Both corresponding VL1 and VH1 domains were at the N-terminus of the light and heavy chains in the dual variable region configuration. In contrast, in the cross-cross configuration, one half of the antibody variable region pair was spatially separated in the protein chain in the cross-cross configuration. In the cross-cross configuration, the V _L 1 domain may be at the N-terminus of the light chain of the protein, but the paired VH1 domain is at the C-terminus of the heavy chain in the cross-cross configuration. The spatial relationship between VL1 and VH1 found in the dual variable region configuration represents the arrangement found in natural antibodies.

One potential drawback of the dual Fv configuration is that the LL linker separating the two variable regions protrudes into the antigen binding region of the Fv2 domain (see Figure 1). Such protrusion may interfere with antigen binding and result in impaired accessibility of antigen 2 to Fv2. Such impaired accessibility or hindrance may prevent antigen binding. Furthermore, such hindrance may be more pronounced the larger the size of antigen 2. Indeed, US Patent No. 7,612,181 documents that the binding affinity and neutralizing ability of a DVD-Ig molecule depends on whether the antigen specificity is present at the N-terminus or the C-terminus. See US Patent No. 7,612,181, Example 2.

Thus, in order to create more stable antibody-like binding proteins that are not subject to loss of affinity for the antigen compared to the parent antibody, molecules with dual variable regions in a crosswise orientation, having a CL domain in the light chain and an Fc region in the heavy chain, have been designed and constructed. The polypeptides that form such antibody-like proteins have the structures shown below, in which the following orientation of the polypeptide chains occurs from the amino terminus to the carboxyl terminus:

(e) light chain: NH2-VL1-linker-VL2-CL-COOH

(f) heavy chain: NH2-VH2-linker-VH1-CH1-Fc-COOH

To evaluate whether the bispecific antibody-like protein of this design can bind two different antigens, two previously generated and humanized variable regions from antibodies specific for IL4 (original humanized anti-IL4) and IL13 (original humanized anti-IL13) were used to construct the bispecific antibody-like molecules shown in Table 1. The sequencing of the mouse antibodies and the humanization method are described in International Publication No. WO 2009/052081 (TBTI). Briefly, the amino acid sequences of the variable regions of the heavy and light chains of mouse anti-IL13 antibody clone B-B13 and mouse anti-IL4 antibody clone 8D4-8 were determined using amino acid sequencing. The mouse sequences were humanized and then reverse translated into nucleotide sequences as described in Example 5 of International Publication No. WO 2009/052081, which is incorporated herein by reference in its entirety. The VH and VL sequences of the parent humanized mouse anti-IL4 antibodies and the VH and VL of the parent humanized mouse anti-IL13 antibodies were combined and arranged as described in Table 1. The shortened code names in column one of Table 1 were created to simplify the discussion of such antibody-like binding proteins. The antibody-like binding proteins differ in the size of the linker inserted between the two variable regions, as shown in Table 1. DNA molecules encoding the polypeptides shown in Table 1 were created based on the reverse translated parent mouse anti-IL4 antibodies and mouse anti-IL13 antibodies. The CH1, CL, and Fc domains were obtained from IGHG1 (GenBank accession number 569F4) and IGKC (GenBank accession number Q502W4).

Table 1
Immunoglobulins with two heads in a criss-cross orientation Short code for protein Description of protein SEQ ID NO: Light chain of parent against IL-4 vs IL4 V _L 1 Heavy chain of the original vs. IL-4 against IL4 V _H 2 Light chain of parent against IL-13 against IL13 V _L 3 Heavy chain of the original vs. IL-13 against IL13 V _H 4 Heavy chain codes IL13(G4S)IL4CHl-Fc against IL13 V _H -(G ₄ S)-against IL4 V _H -C _H1 -Fc 5 IL13(G4S2)IL4CHl-Fc against IL13 V _H -(G ₄ S) ₂ -against IL4 V _H -C _H1 -Fc 6 IL4(G4S)IL13CHl-Fc against IL4 V _H -(G ₄ S)-against IL13 V _H -C _H1 -Fc 7 IL4(G4S2)IL13CHl-Fc against IL4 V _H -(G ₄ S) ₂ -against IL13 V _H -C _H1 -Fc 8 Light chain codes IL13(G4S)IL4CL against IL13 V _L -(G ₄ S)-against IL4 V _L -CL 9 IL13(G4S2)IL4CL against IL13 V _L -(G ₄ S) ₂ -against IL4 V _L -CL 10 IL4(G4S)IL13CL against IL4 V _L -(G ₄ S)-against IL13 V _L -CL 11 IL4(G4S2)IL13CL against IL4 V _L -(G ₄ S) ₂ -against IL13 V _L -CL 12

The protein combinations shown in Table 2 were expressed using transient transfection and purified by protein A chromatography. In each case, size exclusion chromatography revealed aggregation of less than 12%, with a best case aggregation of less than 7%; but no immunoglobulin with the two heads in a crosswise orientation was detected with any ability to bind either IL4 or IL13. However, no functional antibody-like binding could be detected, and no reason for the lack of such activity could be elucidated. It has been previously suggested that this arrangement may provide superior stability compared to the dual variable domain antibodies described in U.S. Patent No. 7,612,181 and International Publication No. WO 2009/052081.

Table 2
Binding of CODH-Ig to IL4 and IL13 Protein Combination Aggregation IL-4 binding IL-13 binding against IL13 V _H -(G ₄ S)-against IL4 V _H -C _H1 -Fc
against IL4 V _L -(G ₄ S)-against IL13 V _L -C _L 5.4% ND* N.D. against IL13 V _H -(G ₄ S)-against IL4 V _H -C _H1 -Fc
against IL4 V _L -(G ₄ S) ₂ -against IL13 V _L -CL 6.3% N.D. N.D. against IL13 V _H -(G ₄ S) ₂ -against IL4 V _H -C _H1 -Fc
against IL4 V _L -(G ₄ S)-against IL13 V _L -CL 11.5% N.D. N.D. against IL13 V _H -(G ₄ S) ₂ -against IL4 V _H -C _H1 -Fc
against IL4 V _L -(G ₄ S) ₂ -against IL13 V _L -CL 10.1% N.D. N.D. against IL4 V _H -(G ₄ S)-against IL13 V _H -C _H1 -Fc
against IL13 V _L -(G ₄ S)-against IL4 V _L -CL 2.7% N.D. N.D. against IL4 V _H -(G ₄ S)-against IL13 V _H -C _H1 -Fc
against IL13 V _L -(G ₄ S) ₂ -against IL4 V _L -CL 3.6% N.D. N.D. against IL4 V _H -(G ₄ S) ₂ -against IL13 V _H -C _H1 -Fc
against IL13 V _L -(G ₄ S)-against IL4 V _L -CL 2.9% N.D. N.D. against IL4 V _H -(G ₄ S) ₂ -against IL13 V _H -C _H1 -Fc
against IL13 V _L -(G ₄ S) ₂ -against IL4 V _L -CL 10.8% N.D. N.D. *ND means not determined

Example 2. Construction of CODV-Ig proteins using molecular modeling

In order to obtain fully functional antibody-like proteins using the two-headed criss-cross configuration that can incorporate the Fc and CL1 domains, a molecular modeling protocol was developed to incorporate and evaluate different linkers between the constant and variable domains and between the two variable domains on both the light and heavy chains. The question was whether the addition of unique linkers between the constant/variable domain interfaces and between the two variable domain interfaces (variable/variable) on the heavy and light chains could ensure proper protein folding and the production of functional antibody-like molecules in the two variable regions criss-cross configuration (see Figure 2). In other words, a total of four independent and unique linkers were evaluated (see Figure 2). This molecular modeling protocol was based on protein-protein docking in homology models and experimental models of the _IL4 Fv and _IL13 Fv regions, respectively, in combination with suitable linkers between the _IL4 Fv and _IL13 Fv regions and between Fv and the constant or Fc regions.

The independent linkers are given individual names: L1 refers to the linker between the N-terminal VL and the C-terminal VL in the light chain; L2 refers to the linker between the C-terminal VL and CL in the light chain; L3 refers to the linker between the N-terminal VH and the C-terminal VH in the heavy chain; L4 refers to the linker between the C-terminal VH and CH1 (and Fc) in the heavy chain. It should be noted that the VH and VL designations refer only to the position of the domains in a particular protein chain in the final form. For example, VH1 and VH2 can be derived from the VL1 and VL2 domains of the parent antibodies and placed at the VH1 and VH2 positions in CODV-Ig. Similarly, VL1 and VL2 can be derived from the VH1 and VH2 domains of the parent antibodies and placed at the VH1 and VH2 positions in CODV-Ig. Thus, the VH and VL designations refer to this position, not to the original position in the parent antibody.

In more detail, a homology model of FVIL4 was constructed based on the data entered in the PDB, 1YLD (light chain) and 1IQW (heavy chain). The FvIL4 dimer was reconstructed on a self-generated crystal structure of the IL13/anti-IL13 FabIL13 complex and optimized. To estimate the volume required for IL4 to bind to FvIL4, the IL4 crystal structure (1RCB.pdb) was docked to the homology model for FvIL4. Twenty-two putative models of the complex were then generated that merited further consideration.

In parallel, the FvIL4 homology model was docked with FvIL13 extracted from an in-house crystal structure of the IL13/FabIL13 complex. One excellent solution was found that allowed the construction of relatively short linkers without introducing steric interference into antigen binding and the placement of the constant domains, as is the case for dual variable region immunoglobulins (see Figure 3). In this arrangement, FvIL4 (VL1) was placed at the N-terminus of the light chain, followed by FvIL13 (VL2) and Fc (CL1) at the C-terminus of the light chain. In the heavy chain, FvIL13 (VH2) was placed at the N-terminus, followed by FvIL4 (VH1) and the constant regions (CH1-CH2-CH3).

As shown in Table 3, the light chain models suggest that the L1 linker between the VL1 and VL2 domains and the L2 linker between the VL2 and CL1 domains should be one to three and zero to two glycine residues in length, respectively. The heavy chain models suggest that the L3 linker between the VH2 and VH1 domains and the L4 linker between the VH1 and CH1 domains should be two to six and four to seven glycine residues in length, respectively (see Table 3 and Figure 2). In this example, glycine was used as the prototypical amino acid for the linkers, but other amino acid residues can also serve as linkers. The structural stability of the proposed models was confirmed using linker conformation optimization, minimization, and molecular dynamics calculations. Systemic combination of four light chain and six heavy chain constructs resulted in 24 possible dual variable region in a cross-like orientation bispecific anti-IL4 and anti-IL13 antibody-like binding proteins (see Table 4).

Table 3
Suggested Linker Lengths Linker between Maximum linker insertion Minimal linker insertion Linker name V _L1 -V _L2 Gly ₃ Gly Li V _L2 -C _L Gly ₂ No L ₂ V _H2 -V _H1 Gly ₆ Gly ₂ L ₃ V _H1 -C _H1 Gly ₇ Gly ₄ L ₄

Table 4
CODV-Ig for expression Code* Heavy chains (N-terminus to C-terminus) SEQ ID NO: HC1 IL13 V _H -(Gly6)-IL4 V _H -(Gly7)-C _H1 -Fc 13 HC2 IL13 V _H -(Gly6)-IL4 V _H -(Gly4-C _H1 -Fc 14 HC3 IL13 V _H -(Gly2)-IL4 V _H -(GlyV)-C _H1 -Fc 15 HC4 IL13 V _H -(Gly2)-IL4 V _H -(Gly4)-C _H1 -Fc 16 HC5 IL13 V _H -(Gly4)-IL4 V _H -(Gly7)-C _H1 -Fc 17 HC6 IL13 V _H -(Gly4)-IL4 V _H -(Gly4)-C _H1 -Fc 18 Code* Light chains (N-terminus to C-terminus) LCI IL4 V _L -(Gly3)-IL13 V _L -C _L1 19 LC2 IL4V _L -(Gly)-IL13V _L -C _L1 20 LC3 IL4 V _L -(Gly3)-IL13 V _L -(Gly2)-C _L1 21 LC4 IL4 V _L -(Gly)-IL13 V _L -(Gly2)-C _L1 22 C _L1 Human light chain constant domain C _L1 23 C _H1 -FC Human C _H1 heavy chain constant domain and Fc region 24 Gly4 Peptide linker with 4 glycines (GGGG) 25 Gly5 Peptide linker with 5 glycines (GGGGG) 26 Gly6 Peptide linker with 6 glycines (GGGGGG) 27 Gly7 Peptide linker with 7 glycines (GGGGGGG) 28 Gly8 Peptide linker with 8 glycines (GGGGGGGG) 29 *The abbreviated code is designed to denote associated structures. Codes beginning with HC indicate the adjacent heavy chain, and codes beginning with LC indicate the adjacent light chains.

In Table 4, the prefix "anti" is not included, but it is meant that IL13 refers to anti-IL13, and IL4 refers to anti-IL4.

Example 3. Creation of plasmids for CODV-Ig expression

Nucleic acid molecules encoding the heavy and light chain variable regions for the six heavy chains and four light chains described in Table 4 were generated by gene synthesis at Geneart (Regensburg, Germany). The light chain variable domains were fused to the light chain constant domains (IGKC, GenBank accession no. Q502W4) using ApaLI and BsiWI restriction endonuclease digestion and then ligated into the ApaLI/BsiWI sites of the episomal expression vector pFF, an analog of the pTT vector described by Durocher et al. (2002, Nuc. Acids Res. 30(2):E9), creating a mammalian expression plasmid for light chain expression.

The heavy chain variable domains were fused to the “Ted” variant of the human heavy chain constant region (IGHG1, GenBank accession no. 569F4), or alternatively to the 6x His-tagged CH1 domain from the human IGHG1 constant region, to create a bispecific Fab. The VH domain was then digested with restriction endonucleases ApaLI and ApaI and then fused to IGHG1 or the His-tagged CH1 domain, respectively, using ligation into the ApaLI/ApaI sites of the pFF episomal expression vector, creating mammalian expression plasmids for expression of the heavy chains (IgG1 or Fab, respectively).

Example 4. Expression of CODV-Ig

Expression plasmids encoding the heavy and light chains of the respective constructs were propagated in E. coli DH5a cells. Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega kit.

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

CODV-IgG1 constructs were purified by affinity chromatography on Protein A columns (HiTrap Protein A HP columns, GE Life Sciences). After column elution with 100 mM acetate buffer and 100 mM NaCl, pH 3.5, CODV-IgG1 constructs were desalted using HiPrep 26/10 desalting columns, formulated in PBS at a concentration of 1 mg/mL, and filtered using a 0.22 μm membrane.

The bispecific CODV-Fab constructs were purified using IMAC on HiTrap IMAC HP columns (GE Life Sciences). After elution from the column with a linear gradient (elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50-500 mM imidazole, pH 7.4), protein-containing fractions were pooled and desalted using HiPrep 26/10 desalting columns, prepared in PBS at a concentration of 1 mg/mL and filtered using a 0.22 μm membrane.

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.

The Nunc F96-MaxiSorp-Immuno plate was coated with goat anti-human IgG (Fc-specific) antibody [NatuTec A80-104A]. The antibody was diluted to 10 μg/mL in carbonate coating buffer (50 mM sodium carbonate, pH 9.6) and 50 μL was dispensed per well. The plate was sealed with adhesive tape and stored overnight at 4°C. The plate was washed three times with wash buffer (PBS, pH 7.4, and 0.1% Tween 20). 150 μL of blocking solution (1% BSA/PBS) was dispensed into each well to coat the plate. After incubation for 1 h at room temperature, the plate was washed three times with wash buffer. 100 μl of sample or standards (ranging from 1500 ng/ml to 120 ng/ml) were added and allowed to stand for 1 h at room temperature. The plate was washed three times with wash buffer. 100 μl of goat anti-human IgG-FC-HRP conjugate [NatuTec A80-104P-60] diluted 1:10,000 was added using incubation solution (0.1% BSA, PBS, pH 7.4, and 0.05% Tween 20). After incubation for 1 h at room temperature, the plate was washed three times with wash buffer. 100 µl ABTS substrate (10 mg ABTS tablet (Pierce 34026) in 0.1 M Na2HPO4, 0.05 M citric acid, pH 5.0) was added. 10 µl 30% H2O2/10 ml substrate buffer was added before use, dispensed into each well, and allowed to develop color. After color development (approximately 10-15 minutes), 50 µl 1% SDS solution was added to stop the reaction. The plate was read at A405.

Example 5. Characteristics of CODV-Ig variants

To determine whether the heavy and light chains of the CODV-Ig antibody-like protein could pair and fold correctly, the level of aggregation was measured using analytical size exclusion chromatography (SEC). Analytical SEC was performed on the collected pairs using an AKTA 10 analyzer (GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm × 30 cm) and a TSKgel SWXL guard column (Tosoh Bioscience). The analysis was performed at a flow rate of 1 ml/min using 250 mM NaCl, 100 mM Na-phosphate, pH 6.7, and detection at 280 nm. 30 μl of protein sample (0.5-1 mg/ml) was loaded onto the column. For molecular size estimation, the column was calibrated using a standard gel filtration mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using the UNICORN v5.11 computer program.

Table 5 shows the results for the first set of 24 different CODV-Ig molecules generated using the anti-IL4 and anti-IL13 variable region combinations described in Table 4. The codes assigned in Table 4 represent the adjacent structures shown in Table 4. For the light chain and heavy chain pairs, aggregation levels were measured using SEC when protein was produced. The results are shown in Table 5, where LC4 (L1=1; L2=2) was the most successful in pairing with all six heavy chains. LC4 corresponds to the IL4 VL-(Gly)-IL13 VL-(Gly2)-CL1 structure, which has a linker L1 of 1, in which one amino acid residue separated the two dual variable region light chain VL domains. In addition, LC4 had an L2 of 2, which contained a Gly-Gly dipeptide linker between the central VL and the C-terminal CH1.

Table 5
Aggregation levels among heavy and light chain pairs HC1 HC2 HC3 HC4 HC5 HC6 LCI >50% >50% ND* N.D. N.D. N.D. LC2 N.D. N.D. N.D. >50% N.D. N.D. LC3 N.D. N.D. N.D. N.D. N.D. N.D. LC4 7.2% 6.8% 6.8% 7.1% 6.3% 5.9% *ND means no protein was obtained

When CODV-Ig molecules were prepared, a BIACORE experiment was performed using a single concentration corresponding to intermediate concentrations of IL13 and IL4 to confirm binding to target antigens. CODV-Ig antibody-like molecules corresponding to the LC4:HC4 and LC4:HC6 combinations described in Table 4 were selected for evaluation of the full kinetic analysis using surface plasmon resonance.

As shown in Table 5, most CODV-Ig molecules could not be obtained at all or only as aggregates (up to 90%). The heavy chain/light chain combinations that yielded acceptable aggregation levels (5-10%) after a single chromatography step were those with the IL4 V _L -(Gly)-IL13 V _L -(Gly2)-CL1 light chain. This light chain was the most carefully designed chain in these CODV-Ig variants and served as a platform for accepting different heavy chains with different linker compositions.

1. Kinetic analysis

Two pairs of heavy and light chains were selected for full kinetic analysis. Recombinant human IL13 and IL4 were purchased from Chemicon (USA). Kinetic characterization of purified antibodies was performed using surface plasmon resonance technique on BIACORE 3000 (GE Healthcare). A capture assay using a species-specific antibody (e.g. human Fc-specific MAB 1302, Chemicon) was used to capture and target the antibodies of interest. The capture antibody was immobilized via primary amine groups (11,000 arbitrary units) on a CM5 assay chip (GE Life Sciences) using standard methods. The antibody of interest was captured using a flow rate of 10 μl/min, with the adjusted value, expressed in arbitrary units, that would result in maximal analyte binding being 30 arbitrary units. The binding kinetics of human IL4 and IL13 were measured over the concentration range from 0 to 25 nM in HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) at a flow rate of 30 μl/min. The chip surfaces were regenerated using 10 mM glycine, pH 2.5. Kinetic parameters were analyzed and calculated using the BIAevaluation v4.1 software package using the flow cell without capture antibody as a reference.

Table 6 below compares the kinetics of the parental BB13 (anti-IL13) and 8D4 (anti-IL4) antibodies (expressed as IgG) with the corresponding domains in the CODV-Ig form (Table 4, codes LC4:HC4 and LC4:HC6). As shown in Table 6, no decrease in binding properties of the CODV-Ig constructs to the corresponding antigens was observed compared to the parental anti-IL13 and anti-IL4 antibodies. The decrease in the rate of association observed with the DVD-Ig/TBTI form, which used the same Fv sequences, did not occur with the CODV-Ig configuration. The opposing binding sites should allow for binding of large antigens or bridging between different cells using a bispecific antibody-like configuration and may also be suitable for a broader selection of parental antibodies. A further advantage of CODV-Ig was that the linker residues did not protrude into the antigen-binding site and did not reduce antigen availability.

Table 6
Kinetic analysis of LC4:HC4 and LC4:HC6 Code Association speed [1/M sec] Dissociation rate [1/sec] KD[M] Original vs IL-4-mAb 2.49E+07 1,95E-04 7.83E-12 Original vs IL-4-mAb 1.59E+06 1,30E-04 8,18E-11 LC4:HC4 CODV-Ig with IL4 3.16E+07 2,89E-04 9,14E-12 LC4:HC4 CODV-IG with IL13 1.20E+06 1,12E-04 9.34E-11 LC4:HC6 CODV-Ig with IL4 2.97E+07 3.30E-04 1,11E-11 LC4:HC6 CODV-IG with IL 13 1.39E+06 1,63E-04 1,18E-10

2. Co-injection of IL4 and IL13 to demonstrate additive antigen binding by the CODV-Ig molecule

To investigate the additive binding of both antigens, a wizard-driven co-injection method was used in which one antigen was injected immediately after the other antigen after a lag period (IL4 then IL13 and vice versa). The resulting binding level can be compared with that achieved using a 1:1 mixture of both antigens at the same concentration. To demonstrate the additive binding of both antigens IL4 and IL13 by CODV-Ig molecules, a BIACORE experiment with a combination of CODV-Ig [HC4:LC4] was performed using co-injection of both antigens in three different assay runs (see Figure 4). Co-injection was performed using 3.125 nM IL4/25 nM IL13 (and vice versa) and using a 1:1 mixture of 3.125 nM IL4 and 25 nM IL13. Co-injection of HBS-EP buffer was performed as a reference. At the 800 s time point, identical binding levels of 63 arbitrary units were achieved after injection of the antigen mixture or co-injection of the antigens, regardless of the co-injection sequence. 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. This observation was reproducible when the injection sequence of the antigens was reversed. These data demonstrate additive binding and no inhibition of binding of both antigens by the CODV-Ig molecules. Therefore, the CODV-Ig construct is capable of binding both antigens simultaneously (i.e., exhibits bispecificity) with saturation of all binding sites (i.e., exhibits tetravalency).

Example 6. Acceptable linker lengths for CODV-Ig

The feasibility of linkers of different lengths was assessed by constructing CODV-Ig molecules having different combinations of linker lengths of L1, L2 in the light chain and L3 and L4 in the heavy chain. CODV-Ig constructs were created with heavy chain L3 and L4 linkers varying in length from 1 to 8 residues for L3 and either 0 or 1 residue for L4. The heavy chain contained an N-terminal anti-IL4 binding domain and a C-terminal anti-IL13 binding domain followed by CH1-Fc. Light chain L1 and L2 linker lengths varied from 3 to 12 residues for L1 and from 3 to 14 residues for L2. The light chain contained an N-terminal anti-IL13 binding domain and a C-terminal anti-IL4 binding domain followed by CL1.

1. Characteristics of CODV-Ig variants

Determination of the level of aggregation using analytical size exclusion chromatography (SEC). Analytical SEC was performed using an AKTA 10 analyzer (GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm × 30 cm) and a TSKgel SWXL guard column (Tosoh Bioscience). The analysis was carried out at a flow rate of 1 ml/min using 250 mM NaCl, 100 mM Na-phosphate, pH 6.7, and recording at 280 nm. 30 μl of protein sample (0.5-1 mg/ml) were loaded onto the column. For molecular size estimation, the column was calibrated using a standard gel filtration mixture (MWGF-1000, SIGMA Aldrich). Data were evaluated using the UNICORN v5.11 software.

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

Binding kinetics analysis using Biacore was performed as follows. Surface plasmon resonance technology on a Biacore 3000 (GE Healthcare) was used for detailed kinetic characterization of purified antibodies. A capture assay using a species-specific antibody (e.g., human Fc-specific MAB 1302, Chemicon) was used to capture and orient the antibodies of interest. To determine the binding kinetics of IL4 and IL13, the corresponding CODV Fabs as described in Example 10, Table 12, were captured using the Anti-Human Fab Capture Kit (GE Healthcare). The capture antibody was immobilized via primary amine groups (11,000 arbitrary units) on a CM5 Assay Chip (GE Life Sciences) using standard methods. The antibody of interest was captured using a flow rate of 10 μl/min, with the adjusted value, expressed in arbitrary units, that would result in maximal analyte binding being 30 arbitrary units. The binding kinetics of recombinant human IL4 and IL13 were measured over the concentration range from 0 to 25 nM in HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) at a flow rate of 30 μl/min. The chip surfaces were regenerated using 10 mM glycine, pH 2.5. Kinetic parameters were analyzed and calculated using the BIAevaluation v4.1 software package using the flow cell without capture antibody as a reference.

The binding affinities of CODV-Ig, CODV-Fab and TBTI for EGFR and HER2 were measured using the Proteon XPR36 protein interaction array system (Biorad). Antigens were immobilized by amine-reactive binding on GLC sensor chips (Biorad). A dilution series of bispecific antibody variants was run in PBSET buffer (Biorad) using one-shot kinetics with dual standards. Data were analyzed using Proteon Manager v3.0 (Biorad) and either the 1:1 Langmuir mass transfer model or the bivalent analyte model.

Table 7 summarizes the results regarding the yield, aggregation (measured using size exclusion chromatography), and binding affinity of CODV-Ig having combinations of linker sizes. The results showed that CODV-Ig molecules in which L2 was zero generally could not be obtained or, if protein was obtained, they gave high levels of aggregation (see Lot IDs 101, 102, 106-111, and 132-137 in Table 7). Thus, in contrast to the molecular modeling prediction described in Example 2, which suggested that an L2 of zero was within the acceptable range, these results indicate that the VL2-CL (or L2) transition requires a linker of at least one residue in length (see Table 7).

Table 7

Optimization of linker sizes for CODV-Ig

* The location on the heavy chain should be IL13V _H -L ₃ -IL4VH-L ₄ -C _H1 -FC

¹ np means the construct cannot be obtained.

In addition, the CODV-Ig linker lengths described above were found to be more sensitive to increases of 1 amino acid residue than increases of 2 amino acid residues. For example, although lots #103 and 104 differ by 1 amino acid residue in L2, lot #103 showed 6-fold greater aggregation and lot #104 showed less aggregation and twice the yield. In contrast, lots #104 and 105, which differ by two residues in L2, showed similar profiles with respect to yield, aggregation, and binding.

Example 7. Heavy chain as a template chain for CODV-Ig

In Examples 1-5, the optimal sizes of the short linkers in the light chain indicated that the light chain served as a template in maintaining a linear arrangement, and that longer linkers in the heavy chain were required to allow the heavy chain to fold correctly into a crisscross configuration to fit the template light chain (see Figure 5, panel A). It was then assessed whether the short linkers in the heavy chain, specifically placed to maintain a linear arrangement in the heavy chain, made that heavy chain the "template" chain and whether the pattern could be repeated on its own, and whether larger linkers were required to allow the non-template chain to fold correctly and accommodate the new template heavy chain (see Figure 5, panel B).

Figure 6 illustrates such design principles for CODV-Ig based on either the light chain or the heavy chain being used as a template. To evaluate the general gist of the concept, CODV-Ig constructs were generated having heavy chain linkers L3 and L4 that varied in length from 1 to 8 residues for L3 and either 0 or 1 residue for L4. The heavy chain contained an N-terminal anti-IL4 binding domain and a C-terminal anti-IL13 binding domain followed by CH1-Fc. The light chain linkers L1 and L2 varied in length from 3 to 12 residues for L1 and from 3 to 14 residues for L2. The light chain contained an N-terminal anti-IL13 binding domain and a C-terminal anti-IL4 binding domain followed by CL1.

Table 8 summarizes the results regarding the yield, aggregation (measured using size exclusion chromatography) and binding affinity of CODV-Ig having combinations of linkers of different sizes and in which the heavy chain was maintained in a linear arrangement as the template chain and the light chain was allowed to fold in a criss-cross configuration. The results showed that CODV-Ig molecules in which L4 was zero generally could not be obtained or, if protein was obtained, they gave high levels of aggregation (similar to molecules in which L2 was zero) (see Lot IDs 207-209, 211-212, 219-224, 231-236, 243-252 and 263-266 in Table 8). One exception was lot #210, in which L1 was 7, L2 was 5, L3 was 2, and L4 was zero. This arrangement yielded sufficient protein and acceptable levels of aggregation and binding, indicating that in some cases a particular combination of linker sizes may be found to compensate for a zero-length linker at position L4.

Table 8

Optimization of linker sizes in the case of heavy chain as a template

* The arrangement on the light chain should be IL13V _L -L ₁ -IL4V _L -L ₂ -C _L1

The results shown in Tables 7 and 8 clearly show that linkers between the variable and constant domains are required to ensure optimal folding. Only in rare arrangements is a linker of zero acceptable (see lots #103-105, in which L1 (LC) is zero, and lot #210, in which L4 is zero). However, in each case the corresponding transition linker between the variable region and the constant region in the other chain cannot be zero.

The above results show that the combinations of L1=7, L2=5, L3=1 and L4=2 were a good starting point for the optimization of the new CODV-Ig in which the heavy chain is the template. The ranges shown in Table 9 were shown to be acceptable ranges for the successful construction of the new CODV-Ig from the two parent antibodies.

Table 9
Linker size ranges for either LC or HC as matrix Linker LC as a matrix HC as a matrix Maximum range Minimum range Maximum range Minimum range L ₁ 1-3 1-2 3-12 5-10 L ₂ 1-4 1-2 3-14 5-8 L ₃ 2-15 4-12 1-8 1-5 L ₄ 2-15 2-12 1-3 1-2

Example 8. Universal applicability of the CODV-Ig form

To evaluate the suitability of the CODV-Ig form for the design of novel antibody-like binding proteins, variable regions from numerous existing human and humanized antibodies specific for insulin-like growth factor 1 receptor (IGF1R(1)), insulin-like growth factor 1 receptor 2 (IGF1R(2)), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), tumor necrosis factor-alpha (TNFα), interleukin 12 and 23 (IL-12/23), and interleukin 1 beta (IL-1β) were incorporated into the CODV-Ig form (see Table 10).

Table 10
Descriptive codes for heavy and light chains used in bispecific CODV-Ig Code* Heavy chains (N-terminus to C-terminus) SEQ ID NO: HC10 IGFlR(l) V _H -(Gly)-HER2 V _H -(Gly2)-C _H1 -Fc 32 HC11 HER2 V _H -(Gly)-IGFlR(l) V _H -(Gly2)-C _H1 -Fc 33 NS 12 IGF1R(2) V _H -(Gly)-EGFR V _H -(Gly2)-C _H1 -Fc 34 HC13 EGFR V _H -(Gly)-IGF1R(2) V _H -(Gly2)-C _H1 -Fc 35 NS 14 TNFa V _H -(Gly)-IL 12/23 V _H -(Gly2)-C _H1 -Fc 36 NS15 IL 12/23 V _H -(Gly)-TNFa V _H -(Gly2-C _H1 -Fc 37 NS16 TNFα V _H -(Gly)-ILlβ V _H -(Gly2)-C _H1 -Fc 38 NS17 ILlβ V _H -(Gly)-TNFα V _H -(Gly2)-C _H1 -Fc 39 Code* Light chains (N-terminus to C-terminus) LC10 HER2 V _L -(Gly7)-IGFlR(l) V _L -(Gly5)-c _l1 40 LC11 IGFlR(l) V _L -(Gly7)-HER2 V _L -(Gly5)-c _l1 41 LC12 EGFR V _L -(Gly7)-IGFlR(2) V _L -(Gly5)-c _l1 42 LC13 IGF1R(2) V _L -(Gly7)-EGFR V _L -(Gly5)-c _l1 43 LC14 IL12/23 V _L -(Gly7)-TNFα V _L -(Gly5)-C c _l1 44 LC15 TNFα V _L -(Gly7)-IL12/23 V _L -(Gly5)-c _l1 45 LC16 ILlβ V _L -(Gly7)-TNFα V _L -(Gly5)-c _l1 46 LC17 TNFα V _L -(Gly7)-ILlβ V _L -(Gly5)-c _l1 47 c _l1 Human light chain constant domain C _L1 23 C _H1 -Fc Human C _H1 heavy chain constant domain and Fc region 24 Gly4 Peptide linker with 4 glycines (GGGG) 25 Gly5 Peptide linker with 5 glycines (GGGGG) 26 Gly6 Peptide linker with 6 glycines (GGGGGG) 27 Gly7 Peptide linker with 7 glycines (GGGGGGG); 28 Gly8 Peptide linker with 8 glycines (GGGGGGGG) 29 *The abbreviated code is designed to denote associated structures. Codes beginning with HC indicate the adjacent heavy chain, and codes beginning with LC indicate the adjacent light chains.

Antibody variable regions from known human antibodies and humanized antibodies were used to test the universal applicability of the CODV-Ig form in the construction of bispecific antibody-like binding proteins. In addition, the possibility of positional effects was investigated with respect to the placement of some antibody variable regions at either the N-terminus or the C-terminus of the heavy chain or light chain. Based on the design of CODV-Ig molecules having linker compositions L1=7, L2=5, L3=1, and L4=2, different antibody sequences were introduced into the CODV-Ig form.

The activities of bispecific antibodies or derivatives against IL1β and TNFα were determined using commercially available HEK-Blue TNFα/IL1β reporter cells (InvivoGen). To determine the activities of antibodies against TNFα and IL1β, cytokines were preincubated for 1 h with different concentrations of antibodies and added to 50,000 HEK Blue TNFα/IL1β cells. Cytokine-mediated SEAP induction was measured after 24 h in the culture supernatant using the QUANTI-Blue assay (InvivoGen).

As shown in Table 11, all constructs showed good to excellent protein yields and acceptable levels of aggregation (see in particular lots #301 and 302 in Table 11). The measured affinities for each antibody variable domain were within the published or expected affinity values. No positional effects were observed in cases where affinity was assessed. Thus, as shown in the following tables, no positional effects were observed for any of the antibody variable domains used, nor for the use of such domains in any of the antibody chains.

Example 9. Preservation of the affinity of the original antibody in the form of CODV-Ig

Identical anti-IL4 and anti-IL13 antibody sequences were incorporated into either the TBTI/DVD-Ig or CODV-Ig form to directly compare the configurations, linker placement, and affinities of the resulting molecules. As shown in Figure 7, the original affinity of each antibody was maintained in the CODV form. As shown in the upper panel of Figure 7, when the variable regions were incorporated into the TBTI/DVD-Ig form, the decrease in affinity of the IL4 antibody located at the internal Fv2 position was manifested as a decrease in the rate of association of the antibody with the antigen. In contrast, there was no loss of affinity with the CODV-Ig form compared to the original antibodies (see Figure 7, lower panel).

Example 10 Applicability of CODV-Ig to Fab Form

The ability of the CODV-Ig form to yield fragments such as Fab fragments was then assessed. Two different heavy chain variable regions were fused together via linker L3 and extended at the C-terminus with linker L4. This VH complex was then fused to the CH1 domain of IGHG1 (GenBank accession no. Q569F4) bearing at the C-terminus the five amino acid sequence DKTHT (SEQ ID NO: 60) from the hinge region followed by six histidine residues. Different light chain variable regions were fused together in a criss-cross configuration with the corresponding heavy chain via linker L1 and extended at the C-terminus with linker L2 and then fused to the kappa constant chain (IGKC, GenBank accession no. Q502W4).

Fabs were expressed by transient transfection as described previously. Seven days after transfection, cells were removed by centrifugation, 10% v/v 1 M Tris-HCl, pH 8.0 was added, and the supernatant was passed through a 0.22 μm filter to remove particles. Fabs were captured using HisTrap high-performance columns (GE Healthcare) and eluted with an imidazole gradient. Protein-containing fractions were pooled and desalted using PD-10 or Sephadex columns. Concentrated and sterile-filtered (0.22 μm) protein solutions were adjusted to a concentration of 1 mg/ml and stored at 4°C until use.

The immediate observed advantages were that the Fab-like molecules in the CODV orientation did not exhibit a tendency to aggregate and retained the affinities of the parent antibodies (see Table 12). Using protein binding constructs from lots #401–421, antibody-like proteins in which the variable regions of the antibodies were arranged as in CODV-Ig molecules with the heavy chain as a template (401, 402, 406, and 407), CODV Fab-like fragments (402, 408, 413, 418, and 421), four-domain antibody-like molecules in the form of TBTI/DVD-Ig (404, 409, 414, and 419), and CODV-Ig without linkers (405, 410, 415, and 420) were directly compared. As shown in Table 12, the results of this comparison indicated that there was likely a loss of affinity compared to the parent antibodies when the variable region was incorporated into the TBTI or DVD-Ig form. In contrast, the CODV-Ig and CODV-Ig Fab-like forms were better able to retain the parent affinities. The results further confirmed that linkers between the variable regions and between the variable regions and the constant domains were required in the case of CODV-Ig molecules (see Table 12).

Example 11. Replacement of variable domains in CODV-Ig and CODV-Fab

To characterize the CODV form in its mode of T cell recruitment, bispecific CODV-like binding protein Fabs (CODV-Fabs) containing a TCR binding site (CD3epsilon) and a CD19 binding site were generated and compared with a bispecific Fab derived from the TBTI/DVD-Ig form (B-Fab). To investigate the importance of binding site orientation (TCR x CD19 versus CD19 x TCR), both orientations were assessed for each binding protein.

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

The CD3-CD19 form of B-Fab (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 CD19-CD3 CODV-Fab (1109) with an EC50 of 3.2 ng/ml (see Figure 8).

Changing the configuration of the B-Fab molecule (Fab form TBTI/DVD-Ig) to the CD19-CD3 orientation resulted in a significant loss of activity (see Figure 8). The converted B-Fab molecule was inactive at concentrations that were maximal for both CODV-Ig Fab orientations and the other B-Fab orientation. In the case of CODV-Ig Fab and one B-Fab orientation, the maximal response was observed (in the range from 1 to 100 ng/ml). In the case of CD19-CD3 B-Fab orientation, even at the maximal concentration (30 μg/ml) the optimal cytotoxic response was not achieved. Conversely, changing the domain orientation in CODV-Fab to CD3-CD19 (1108) resulted in a molecule that had significant activity in this assay (see Figure 8). Although domain swapping in CODV-Fab also reduced the induction of T-cell-mediated cytotoxicity (100-fold increase in EC50), this effect was much less pronounced than that observed with the B-Fab form and the molecule was able to induce cytotoxicity to a maximal level. Data were representative and obtained from three independent experiments.

Example 12. Effect of individual amino acid sequence composition on CODV-Ig linkers

The optimized construct corresponding to lot #204 (see Example 7 and Table 8) was chosen to study the effect of linker composition on the L1-L4 linkers. The linker lengths were 7, 5, 1, and 2 residues for L1, L2, L3, and L4, respectively (see Table 13). The test sequences were derived from naturally occurring linkers at the transitions between the VH and CH1 domains of natural antibodies or between the Fv and CL domains of the kappa or lambda light chains of antibodies. The sequences selected for the study were ASTKGPS (SEQ ID NO: 48), which is derived from the transition region between the VH and CH1 domains, RTVAAPS (SEQ ID NO: 49), and GQPKAAP (SEQ ID NO: 50), which are derived from the transition regions between the Fv and CL domains of the kappa and lambda light chains, respectively. In addition, one construct with a random linker composition was generated to demonstrate that any sequence could potentially be used in the L1-L4 linkers. This linker composition was obtained by randomly distributing the amino acids valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline at 15 positions of the four linkers. The aromatic amino acids phenylalanine, tyrosine and tryptophan, as well as the amino acids methionine and cysteine were previously excluded to avoid possible increase in aggregation.

A 3D model of the design for lot #204 was created to verify the suitability or to refine the linker composition choices. Thus, serine was chosen for linker L3 because positively and negatively charged residues were observed in the 3D model in close proximity. Residues in linker L4 were chosen to be compatible with solvent exposure at positions predicted in the model. Similarly, no problems were predicted or anticipated with the composition of linkers L1 and L2. 3D models of the selected linker composition predictions were constructed.

As shown in Table 12, the composition of the linkers can have a strong impact on the yield. L1 sequences that were derived from the lambda chain (compare lots #505-507 with lots #501-503) were more productive protein generators (up to 8-fold increase). Indeed, linkers based on random generation also gave good yields, as shown in Table 13, lot #508. Thus, the composition of the linkers should be one of the parameters taken into account when optimizing CODV-Ig.

The activities of bispecific antibodies or derivatives against IL4 and IL13 cytokines were determined on commercially available HEK-Blue IL-4/IL-13 reporter cells (InvivoGen). HEK-Blue IL-4/IL-13 cells are designed to monitor STAT6 pathway activation by IL-4 or IL13. Stimulation of cells with either cytokine results in the production of secreted embryonic alkaline phosphatase (SEAP) reporter gene, which can be measured in culture supernatant using the QUANTI-Blue assay. To test the activities of antibodies against IL4 or IL13, cytokines were preincubated for 1 h with different concentrations of antibodies and added to 50,000 HEK-Blue IL-4/IL-13 cells. Cytokine-mediated SEAP induction was measured after 24 h incubation in cell culture supernatant using the QUANTI-Blue assay (InvivoGen).

Example 13. Introduction of cysteines into CODV-Ig linkers

Published data indicate that the stability of antibodies and antibody-derived proteins can be enhanced by the introduction of non-naturally occurring disulfide bonds (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 investigate whether the equivalent human IgG1-derived Fc fragment engineered as CODV-Ig could be stabilized by the introduction of interchain and intrachain disulfide bonds, the equivalent Fc positions in the CODV-Ig lot #204 construct (from Example 7) were mutated to cysteine residues and the mutant proteins were overexpressed, purified, and characterized (see Table 14).

As shown in Table 14, all mutant CODV-Ig molecules containing additional cysteine residues had melting temperatures similar to the melting temperature of CODV-Ig construct #204.

In addition, two cysteines were simultaneously introduced at Kabat numbering positions 100 for the light chain and 44 for the heavy chain in each of the variable domains, as described by Brinkmann et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 7538-42. Such positions were shown to be structurally conserved in the antibody fold and therefore allow substitution by cysteine without interfering with the integrity of the individual domains.

As shown in Table 15, CODV and CODV-Ig constructs in which cysteine residues were introduced at Kabat numbering positions 100 for the light chain and 44 for the heavy chain in each of the variable domains had higher melting temperatures than CODV and CODV-Ig constructs in which cysteine residues were not introduced at such positions (see, e.g., lots #704 and 706 and lots #713 and 714).

1. Thermal stability measurements of CODV and TBTI variants

Melting points (Tm) of CODV and TBTI variants were determined using differential scanning fluorimetry (DSF). Samples were diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 μg/μl, and 2 μl of a 40-fold concentrated solution of SYPRO-Orange dye (Invitrogen) in D-PBS were added to 96-well white half-bordered plates. All measurements were performed in duplicate using the MyiQ2 real-time PCR instrument (Biorad). Tm values were obtained from the negative first derivative of the melting curves using the iQ5 v2.1 software.

The effect of introducing cysteine residues directly into the linkers or into the variable region was then investigated. In this example, lot #204 (from Example 7 and Table 8) was used as a model CODV-Ig binding protein, and cysteine was substituted for glycine in L1, L, or the variable region based on the 3D model. As shown in Table 16 below, the results show how introducing a pair of cysteine residues can affect yield and aggregation. All putative mutations were simulated to ensure that the disulfide bonds were formed correctly and that the correct geometry of the linkers and their environment was maintained in the models. However, lot #808 showed good yield and little aggregation, indicating that a suitable cysteine bridge could be formed.

Although the invention has been described with reference to various embodiments, it is understood that changes and modifications may be made by those skilled in the art. It is therefore intended that the appended claims cover all such equivalent variations that fall within the scope of the claimed invention. In addition, the section headings used in this specification are for organizational purposes only and are not to be construed as limiting the subject matter described.

Each variant described in this publication may be combined with any other variant or variants, unless otherwise clearly stated. In particular, any feature or variant stated as preferred or advantageous may be combined with any other feature or features or variant or variants stated as preferred or advantageous, unless otherwise clearly stated.

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

--->

LIST OF SEQUENCES

<110> Rao, Ercole

Nicholas, Vincent

Corvey, Carsten

Beil, Christian

Lange, Christian

Steinmetz, Anke

Baurin, Nicholas

Li, Danxi

<120> Antibody-like binding proteins with dual variable

regions having the orientation of the connecting regions

criss-cross

<130> 11-414-WO

<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

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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

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

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Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr

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crosswise IL4(G4S2)IL13CHFc

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Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser

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Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His Trp Ile

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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

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Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Arg Ser

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Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Leu Lys Glu

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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

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Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

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Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

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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> Protein

<213> Artificial sequence

<220>

<223> Light chain design with two heads in orientation

crosswise 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> Protein

<213> Artificial sequence

<220>

<223> Light chain design with two heads in orientation

crosswise 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> Protein

<213> Artificial sequence

<220>

<223> Light chain design with two heads in orientation

crosswise 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> Protein

<213> Artificial sequence

<220>

<223> Light chain design with two heads in orientation

crosswise 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientations

crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientations

crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientations

crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientations

crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientations

crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain for Ig with double V in crosswise orientation,

having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for Ig with double V in orientation

crosswise, having the 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

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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for Ig with double V in orientation

crosswise, having the 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> 21

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for Ig with double V in orientation

crosswise, having the code LC3

<400> 21

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> 22

<211> 328

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for Ig with double V in orientation

crosswise, having the code LC4

<400> 22

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> Protein

<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> Protein

<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> Protein

<213> Artificial sequence

<220>

<223> Peptide linker with four glycine residues

<400> 25

Gly Gly Gly Gly

1

<210> 26

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker with 5 amino acid glycine residues

<400> 26

Gly Gly Gly Gly Gly

1 5

<210> 27

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker with 6 amino acid glycine residues

<400> 27

Gly Gly Gly Gly Gly Gly

1 5

<210> 28

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker with seven amino acid glycine residues

<400> 28

Gly Gly Gly Gly Gly Gly Gly

1 5

<210> 29

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker with eight glycine amino acid residues

<400> 29

Gly Gly Gly Gly Gly Gly Gly Gly

1 5

<210> 30

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial peptide linker

<400> 30

Gly Gly Gly Gly Ser

1 5

<210> 31

<211> 10

<212> Protein

<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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientation crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

orientation crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Heavy chain sequence for Ig with double V in

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

orientation crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

orientation crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

crosswise orientation, having the code LC14

<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 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

crosswise orientation, having the 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> Protein

<213> Artificial sequence

<220>

<223> Light chain sequence for double-V Ig

orientation crosswise, having the 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> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 48

Ala Ser Thr Lys Gly Pro Ser

1 5

<210> 49

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 49

Arg Thr Val Ala Ala Pro Ser

1 5

<210> 50

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 50

Gly Gln Pro Lys Ala Ala Pro

1 5

<210> 51

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 51

His Ile Asp Ser Pro Asn Lys

1 5

<210> 52

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 52

Thr Lys Gly Pro Ser

1 5

<210> 53

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 53

Thr Val Ala Ala Pro

1 5

<210> 54

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 54

Gln Pro Lys Ala Ala

1 5

<210> 55

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 55

Gln Arg Ile Glu Gly

1 5

<210> 56

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial peptide linker

<400> 56

Gly Gly Cys Gly Gly Gly Gly

1 5

<210> 57

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial peptide linker

<400> 57

Gly Gly Gly Cys Gly Gly Gly

1 5

<210> 58

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial peptide linker

<400> 58

Gly Gly Gly Gly Cys Gly Gly

1 5

<210> 59

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial peptide linker

<400> 59

Gly Gly Gly Gly Gly Cys Gly

1 5

<210> 60

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Amino acid sequence of the hinge region of IGHG1

<400> 60

Asp Lys Thr His Thr

1 5

<---

Claims (76)

1. An antibody-like binding protein construct that binds a first antigen and a second antigen, having interchangeable variable domains comprising two polypeptide chains that form two antigen-binding sites, where the first polypeptide chain contains the structure V1-L1-V2-L2-CL, and and the second polypeptide chain contains the structure V3-L3-V4-L4-CH1; Where: (a)(i) V ₁ is the first immunoglobulin variable domain of the heavy chain (VH1), V ₂ is the second immunoglobulin variable domain of the light chain (VL2), V ₃ is the second immunoglobulin variable domain of the heavy chain (VH2), and V ₄ is the first immunoglobulin variable domain of the heavy chain (VL1); (ii) V ₁ is the first immunoglobulin variable domain of the light chain (VL1), V ₂ is the second immunoglobulin variable domain of the heavy chain (VH2), V ₃ is the second immunoglobulin variable domain of the light chain (VL2), and V ₄ is the first immunoglobulin variable domain of the heavy chain (VH1); or (iii) V ₁ is the first immunoglobulin variable domain of the heavy chain (VH1), V ₂ is the second immunoglobulin variable domain of the heavy chain (VH2), V ₃ is the second immunoglobulin variable domain of the light chain (VL2), and V ₄ is the first immunoglobulin light chain variable domain (VL1); where V _L1 and V _H1 are associated to form the first antigen-binding site, and V _L2 and V _H2 are associated to form the second antigen-binding site; (b) C _L is the immunoglobulin light chain constant domain; (c) _CH1 is the immunoglobulin constant domain of the heavy chain _CH1 ; and (d) L ₁ , L ₂ , L ₃ and L ₄ are amino acid linkers; wherein L ₂ is at least 1 amino acid residue long and L ₄ is at least 1 amino acid residue long; and where the length of one or more L1 and L3 may be equal to or greater than zero amino acids; where the first and second polypeptides form a light chain-heavy chain cross-pair. 2. An antibody-like binding protein construct according to claim 1, wherein: L ₁ has a length of 3-12 amino acid residues; L ₂ has a length of 3-14 amino acid residues; L ₃ is 1-8 amino acid residues long; and L ₄ is 1-3 amino acid residues long. 3. An antibody-like binding protein construct according to claim 1, wherein: L ₁ has a length of 5-10 amino acid residues; L ₂ has a length of 5-8 amino acid residues; L ₃ is 1-5 amino acid residues long; and L ₄ is 1 or 2 amino acid residues long. 4. An antibody-like binding protein construct according to claim 1, wherein: L ₁ is 7 amino acid residues long; L ₂ is 5 amino acid residues long; L ₃ is 1 amino acid residue long; and L ₄ is 2 amino acid residues long. 5. An antibody-like binding protein construct according to claim 1, wherein: L ₁ has a length of 1-3 amino acid residues; L ₂ has a length of 1-4 amino acid residues; L ₃ is 2-15 amino acid residues long; and L ₄ has a length of 2-15 amino acid residues. 6. An antibody-like binding protein construct according to claim 1, wherein: L ₁ is 1 or 2 amino acid residues long; L ₂ is 1 or 2 amino acid residues long; L ₃ is 4-12 amino acid residues long; and L ₄ has a length of 2-12 amino acid residues. 7. An antibody-like binding protein construct according to claim 1, wherein: L ₁ has a length of 1 amino acid residue; L ₂ is 2 amino acid residues long; L ₃ is 7 amino acid residues long; and L ₄ is 5 amino acid residues long. 8. The antibody-like binding protein construct of claim 1, wherein L ₁ is absent and L ₃ is 2 or more amino acid residues in length. 9. The antibody-like binding protein construct of claim 1, wherein _L3 is absent and _L1 is 1 or more amino acid residues in length. 10. The antibody-like binding protein construct of claim 1, wherein _L1 is absent and each _L2 , _L3 and _L4 is at least 1 amino acid residue in length. 11. The antibody-like binding protein construct of claim 1, wherein L ₃ is absent and each L ₁ and L ₂ is at least 1 amino acid residue in length. 12. An antibody-like binding protein construct according to claim 1, wherein: a) L ₂ is at least twice as long as L ₄ ; or b) L ₄ is at least twice as long as L ₂ . 13. The antibody-like binding protein construct of claim 1, wherein both L ₁ and L ₃ are at least one amino acid long; and where L ₁ is at least twice as long as L ₃ if L ₂ is at least twice as long as L ₄ ; or L ₃ is at least twice as long as L ₁ if L ₄ is at least twice as long as L ₂ . 14. The antibody-like binding protein construct of claim 1, wherein at least the first or second polypeptide chain comprises an Fc, i.e., an immunoglobulin hinge region, and immunoglobulin heavy chain constant domains _CH2 and _CH3 derived from the Fc domain. 15. The antibody-like binding protein construct of claim 14, wherein the Fc is linked to the carboxyl terminal region of the polypeptide chain. 16. The antibody-like binding protein construct of claim 14, wherein the Fc is linked to C _L and/or C _H1 . 17. The antibody-like binding protein construct of claim 16, wherein the Fc of the first polypeptide is linked to C _L and/or the Fc of the second polypeptide is linked to C _H1 . 18. An antibody-like binding protein construct according to claim 1, wherein the binding protein is capable of specifically binding to one or more target antigens. 19. The antibody-like binding protein construct of claim 18, wherein the one or more target antigens are selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-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, 5, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (integrin b4), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNF-α, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5/CCXCR1), DNGR-1(CLEC91) and HMGB1. 20. An antibody-like binding protein construct according to claim 1, wherein the binding protein is bispecific and is capable of binding to two different target antigens. 21. The antibody-like binding protein construct of claim 20, wherein the two different target antigens 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. 22. An antibody-like binding protein construct according to claim 1, wherein the binding protein is capable of inhibiting the function of one or more target antigens. 23. The antibody-like binding protein construct of claim 1, wherein at least one of the linkers selected from the group consisting of L ₁ , L ₂ , L ₃ and L ₄ comprises at least one glycine residue. 24. An isolated nucleic acid molecule encoding an antibody-like binding protein construct according to any one of claims 1 to 23. 25. An expression vector containing a nucleic acid molecule according to item 24. 26. An isolated host cell for producing an antibody-like binding protein construct containing a nucleic acid molecule according to claim 24, or an expression vector containing a nucleic acid molecule according to claim 25.