Dual variable domain antibody-like binding proteins with cross-linking domain orientation - Patents.com

Abstract

To provide genetically engineered bispecific antigen-like binding proteins with superior properties that can be readily produced in high yield from mammalian cell culture.SOLUTION: The invention provides antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, where each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an antibody-like binding protein comprising four polypeptide chains forming four antigen-binding sites, where each pair of polypeptides forming the antibody-like binding protein has dual variable domains with crossed orientation. The present invention also relates to methods for producing such antigen-like binding proteins. [Background technology]

[0002] Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies, with binding specificities for two different antigens, can be produced using recombinant techniques and are predicted to have broad clinical applications. An intact IgG antibody molecule is a Y-shaped molecule that contains four polypeptide chains: two heavy chains and two light chains. Each light chain contains two domains: the variable or V L The N-terminal domain and the constant (or C L ) domains (known as constant kappa (Cκ) or constant lambda (Cλ) domains) Each heavy chain consists of four or five domains, depending on the class of antibody. The N-terminal domain is the variable (or V H ) domain (or area) Then, the first steady state (or C H1 ) domain, the hinge region, and then the second and third constant (or C H2 and C H3 ) domains. In assembled antibodies, the V L and V H The domains associate together to form the antigen-binding site. L and C. H1 Domains meet together The two heavy-light chain heterodimers are C H2 and C. H3 They are associated together by domain interactions and by interactions between the hinge regions on the two heavy chains.

[0003] It is known that proteolytic digestion of antibodies can lead to the production of antibody fragments (Fab and Fab2). Such fragments of intact antibodies can exhibit antigen-binding activity. Antibody fragments can also be produced recombinantly. F consists of only the variable regions of the heavy and light chains linked together. V Fragments may be obtained. These F V The fragments are monovalent with respect to antigen binding. Most naturally occurring antibodies In order to maintain full bonding strength, generally V H and V L Although both are required, smaller fragments such as individual variable domains (domain antibodies or dABs: Non-Patent Document 1) and individual complementarity determining regions or CDRs (Non-Patent Document 2) have also been shown to retain the binding properties of the parent antibody.

[0004] Single-chain variable fragment (scF V The composition is an antibody contained in a single polypeptide chain. V H and V L These small proteins (MW: ∼25,000 Da) generally maintain their specificity and affinity for their antigens within a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules.

[0005] The advantage of using antibody fragments rather than complete antibodies in diagnosis and therapy is their small size. They are less likely to be immunogenic than complete antibodies and are more able to penetrate tissues. The disadvantage with the use of such fragments is that they only have one antigen binding site, leading to reduced avidity. Also, due to their small size, they are cleared very quickly from serum and therefore exhibit a short lifetime.

[0006] It has been of interest to create bispecific antibodies (BsAbs) that combine the antigen-binding sites of two antibodies in one molecule and thus can bind two different antigens simultaneously. Besides application for diagnostic purposes, such molecules pave the way for new therapeutic applications, for example by redirecting powerful effector systems to diseased sites (where cancerous cells often express mechanisms that suppress normal immune responses, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) induced by monoclonal antibodies) or by enhancing the neutralizing or stimulating activity of antibodies. This possibility was recognized at an early stage and led to many efforts to obtain such bispecific antibodies. Early attempts to link the binding specificities of two complete antibodies against different target antigens for therapeutic purposes used chemically fused heteroconjugate molecules (Non-Patent Document 4).

[0007] Bispecific antibodies were initially made by fusing two hybridomas, each capable of producing a different immunoglobulin (Non-Patent Document 5), but the complexity of the types (up to 10 different types) produced in cell culture made purification difficult and expensive (Non-Patent Document 6). Using this format, mouse IgG2a and rat IgG2b antibodies can be produced together in the same cell (e.g., as a quadroma fusion of the two hybridomas, or genetically engineered). The antibodies were produced in recombinant CHO cells (either in IgG2a or in recombinant human CHO cells). Since the light chain of each antibody preferentially associates with the cognate heavy chain, three main species of antibodies are assembled: the two parent antibodies and a heterodimer of two antibodies, each containing one light / heavy chain pair associated via the Fc region. The desired heterodimer can be purified from this mixture because its binding properties for Protein A differ from those of the parent antibodies: rat IgG2b does not bind Protein A, whereas mouse IgG2a does. As a result, the mouse-rat heterodimer binds Protein A but elutes at a higher pH than the mouse IgG2a homodimer, which allows the selective purification of the bispecific heterodimer (Non-Patent Document 7). The resulting bispecific The heterodimers are completely non-human and therefore highly immunogenic, which may have adverse side effects (e.g., "HAMA" or "HARA" reactions) and / or render the treatment ineffective. There remains a need for engineered bispecific agents with superior properties that can be readily produced in high yields from mammalian cell culture.

[0008] As mentioned above, despite the promising results obtained with heteroconjugates or bispecific antibodies produced from cell fusions, several factors make their large-scale therapeutic application impractical, including: the rapid clearance of heteroconjugates in vivo, the laboratory-intensive techniques required to generate either type of molecule, the need for large-scale purification of the heteroconjugates away from the homoconjugates or monospecific antibodies, and the generally low yields obtained.

[0009] Genetic engineering is being used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with desired sets of binding specificities and effector functions. A variety of recombinant methods have been developed to efficiently produce BsAbs, both as antibody fragments (Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 10) and in full-length IgG formats (Non-Patent Document 11).

[0010] Combining two different scFvs results in the smallest molecular weight BsAb format, called sc-BsAb or Ta-scFv (Non-Patent Document 12, Non-Patent Document 13). BsAbs were constructed by genetically fusing two scFvs with a dimerization functional group such as a leucine zipper (Non-Patent Document 14, Non-Patent Document 15).

[0011] Diabodies are small bivalent, bispecific antibody fragments. The fragments can be arranged on the same polypeptide chain by using a linker (fewer than 12 amino acid residues) that is too short to allow pairing between the two domains on the same chain. L V linked with H The domains are forced to pair intermolecularly with complementary domains on other strands. These dimeric antibody fragments, or "diabodies", are bivalent and bispecific (Non-Patent Document 16). Diabodies are similar in size to Fab fragments. V is linked by a linker of 3 to 12 amino acid residues. H and V L The polypeptide chains of the domain mainly form dimers (diabodies), whereas For linkers of 0 to 2 amino acid residues, trimers (triabodies) and tetrabodies predominate. In addition to the length of the linker, the exact pattern of oligomerization appears to depend on the composition and orientation of the variable domains (Non-Patent Document 17). The final structure of the diabody molecule is very poorly predictable.

[0012] Although constructs based on sc-BsAbs and diabodies show interesting clinical potential, such non-covalently linked molecules are not sufficiently stable under physiological conditions. The overall stability of scFv fragments is largely dependent on the V L and V HThe V of scFv fragments depends on the intrinsic stability of the domains as well as the stability of the domain interfaces. H -V L Insufficient safety of the interface has often been suggested as the main cause of irreversible inactivation of scFvs, and it is one of the interfaces enabled by peptide linkers. This is because the temporary release exposes hydrophobic patches that favor aggregation and therefore lead to instability and poor productivity (Non-Patent Document 18).

[0013] V H and V L An alternative method for producing bispecific, bivalent antigen-binding proteins from domains is described in US Pat. No. 5,399,663. Such a double-headed, dual Fv configuration is obtained by expressing a bicistronic vector encoding two polypeptide chains. In the dual Fv configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy and one light), where one polypeptide chain contains the V domains separated by a peptide linker. H (V H1 -Linker-V H2 ) twice in a row, and The polypeptide chain of is composed of complementary V L domain( V L1 -Linker-V L2 In the crossed double-headed configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate polypeptide chains (one heavy and one light), where one polypeptide chain is bound to a V H ( V H1 -Linker-V H2 ) twice in succession, and the other polypeptide chain is a complementary V linked in succession in the opposite orientation by a peptide linker. L Domain (V L2 -Linker-V L1 )from Molecular modeling of the construct suggested that the linker size was sufficient, with a total length of 30-40 Å (15-20 amino acid residues).

[0014] Increasing the valency of an antibody is of interest because it enhances the functional affinity of the antibody through avidity effects. A multivalent protein complex (PPC) with increased valency is described in US Pat. No. 5,999,336. A PPC generally comprises two polypeptide chains arranged laterally to each other. Each polypeptide chain usually comprises three or four "v-regions", which contain amino acid sequences that can form an antigen-binding site when matched with corresponding v-regions on the opposite polypeptide chain. Up to about six "v-regions" can be used on each polypeptide chain. The v-regions of each polypeptide chain are linearly linked to each other and can be interspersedly linked. The v-regions on each polypeptide chain may be linked by a linking region. When arranged in the form of a PPC, the v-regions on each polypeptide chain form separate antigen binding sites. The complex may contain one or more binding specificities.

[0015] C of Fc H3 A strategy to create Fc heterodimers was proposed by Carter et al. using a series of “knob-into-hole” mutations in the domains (Non-Patent Document 19, Non-Patent Document 20). These mutations are located in the C H3 This results in altered residue packing complementarity between the domains, which in turn favors the formation of heterodimers compared to homodimers, leading to superior hematological properties from mammalian cell culture. To achieve heterodimer expression, the strategy resulted in high heterodimer production, but homodimers were not completely suppressed (Non-Patent Document 21).

[0016] Gunasekaran et al. have demonstrated that Fc heterodimer formation is promoted while at the same time C H3We explored the possibility of maintaining the integrity of the hydrophobic core by altering the charge complementarity of the domain interface (Non-Patent Document 22). Taking advantage of the electrostatic steering mechanism, these constructs efficiently promoted Fc heterodimer formation with minimal homodimer contamination by mutation of two pairs of peripherally located charged residues. In contrast to the knobs-into-holes design, homodimers were equally suppressed but not completely eliminated due to the nature of the electrostatic repulsion mechanism.

[0017] Davis et al. H3 have described an antibody engineering method to convert Fc homodimers into heterodimers without introducing extra interchain disulfide bonds by combining β-strand portions of the domains with each other (Non-Patent Document 23). Expression of SEEDbody (Sb) fusion proteins in mammalian cells produces high yields of Sb heterodimers, which are easily purified to remove trace by-products.

[0018] Patent Document 3 discloses H3 describes bispecific antibodies based on heterodimerization of C domains. H3 The mutations H95R and Y96F were introduced into one heavy chain in the domain. The amino acid substitutions in H3 It occurs in the C domain and heterodimerizes with the IgG1 backbone. The common light chain that tends to pair with all heavy chains is the C H3 All formats based on heterodimerization via domains are prerequisites. A total of three types of antibodies are produced: 50% with a pure IgG1 backbone, one third with a pure H95R and mutated Y96F backbone, and one third with two different heavy chains (bispecific). The heterodimer of interest can be generated from this mixture because its binding properties to Protein A differ from those of the parent antibody: i.e., the C from IgG3 H3The domain does not bind to Protein A, whereas IgG1 does. Consequently, the heterodimer binds to Protein A but elutes at a higher pH than the pure IgG1 homodimer, and this allows the selective purification of the bispecific heterodimer.

[0019] Patent document 4 describes dual variable domain IgG (DVD-IgG) bispecific antibodies based on the dual Fv format described in Patent document 1. A similar bispecific format was also described in Patent document 5. The addition of constant domains to each chain of the dual Fv (C to the heavy chain) H1 -Fc and kappa or lambda constant domains to the light chain) resulted in functional bispecific antibodies without the need for any further modifications (i.e., obvious addition of constant domains to enhance stability). Some antibodies expressed in DVD-Ig / TBTI format show position effects for the second (i.e., innermost) antigen binding site (Fv2). Depending on the sequence and nature of the antigen recognized by the Fv2 site, this antibody domain This is due to a decrease in affinity for the antigen (i.e., loss of on-rate compared to the parent antibody). One possible explanation for this observation is that L1 and V L2 The linker between Fv2 , protruding into the CDR regions, making Fv2 somewhat less accessible to larger antigens.

[0020] The second configuration of the bispecific antibody fragment described in WO 2005 / 023991 is the crossed double headed (CODH) configuration, in which the variable domains expressed on the two chains have the following orientation: For the light chain, V L1 -Linker-V L2 ; For the heavy chain, V H2 -Linker-V H2 Patent document 1 describes a bispecific cross-linked double-headed antibody fragment (construct GOSA.E). disclose that this retains higher binding activity than dual Fvs (see page 20, lines 20-50 of Patent Document 1), and further disclose that this format is less affected by the linker used between the variable domains (see pages 20-21 of Patent Document 1). [Prior art documents] [Patent documents]

[0021] [Patent Document 1] U.S. Patent No. 5,989,830 [Patent Document 2] US Patent No. 2005 / 0003403 A1 [Patent Document 3] US Patent No. 2010 / 331527 A1 [Patent Document 4] U.S. Patent No. 7,612,181 [Patent Document 5] US Patent No. 2010 / 0226923 A1 [Non-patent literature]

[0022] [Non-Patent Document 1] Ward et al., 1989, Nature 341(6242): 544-46 [Non-Patent Document 2] Williams et al., 1989, Proc. Natl. Acad. Sci. USA 86(14): 5537-41 [Non-Patent Document 3] Bird et al., 1988, Science 242(4877): 423-26 [Non-Patent Document 4] Staerz et al., 1985, Nature 314(6012): 628-31 [Non-Patent Document 5] Milstein et al., 1983, Nature 305(5934): 537-40 [Non-Patent Document 6] George et al., 1997, THE ANTIBODIES 4: 99-141 (Capra et al., ed., Harwood Academic Publishers)

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[0023] The present invention provides an antibody-like binding protein comprising four polypeptide chains forming four antigen binding sites, wherein two of the polypeptide chains have the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the two polypeptide chains have the formula: V H2 -L3-V H1 -L4-C H1 -Fc [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region of an immunoglobulin, and C H2 , C H3 an immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; And wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.

[0024] The present invention also provides an antibody-like binding protein comprising two polypeptide chains forming two antigen binding sites, wherein the first polypeptide chain has the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the second polypeptide chain has the formula: V H2 -L3-V H1 -L4-C H1 [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the first and second polypeptides form a cross-light-heavy chain pair.

[0025] The present invention further provides a method for making an antibody-like binding protein comprising four polypeptide chains forming four antigen binding sites, each of which is a V L and V H identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen, each of which contains a light chain or a heavy chain as a template chain; L V L1 Assign as;V L2 ,V H1 and V H2 Formulae [I] and [II] below: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 -Fc [II] determining maximum and minimum lengths for L1, L2, L3 and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that, when combined, bind to a first target antigen and a second target antigen to form an antibody-like binding protein; comprising; Where: V L1is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region and C of an immunoglobulin H2 , C H3 is the constant domain of an immunoglobulin heavy chain; and L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair.

[0026] The present invention further provides a method for making an antibody-like binding protein comprising four polypeptide chains forming four antigen binding sites, each of which is a V L and V H identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen; assigning either a light chain or a heavy chain as a template chain; L V L1 Assign as;V L2 ,V H1 and V H2 of Formulas [I] and [II] below: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 [II] determining maximum and minimum lengths for L1, L2, L3 and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that, when combined, bind to a first target antigen and a second target antigen to form an antibody-like binding protein; Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair.

[0027] Specific embodiments of the present invention will become apparent from the following more detailed description of some embodiments and from the claims. [Brief description of the drawings]

[0028] [Figure 1] Schematic representation of the arrangement of antigen-binding domains FV1 and FV2 within a dual V-region configuration and their respective peptide linkers LL and LH in the TBTI format. [Diagram 2] 2D is a schematic diagram of the arrangement of antigen-binding domains FV1 (anti-IL4) and FV2 (anti-IL13) and their respective peptide linkers in a crossover doubly variable (CODV) configuration. [Diagram 3]Schematic representation of FV anti-IL4 and Fab anti-IL13 showing one possible spatial configuration resulting from protein-protein binding of the FVs of anti-IL4 and anti-IL13. [Figure 4] Evaluation of the tetravalent bispecific binding capacity of CODV proteins in a BIACORE assay by sequential or simultaneous injection of two antigens onto a DVD-Ig protein coated chip. The maximum signal observed by sequential injections could be obtained by simultaneous injection of two antigens, demonstrating saturation of all binding sites. [Diagram 5] 2D: Schematic diagram of the antigen-binding domains and their respective peptide linkers LL (L1 and L2) and LH (L3 and L4) in the CODV context. In panel A, the light chains are kept in a "linear or template" sequence, whereas the heavy chains adopt a "crossed" configuration. In panel B, the heavy chains are kept in a "linear or template" sequence, whereas the light chains adopt a "crossed" configuration. [Figure 6] Schematic representation of CODV-Ig design based on using either the light or heavy chain as a "template." [Figure 7] Comparison of TBTI / DVD-Ig or CODV-IG incorporating anti-IL4 and anti-IL13 sequences. [Figure 8] Comparison of CODV-Fab and B-Fab in a cytotoxicity assay using NALM-6 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention provides antibody-like binding proteins comprising four polypeptide chains forming four antigen-binding sites, where each pair of polypeptides forming the antibody-like binding protein has dual variable domains with crossed orientation. The present invention also provides methods for producing such antigen-like binding proteins.

[0030] Computer modeling predicted that the crossed double head (CODH) design of US Patent No. 5,989,830 may result in a complex with both binding sites facing in opposite directions without the limitations suggested for the dual Fv configuration of US Patent No. 7,612,181. In particular, computer modeling showed that the length of the amino acid linker between the variable domains is not critical for the CODH design, but is important to allow full access to both antigen binding sites in the dual Fv design. Similar to the DVD-Ig / TBTI format, an antibody-like binding protein construct was prepared in which constant domains were attached in a CODH configuration to form an antibody-like binding protein containing four polypeptide chains forming four antigen binding sites, where each pair of polypeptides forming the antibody-like binding protein has dual variable domains with crossed orientation (i.e., CODH-Ig). The CODH-Ig molecule is expected to have significantly improved stability compared to the CODH molecule (just as DVD-Ig / TBTI had improved stability over the dual Fv molecule).

[0031] To test the above hypothesis, CODH-Ig molecules were generated using anti-IL4 and anti-IL13 antibody sequences described in US Patent Application Publication No. 2010 / 0226923A1. The CODH-Ig molecules differed from the CODH molecules of US Patent Application Publication No. 2010 / 0226923 with respect to the length of the amino acid linker separating the variable domains on each polypeptide chain. The CODH-Ig molecules were expressed in cells after transient transfection and then purified by protein A chromatography. Although their size exclusion chromatography (SEC) profiles showed aggregation levels of 5-10%, none of the CODH-Ig molecules were functional and therefore capable of binding all of their target antigens. The lack of antigen binding activity could have been due to disrupted dimerization of the heavy and light chain Fv regions due to inappropriate linker length compromising the formation of accurate antigen binding sites. As a result, a protocol was developed to identify suitable amino acid linkers for insertion between the two variable domains on both the heavy and light polypeptides of an antibody-like binding protein, and between the second variable domain and the constant domain, based on homologous protein-protein binding and experimental models of the FvIL4 and FvIL13 regions, respectively, inclusion of the Fc1 domain into the models, and construction of suitable linkers between the FvIL4 and FvIL13 regions and between the Fv and the constant Fc1 region.

[0032] Standard recombinant DNA technology methodologies can be used to construct polynucleotides encoding polypeptides forming the antibody-like binding proteins of the invention, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Unless specific definitions are provided, the nomenclature and laboratory methods and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses, pharmaceutical manufacture, formulation, delivery, and treatment of patients.

[0033] 1. general definition As used in accordance with this disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated: Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0034] The term "polynucleotide" as used herein refers to a single- or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In some embodiments, the nucleotides comprising a polynucleotide may be any type of nucleotide, either ribonucleotides or deoxyribonucleotides or modified forms. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates, and phosphoroamidates. The term "polynucleotide" specifically includes single- and double-stranded forms of DNA.

[0035] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that, by its origin, is an isolated polynucleotide: (1) it is not associated with all, or a portion, of polynucleotides found in nature, (2) it is linked to polynucleotides with which it is not linked in nature, or (3) it is not found in nature as part of a larger sequence.

[0036] An "isolated polypeptide" is: (1) a polypeptide that is not normally found with at least some other polypeptide. An "isolated polypeptide" is one that is: (1) free of polypeptides; (2) essentially free of other polypeptides from the same source, e.g., the same species; (3) expressed by cells from a different species; (4) separated from at least 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) not associated (covalently or noncovalently) with portions of polypeptides with which it is naturally associated; (6) operably associated (covalently or noncovalently) with polypeptides with which it is not naturally associated; or (7) not found in nature. Such isolated polypeptides may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, an isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

[0037] As used herein, the term "human antibody" 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 yet other embodiments, human antibodies are produced recombinantly.

[0038] Naturally occurring antibodies usually comprise a tetramer. Each such tetramer usually consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (generally having a molecular weight of about 25 kDa) and one full-length "heavy" chain (generally having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each of the light and heavy chains usually contains a variable domain of about 100-110 or more amino acids involved in antigen recognition. The carboxy-terminal portion of each chain usually defines a constant domain involved in effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ) and three stationary domains In (C H1 , C H2 and C H3 ), where V H The domain is the amino terminus of a polypeptide. exists in C H3 The variable domain is located at the carboxyl terminus, and a full-length light chain immunoglobulin polypeptide contains a variable domain (V L ) and the constant domain (C L ), where V L Domestic C is present at the amino terminus of a polypeptide L The domain is located at the carboxyl terminus .

[0039] Human light chains are usually classified as kappa and lambda light chains, while human heavy chains are usually classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's 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 subdivided into subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains usually consist of a "J" region of about 12 or more amino acids. The heavy chains also contain a "D" region of about 10 or more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair usually form the antigen-binding site. The variable domains of naturally occurring antibodies usually exhibit the same general structure of relatively conserved framework (FR) regions joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned by the framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, both the light and heavy chain variable regions usually contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0040] The term "native Fc" as used herein refers to a molecule, whether in monomeric or multimeric form, that includes a non-antigen-binding fragment sequence resulting from antibody digestion or produced by other means, and may include a hinge region. The initial immunoglobulin source of the native Fc is preferably of human origin, preferably IgG1 and IgG2, but can be any immunoglobulin. Native Fc molecules are composed of monomeric polypeptides that can be linked covalently (i.e., disulfide bonds) and non-covalently into dimeric or multimeric forms. 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 IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is a generic term referring to monomeric, dimeric and multimeric forms.

[0041] The term "Fc variant" as used herein refers to a molecule or sequence that is altered from a native Fc, but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can include a molecule or sequence that is humanized from a non-human native Fc. Additionally, a native Fc may be necessary for the antibody-like binding proteins of the present invention. The term "Fc variant" includes regions that can be removed for reasons that result in unnecessary structural features or biological activity. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or that have been modified in one or more Fc sites or residues that affect or are involved in the following: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0042] As used herein, the term "Fc domain" encompasses native Fc and Fc variants, as well as sequences as defined above. As with Fc variants and native Fc molecules, the term "Fc domain" includes the monomeric or multimeric form of the molecule, whether it is digested from a whole antibody or produced by other means.

[0043] 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, where two of the polypeptide chains have the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the two polypeptide chains have the formula: V H2 -L3-V H1 -L4-C H1 -Fc [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region of an immunoglobulin, and C H2 , C H3 an immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair. As used herein, the term "antibody-like binding protein" also refers to a non-naturally occurring (or recombinant) molecule that specifically binds to at least one target antigen and which comprises two polypeptide chains that form two antigen-binding sites, wherein the first polypeptide chain has the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the second polypeptide chain has the formula: V H2 -L3-V H1 -L4-C H1 [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the first and second polypeptides form a cross-linked light chain-heavy chain pair. A "molecule" is one prepared, expressed, produced, or isolated by recombinant means.

[0044] One embodiment of the invention provides antibody-like binding proteins having biological and immunological specificity for one to four target antigens. Another embodiment of the invention provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains forming such antibody-like binding proteins. Another embodiment of the invention provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains forming such antibody-like binding proteins. Yet another embodiment of the invention provides host cells that express such antibody-like binding proteins (i.e., comprise nucleic acid molecules or vectors encoding polypeptide chains forming such antibody-like binding proteins).

[0045] As used herein, the term "swapability" refers to the ability to fold and "Full interchangeability" refers to the interchangeability of variable domains in the CODV format while retaining binding affinity. H1 and V H2 The order of domains and hence V L1 and V L2 This refers to the ability to interchange (i.e., reverse) the order of the domains while maintaining full functionality of the antibody-like binding protein as evidenced by retention of binding affinity. Additionally, it should be noted that the V H and V L simply refers to the location of the domain on a particular protein chain in the final format. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2 Derived from the V domain, an antibody-like binding protein H1 and V H2 Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 Derived from the V domain, an antibody-like binding protein H1 and V H2 Therefore, V Hand V L The designation refers to the current position, not the original position in the parent antibody. H Domain and V L Domains are therefore "interchangeable".

[0046] An "isolated" antibody-like binding protein is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody-like binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody-like binding protein is purified to: (1) greater than 95% by weight of the antibody, most preferably greater than 99%, as measured by the Lowry method; (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or, preferably, silver staining. An isolated antibody-like binding protein includes the antibody-like binding protein in situ, within a recombinant cell, since it is the only component of the antibody-like binding protein's natural environment.

[0047] As used herein, the terms "substantially pure" or "substantially purified" refer to a compound or species that is the predominant species present (i.e., more abundant, on a molar basis, than any other individual species in a composition). In some embodiments, a substantially purified fraction is a composition in which the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In yet other embodiments, the composition is purified to essential homogeneity (no contaminant species are detectable in the composition by conventional detection methods) consisting essentially of a single macromolecular species.

[0048] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule that is bound by an antibody-like binding protein and can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. A target antigen is one or more The antibody-like binding protein may have an epitope that is identical to that of the target antigen. For each target antigen recognized by the antibody-like binding protein, the antibody-like binding protein can compete with an intact antibody that recognizes the target antigen. A "bivalent" antibody-like binding protein is considered to contain antigen-binding sites with the same antigen specificity, in addition to "multispecific" or "multifunctional" antibody-like binding proteins.

[0049] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites or epitopes. Bispecific antibodies may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of F(ab') fragments.

[0050] Typically, an F(ab) fragment contains one light chain and one heavy chain V H and C H1 Domain where V of the F(ab) fragment H -C H1 The heavy chain portion is a separate heavy chain polypeptide. As used herein, F(ab) also refers to one light chain containing two variable domains separated by an amino acid linker, and one light chain containing two variable domains and a C H1 The antibody may comprise one heavy chain comprising the domain.

[0051] Typically, an F(ab') fragment comprises one light chain and two F(ab') molecules. To allow the formation of interchain disulfide bonds between the two heavy chains, more constant regions (C H1 and C. H2 The heavy chain comprises a portion of one heavy chain that contains a nucleotide sequence between the nucleotides (between the nucleotides).

[0052] In the context of the antibody-like binding proteins of the invention, the phrases "biological properties", "biological characteristics" and the term "activity" are used interchangeably herein and include, but are not limited to, epitope affinity and specificity, the ability to antagonize the activity of the antigenic target (or target polypeptide), the stability of the antibody-like binding protein in vivo, and the immunogenicity of the antibody-like binding protein. Other distinguishable biological properties or characteristics of antibody-like binding proteins include, for example, cross-reactivity (i.e., to non-human homologs of the antigenic target, or generally to other antigenic targets or tissues), and the ability to preserve high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis, in vitro and in vivo neutralization assays, and immunohistochemistry using tissue sections from different sources, including humans, primates, or any other source as appropriate.

[0053] As used herein, the term "immunologically functional immunoglobulin fragment" refers to a polypeptide fragment that contains at least the CDRs of an immunoglobulin heavy or light chain from which the polypeptide fragment is derived. An immunologically functional immunoglobulin fragment is capable of binding to a target antigen.

[0054] As used herein, a "neutralizing" antibody-like binding protein refers to a molecule that can block or substantially reduce an effector function of a target antigen to which it binds. As used herein, "substantially reduce" means reducing the effector function of the target antigen by 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%, and most preferably at least about 90%.

[0055] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In some embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, specific three-dimensional structural characteristics and An epitope is the 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 selectively recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In preferred embodiments, an antibody-like binding protein has an equilibrium dissociation constant of ≦10 -8 M, more preferably the equilibrium dissociation constant is ≦10 -9 M, and most preferably when the equilibrium dissociation constant is ≦10 -10 When M, the antigen is specific is said to bind to

[0056] Dissociation constant (K D ) is determined, for example, by surface plasmon resonance. In general, surface plasmon resonance analysis involves the determination of binding interactions between a ligand (a target antigen on a biosensor matrix) and an analyte (an antibody-like binding protein in solution) using a surface probe with a BIAcore system (Pharmacia Biosensor; Piscataway, NJ). The surface plasmon resonance (SPR) is used to measure the concentration of the analyte in real time. Surface plasmon resonance analysis can also be performed by immobilizing the analyte (an antibody-like binding protein on a biosensor matrix) and presenting the ligand (target antigen). D " The term refers to the dissociation constant of the interaction between a particular antibody-like binding protein and a target antigen.

[0057] As used herein, the term "specifically binds" refers to an antibody-like binding protein or antigen-binding fragment thereof that specifically binds to an antigen containing an epitope 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. "Kd" refers to the ability to bind to an epitope with a Kd of or greater, and / or with an affinity that is at least two-fold greater than the affinity for a nonspecific antigen.

[0058] The term "linker" as used herein refers to one or more amino acid residues inserted between the light and heavy chain domains of an immunoglobulin to give them sufficient flexibility to fold into a crossed dual variable region immunoglobulin. The linker is inserted at the transition between the variable domains or between the variable and constant domains, respectively, at the sequence level. The transition between the variable domains can be identified because their approximate size is well understood. The exact location of the domain transition can be determined by positioning a peptide stretch that does not form an element of secondary structure such as a beta sheet or an alpha helix, as evidenced by experimental data or as can be predicted by modeling or secondary structure prediction techniques. The linker described herein is inserted between the N-terminal V on the light chain and the constant domain. L1 and V L2 L1 located between the domains; and V on the light chain L2 and C-terminal C L Located between the domains is called L2. The chain linker is an N-terminal V H2 and V H1 L3, located between the domains; and V H1 and C. H1 -FcDo The linkers L1, L2, L3, and L4 are independently Although they may have the same sequence and / or length in some cases.

[0059] As used herein, the term "vector" refers to any molecule (e.g., a nucleic acid, a plasmid, or a virus) used to transfer coding information into a cell. The term "vector" includes a nucleic acid molecule capable of transferring another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, in which additional DNA segments may be inserted into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-E. coli vectors) are capable of replication in a host cell. Recombinant expression vectors (recombinant mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are also 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 of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as plasmids are the most commonly used form of vector, however, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0060] The term "operably linked" as used herein refers to the arrangement of flanking sequences, such as those described herein, that are constructed or assembled to perform their normal function. Thus, a flanking sequence operably linked to a coding sequence may enable replication, transcription and / or translation of the coding sequence to be accomplished. For example, a coding sequence is operably linked to a promoter if the promoter is capable of directing the transcription of that coding sequence. A flanking sequence need not be contiguous with a coding sequence as long as it functions correctly. Thus, for example, an intervening transcribed but untranslated sequence can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0061] The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain changes may occur over time, either due to mutation or environmental influences, but such cells are nevertheless included 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 present invention, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the antibody-like binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the antibody-like binding protein, such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured, from which the antibody-like binding protein can be recovered.

[0062] The term "transformation" as used herein refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been altered to contain new DNA. For example, a cell is transformed when it is genetically altered from its original state. After transformation, the transforming DNA may recombine with the cell's DNA by being physically integrated into the cell's chromosome, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered stably transformed when the DNA is replicated with cell division. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell; a cell is transfected when exogenous DNA is introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into a suitable host cell.

[0063] The term "natural origin" as used herein and applied to an object refers to an object that can be found in nature and has not been processed by humans. For example, a polynucleotide or polypeptide present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans is of natural origin. Similarly, "non-natural origin" as used herein refers to something that is not found in nature, or that has been structurally modified by humans, or that has been synthesized.

[0064] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids); (,(-disubstituted amino acids Non-naturally occurring amino acids, such as , N-alkylamino acids, lactic acid, and other non-conventional amino acids, may also be suitable components of the antibody-like binding proteins of the invention. Examples of non-conventional amino acids include 4-hydroxyproline, (-carboxyglutamate, (-N,N,N-trimethyllysine, (-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formaldehyde, N-acetylglucosamine ... Myrmethionine, 3-methylhistidine, 5-hydroxylysine, (-N-methylarginine Examples of amino acids include amino acids, imino acids, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0065] Naturally occurring residues may be divided into classes based on common side chain properties as follows: (1) Hydrophobicity: 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 hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues that affect chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyl, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyl.

[0066] Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are generally incorporated by chemical peptide synthesis rather than synthesis in living systems. These include peptidomimetics and inverted or reversed forms of other amino acid residues. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

[0067] A skilled artisan can use conventional techniques to determine suitable variants of the polypeptide chain of an antibody-like binding protein of the invention. For example, the skilled artisan can identify suitable regions of the polypeptide chain that may be altered without destroying activity by targeting regions that are not likely to be important for activity. Alternatively, the skilled artisan can identify residues and portions of the molecule that are conserved among similar polypeptides. Areas that may be important for biological activity or structure may also be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting the structure of the polypeptide.

[0068] As used herein, the term "patient" includes human and animal subjects.

[0069] A "disease" is any condition that would benefit from treatment with an antibody-like binding protein of the invention. "Disease" and "condition" are used interchangeably herein and include chronic and acute diseases or illnesses, including pathological conditions that predispose a patient to the disease in question.

[0070] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with the disease as well as those susceptible to the disease or those in which the disease is to be prevented.

[0071] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a composition that is suitable for administration to a patient. It refers to a compound or composition that, when administered, is capable of producing a desired therapeutic effect.

[0072] As used herein, the term "pharmaceutical acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for effecting or facilitating the delivery of an antibody-like binding protein.

[0073] "Effective amount" and "therapeutically effective amount" when used in reference to pharmaceutical compositions containing one or more antibody-like binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of antibody-like binding protein sufficient to inhibit one or more clinically defined pathological processes related to the condition being treated for a period of time. The effective amount may vary depending on the particular antibody-like binding protein used and will depend on various factors and conditions related to the patient being treated, as well as the severity of the disease. For example, if an antibody-like binding protein is to be administered in vivo, factors such as the age, weight, and health condition of the patient, as well as dose-response curves and toxicity data obtained from preclinical animal studies, will be among those factors considered. The determination of an effective or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

[0074] One embodiment of the invention provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of an antibody-like binding protein.

[0075] 2. Antibody-like binding proteins In one embodiment of the invention, the antibody-like binding protein comprises four polypeptide chains forming four antigen binding sites, wherein two of the polypeptide chains have the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the two polypeptide chains have the formula: V H2 -L3-V H1 -L4-C H1 -Fc [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region of an immunoglobulin, and C H2 , C H3 an immunoglobulin heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light-heavy chain pair.

[0076] In another embodiment of the invention, the antibody-like binding protein comprises two polypeptide chains which form two antigen binding sites, wherein the first polypeptide chain has the formula: V L1 -L1-V L2 -L2-C L [I] The compound has a structure represented by and the second polypeptide chain has the formula: V H2 -L3-V H1 -L4-C H1 [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulinH1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the first and second polypeptides form a cross-light-heavy chain pair.

[0077] Antibody-like binding proteins of the invention may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0078] In some antibody-like binding proteins of the invention, the length of L3 is at least twice as long as L1. In other antibody-like binding proteins of the invention, the length of L4 is at least twice as long as L2. In some antibody-like binding proteins of the invention, the length of L1 is at least twice as long as L3. In other antibody-like binding proteins of the invention, the length of L2 is at least twice as long as L4.

[0079] In some antibody-like binding proteins of the invention, L1 is 3 to 12 amino acid residues in length. In another antibody-like binding protein, L1 is 5-10 amino acid residues long, L2 is 5-8 amino acid residues long, L3 is 1-5 amino acid residues long, and L4 is 1-2 amino acid residues long. In the somatoid binding protein, L1 is 7 amino acid residues long, L2 is 5 amino acid residues long, L3 is 1 amino acid residue long, and L4 is 2 amino acid residues long.

[0080] In some antibody-like binding proteins of the invention, L1 is 1 to 3 amino acid residues in length. In one embodiment, L1 is 1-4 amino acid residues in length, L2 is 1-4 amino acid residues in length, L3 is 2-15 amino acid residues in length, and L4 is 2-15 amino acid residues in length. In another antibody-like binding protein, L1 is 1-2 amino acid residues in length, L2 is 1-2 amino acid residues in length, L3 is 4-12 amino acid residues in length, and L4 is 2-12 amino acid residues in length. In an antibody-like binding protein, L1 is 1 amino acid residue long, L2 is 2 amino acid residues long, L3 is 7 amino acid residues long, and L4 is 5 amino acid residues long.

[0081] In some antibody-like binding proteins of the invention, L1, L3, or L4 may be equal to zero. However, in antibody-like binding proteins where L1, L3, or L4 are zero, the variable region The linker between the constant region and the corresponding transition region between dual variable domains on separate chains cannot be zero. In some embodiments, L1 is zero and L3 is two or more amino acid residues, L3 is zero and L1 is one or more amino acid residues, or L4 is zero and L2 is three or more amino acid residues.

[0082] 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 contains at least one cysteine ​​residue.

[0083] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 25); peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:27); peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:28); peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:29). Other combinations of amino acid residues may also be used, such as the peptide Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:30) and the peptide Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:31). Other suitable linkers include single Ser and Val residues; the dipeptides Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO:52), Thr-Val-Ala-Ala-Pro (SEQ ID NO:53), Gln-Pro-Lys-Ala-Ala (SEQ ID NO:54), Gln-Arg-Ile-Glu-Gly (SEQ ID NO:55); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO:48), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO:49), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO:50), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO:51). The above examples are not intended to limit the scope of the invention in any way, but linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable for antibody-like binding proteins of the invention (see Example 12).

[0084] The identity and sequence of the amino acid residues of the linker may vary depending on the type of secondary structure elements that need to be achieved in the linker. For example, glycine, serine and alanine are best for linkers with maximum flexibility. Some combinations of glycine, proline, threonine and serine are useful when a more rigid and extended linker is required. Depending on the desired properties, any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as needed.

[0085] In some antibody-like binding proteins of the invention, V L1 comprises the amino acid sequence of SEQ ID NO:1; L2 comprises the amino acid sequence of SEQ ID NO:3; H1 comprises the amino acid sequence of SEQ ID NO:2; V H2 comprises the amino acid sequence of SEQ ID NO:4.

[0086] In some embodiments of the invention, the antibody-like binding protein is capable of specifically binding to one or more antigen targets. In preferred embodiments of the invention, the antibody-like binding protein is 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 (MIP-5), CCL6 (MIP-6), CCL7 (MIP-8), CCL8 (MIP-9), CCL9 (MIP-1b), CCL10 (MIP-1c), CCL11 (MIP-1b), CCL12 (MIP-1c), CCL13 (MIP-1b), CCL14 (MIP-1c), CCL15 (MIP-1d), CCL16 (MIP-1b), 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 (MIP-1c), CCL5 ...c), CCL5 (M 1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2) , CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3(GCSF), CTLA4, CX3CL1(SCYD1), CXCL12(SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL 8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b4 integrin), LEP (leptin), MHC class II, TLR2, TLR4 , TLR5, TNF, TNF-a, TNFSF4 (OX40 ligand), TNFSF5 ( In another embodiment of the invention, the antibody-like binding protein is capable of specifically binding to at least one antigen target selected from the group consisting of CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5 / CCXCR1), DNGR-1 (CLEC91), and HMGB1. In another embodiment of the invention, the antibody-like binding protein is capable of inhibiting one or more functions of the antigen target.

[0087] In some embodiments of the invention, the antibody-like binding protein is bispecific and can bind to two different antigen targets or epitopes. In preferred embodiments of the invention, the antibody-like binding protein is bispecific and each light chain-heavy chain pair can bind to two different antigen targets or epitopes. In more preferred embodiments, the antibody-like binding protein can bind to two different antigen targets selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL-1β, TNFα and IL-23, and IL17 and IL23. In even more preferred embodiments, the antibody-like binding protein can bind to the antigen targets IL4 and IL13.

[0088] In some embodiments of the invention, the antibody-like binding protein specifically binds IL4 with an on-rate of 2.97E+07 and an off-rate of 3.0E-04, and specifically binds IL13 with an on-rate of 1.39E+06 and an off-rate of 1.63E-04. In other embodiments of the invention, the antibody-like binding protein specifically binds IL4 with an on-rate of 3.16E+07 and an off-rate of 2.89E-04, and specifically binds IL13 with an on-rate of 1.20E+06 and an off-rate of 1.12E-04.

[0089] In one embodiment of the invention, an antibody-like binding protein comprises four polypeptide chains forming four antigen-binding sites, each of which is V L and V H identifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen; assigning either a light chain or a heavy chain as a template chain; L V L1 Assign as;V L2 , VH1 , and V H2 The following formula [I] and [II]: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 -Fc [II] determining maximum and minimum lengths for L1, L2, L3, and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that, when combined, bind to a first target antigen and a second target antigen to form an antibody-like binding protein; Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region and C of an immunoglobulin H2 , C H3 is the constant domain of an immunoglobulin heavy chain; and L1, L2, L3, and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II are produced by cross-light-heavy chain pairing.

[0090] In another embodiment of the invention, an antibody-like binding protein comprises four polypeptide chains forming four antigen binding sites, each of which is V L and V Hidentifying a first antibody variable domain that binds to a first target antigen and a second antibody variable domain that binds to a second target antigen; assigning either a light chain or a heavy chain as a template chain; L V L1 Assign as;V L2 ,V H1 and V H2 The following formula [ I] and [II]: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 [II] determining maximum and minimum lengths for L1, L2, L3 and L4; creating polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I and II that, when combined, bind to a first target antigen and a second target antigen to form an antibody-like binding protein; Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; and L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II are produced by cross-light-heavy chain pairing.

[0091] In another embodiment of the invention, an antibody-like binding protein is produced in which the first antibody variable domain and the second antibody variable domain are identical.

[0092] One embodiment of the present invention is a compound represented by the following formulas [I] and [II]: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 -Fc [II] Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; Fc is the hinge region and C of an immunoglobulin H2 , C H3 is the constant domain of an immunoglobulin heavy chain; and L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light chain-heavy chain pair; The present invention provides a method for producing an antibody-like binding protein comprising expressing in a cell one or more nucleic acid molecules encoding a polypeptide having a structure represented by:

[0093] Another embodiment of the present invention is a compound represented by the following formulas [I] and [II]: V L1 -L1-V L2 -L2-C L [I] V H2 -L3-V H1 -L4-C H1 [II] Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is the C of immunoglobulin H1 a heavy chain constant domain; L1, L2, L3 and L4 are amino acid linkers; and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-light chain-heavy chain pair; The present invention provides a method for producing an antibody-like binding protein comprising expressing in a cell one or more nucleic acid molecules encoding a polypeptide having a structure represented by:

[0094] 3. Use for antibody-like binding proteins The antibody-like binding proteins of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantification of one or more target antigens. The antibody-like binding proteins become bound to one or more target antigens with an affinity appropriate for the assay method being used.

[0095] For diagnostic applications, in some embodiments, the antibody-like binding protein can be labeled with a detectable moiety. The detectable moiety can be any that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety can be: 3 H, 14 C. 32P , 35 S, 125 I, 99 Tc, 111 In or 67 It can be a radioisotope, such as Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0096] The antibody-like binding proteins of the present invention are also useful for in vivo imaging. Antibody-like binding proteins labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. The antibody-like binding proteins can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0097] The present invention also relates to kits containing the antibody-like binding proteins and other reagents useful for detecting target antigen levels in a biological sample. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.

[0098] 4. Therapeutic compositions and administration of antibody-like binding proteins Therapeutic or pharmaceutical compositions comprising antibody-like binding proteins are within the scope of the present invention. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of the antibody-like binding protein or antibody-like binding protein-drug conjugate in admixture with pharma- ceutical or physiologically acceptable formulation substances selected for compatibility with the method of administration.

[0099] Acceptable formulation materials preferably are non-toxic to recipients at the dosages and concentrations used.

[0100] Pharmaceutical compositions can include formulation materials to, for example, modify, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, quercetin, glycerol ... In some embodiments, the additives may include, but are not limited to, carboxylic acids, phosphates, phosphoric acids, or other organic acids, bulking agents (e.g., mannitol, or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colors, flavors, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt forming agents, and the like. ions (e.g., sodium, etc.), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide, etc.), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol, etc.), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronic; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxalate). Sapal), stability enhancers (e.g., sucrose or sorbitol), isotonicity enhancers (e.g., Examples of suitable pharmaceutical adjuvants include alkali metal halides, preferably sodium or potassium chloride, or mannitol, sorbitol, etc., delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. (See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990) and successive editions of the same, which are incorporated herein by reference for any purpose).

[0101] The optimal pharmaceutical composition will be determined by the skilled artisan depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody-like binding protein.

[0102] The primary vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection may be water, saline solution, or artificial cerebrospinal fluid supplemented with other substances common to compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other representative pharmaceutical compositions include Tris buffer with a pH of about 7.0-8.5, or acetate buffer with a pH of about 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present invention, the antibody-like binding protein composition may be prepared for storage by mixing the selected composition having the desired purity with any formulation substance in the form of a lyophilized cake or aqueous solution. Additionally, the antibody-like binding protein may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0103] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions can be selected for delivery via the digestive tract, such as by inhalation or orally. The preparation of such pharma- ceutically acceptable compositions is within the skill of the art.

[0104] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, usually in the pH range of about 5 to about 8.

[0105] When parenteral administration is contemplated, the therapeutic compositions used in the present invention may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution containing the antibody-like binding protein of interest in a pharma- ceutically 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, properly preserved. Yet another formulation provides for controlled or sustained release of the product which can then be delivered via depot injection. These may include formulations of the molecule of interest with substances such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. Hyaluronic acid may also be used, which may have the effect of enhancing duration in the blood circulation. Other suitable means for introducing the molecule of interest include implantable drug delivery devices.

[0106] In one embodiment, pharmaceutical compositions can be formulated for inhalation.For example, antibody-like binding protein can be formulated as dry powder for inhalation.Antibody-like binding protein inhalation solution can also be formulated with propellant for aerosol delivery.In yet another embodiment, the solution can be nebulized.

[0107] It is also contemplated that certain formulations may be administered orally. In one embodiment of the invention, antibody-like binding proteins administered in this manner may be formulated with or without carriers customarily used in compounding solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at a time when bioavailability in the gastrointestinal tract is maximized and pre-systemic degradation is minimized. Additional agents can be included to enhance absorption of the antibody-like binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0108] Another pharmaceutical composition can include an effective amount of antibody-like binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, the solution can be prepared in unit dose form. 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 lubricants such as magnesium stearate, stearic acid or talc.

[0109] Further pharmaceutical compositions of the present invention will be clear to those skilled in the art, including formulations related to antibody-like binding proteins in sustained or controlled delivery formulations. Those skilled in the art are also familiar with the techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections. Further examples of sustained release formulations include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl L-glutamic acid, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.

[0110] The pharmaceutical composition of the present invention used for in vivo administration must usually be sterile. This can be achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method can be performed either before lyophilization or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in solution. In addition, the parenteral composition is generally placed in a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0111] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dry or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

[0112] The present invention also includes kits for making single dosage units. The kits can each contain both a first container with a dry protein and a second container with an aqueous formulation. Also included within the scope of the present invention are kits that include single or multi-chambered pre-filled syringes (e.g., liquid syringes and dispersion syringes).

[0113] The effective amount of antibody-like binding protein pharmaceutical composition used for treatment will depend, for example, on the context and purpose of the treatment. Those skilled in the art will therefore understand that appropriate dosage levels for treatment will vary in part depending on the molecule being delivered, the indication for which the antibody-like binding protein is being used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and health) of the patient. Thus, the clinician can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages can range from about 0.1 μg / kg to about 100 mg / kg or more, depending on the factors mentioned above. In another embodiment, the dosage can range from 0.1 μg / kg to at most about 100 mg / kg; or from 1 μg / kg to at most about 100 mg / kg; 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg to at most about 100 mg / kg. can.

[0114] The frequency of administration will depend on the pharmacokinetic parameters of the antibody-like binding protein in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may thus be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the molecule of interest), or as a continuous infusion via an implanted device or catheter. Furthermore, fine adjustment of the dosage is routinely performed by those of ordinary skill in the art and is within the scope of their routine practice. Appropriate dosages can be ascertained using appropriate dose-response data.

[0115] The routes of administration of the pharmaceutical compositions may be, for example, oral; intravenous, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional; sustained release systems; or implanted devices. If desired, the compositions can be administered by bolus injection or continuously by infusion or implantation device.

[0116] The compositions can also be administered locally via implantation of a membrane, sponge, or other suitable material into which the molecule of interest has been absorbed or encapsulated. When an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the molecule of interest can be via diffusion, timed release bolus, or continuous administration.

[0117] 5. Working Example The examples which follow illustrate specific embodiments of the invention and various uses thereof, but are set forth for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0118] Example 1 Design and engineering of bispecific cross-domain dual variable domain antibody-like binding proteins Crossed dual variable regions in an Fv format have been described in U.S. Patent 5,989,830 and have been termed the crossed double head (CODH) configuration. Molecular modeling predicted that the crossed double head (CODH) design would result in a complex with both binding sites facing in opposite directions without the constraints suggested by the dual Fv configuration. The CODH Fv format is a novel approach that allows for the creation of complexes with both binding sites facing in opposite directions without the constraints suggested by the dual Fv configuration. L The addition of a domain to the light chain and an Fc region to the heavy chain results in a complete antibody-like molecule. A similar conversion was successfully performed with the corresponding dual variable domain (DVD-Ig) and TBTI described in U.S. Patent 7,612,181 and International Application WO 2009 / 052081. The sequence of the variable region in the CODH format is shown in the following structure, showing the amino to carboxyl orientation of the peptide chains: (a) Light chain: NH2-V L1 -Linker-V L2 -COOH (b) Heavy chain: NH2-V H2-Linker-V H1 -COOH.

[0119] The amino to carboxyl terminal sequences of the variable regions in (a) and (b) above can be distinguished from the sequences in the dual Fv configuration shown in (c) and (d) below: (c) Light chain: NH2-V L1 -Linker-V L2 -COOH (d) Heavy chain: NH2-V H1 -Linker-V H2 -COOH.

[0120] An important difference worth noting is the relative positioning of the corresponding light and heavy chain variable regions (V H1 / V L1 and V H2 / V L2 ) are different arrangements. The corresponding V L1 and V H1 Both domains were at the N-terminus of the light and heavy chains in a dual variable region configuration. In contrast, in a crossover configuration, the members of a pair of antibody variable regions are spatially separated within the protein chains in the crossover configuration. In the crossover configuration, the V L1 The domain may be at the N-terminus of the protein light chain, but may be paired with a V H1 The V domain is at the C-terminus of the heavy chain in a cross-configuration. L1 and V H1 The spatial relationship to is the sequence found in natural antibodies.

[0121] One potential disadvantage of the dual Fv configuration is the lack of a linker L separating the two variable regions. L The protrusion of the Fv2 domain into the antigen-binding site of the Fv2 domain (see Figure 1). This may interfere with binding and may result in disruption of the accessibility of Fv2 to antigen 2. This disruption or disruption of accessibility may prevent antigen binding. This disruption may also be more pronounced when the size of antigen 2 is larger. In fact, US Patent 7,612,181 describes that the binding affinity and neutralization capacity of DVD-Ig molecules depend on whether the antigen specificity is present at the N-terminus or C-terminus. See US Patent 7,612,181, Example 2.

[0122] Therefore, to create a more stable antibody-like binding protein that is less susceptible to loss of antigen affinity compared to the parent antibody, L Cross-linking domains and Fc regions on the heavy chain Dual variable region molecules have been designed and constructed. The polypeptides that form these antibody-like proteins have the structure shown below, where the amino to carboxyl terminal orientation of the polypeptide chains is indicated: (e) Light chain: NH2-V L1 -Linker-V L2 -C L -COOH (f) Heavy chain: NH2-V H2 -Linker-V H1 -C H1 -Fc-COOH.

[0123] To evaluate whether this bispecific antibody-like protein design could bind to two different antigens, two pre-generated and humanized variable regions from antibodies specific for IL4 (parent humanized anti-IL4) and IL13 (parent humanized anti-IL13) were used to construct the bispecific antibody-like molecules shown in Table 1. The sequencing and humanization process of the murine antibodies was described in International Patent Application WO2009 / 052081 (TBTI). Briefly, the amino acid sequences of the variable heavy and light chains of murine anti-IL13 clone B-B13 and murine anti-IL4 clone 8D4-8 were determined by amino acid sequencing. The murine sequences were humanized and then back-translated into nucleotide sequences as described in Example 5 of International Patent Application WO2009 / 052081, the entire contents of which are incorporated herein by reference. Parent humanized anti-IL4VH and V L , and parent humanized anti-IL13V H and V L The sequences were combined and aligned as shown in Table 1. The abbreviated codes in column 1 of Table 1 were created to simplify the review of these antibody-like binding proteins. The antibody-like binding proteins were identified as follows: The size of the linker inserted between the two variable regions varies. The DNA molecules encoding the polypeptides shown in Table 1 were generated from the back-translated parent anti-IL4 and parent anti-IL13. H1 , C L , and the Fc domain is IGHG1 (GenBank accession no. 569F4) and IGKC (GenBank accession no. Q502W4).

[0124] [Table 1]

[0125] The protein combinations shown in Table 2 were expressed by transient transfection and purified by protein A chromatography. In all cases, size exclusion chromatography showed less than 12% aggregation, with most having less than 7% aggregation; however, none of the cross-double-headed immunoglobulins showed the ability to bind either IL4 or IL13. However, no antibody-like binding could be detected, and the reason for this lack of activity could not be identified. It was previously predicted that this sequence may show superior stability over the dual variable region domain antibodies described in US Pat. No. 7,612,181 and International Application WO2009 / 052081.

[0126] [Table 2]

[0127] Example 2 Design of CODH-Ig protein by molecular modeling Fc and C L1 In order to obtain a fully functional antibody-like protein that exploits the crossed double-headed configuration amenable to domain incorporation, a molecular modeling protocol was developed for the inclusion evaluation of different linkers between the constant and variable domains and between the dual variable domains on both the heavy and light chains. The question was whether the addition of a unique linker between each of the constant / variable domain interfaces and between the two variable / variable domain interfaces on both the heavy and light chains would allow proper protein folding to occur and produce a functional antibody-like molecule in the crossed double variable region configuration (see Figure 2). In other words, four independent and unique linkers were all evaluated (see Figure 2). This molecular modeling protocol was developed to evaluate the inclusion of different linkers between the constant and variable domains and between the two variable / variable domain interfaces on both the heavy and light chains. IL4 and Fv IL13 Appropriate linking between domains and between Fv and constant or Fc domains Combined with the car, Fv IL4 and Fv IL13 Homology model and experimental model of the region The DEL was based on protein-protein docking.

[0128] The independent linkers were uniquely assigned names as follows: L1 is the N-terminal V L and C-terminal V L L2 refers to the terminal V on the light chain; L and C. L Refers to the linker between L3 is the N-terminal V on the heavy chain H and C-terminal V H L4 refers to the linker between the C-terminal V H and C. H1 (and Fc). H and V L refers only to the location of the domain on a particular protein chain in the final format. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2 domain and the V H1 and V H2Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 domain and the V H1 and V H2 Thus, V H and V L The designation refers to the current position and not the original position in the parent antibody.

[0129] More specifically, Fv IL4 The homology model is based on PDB entries 1YLD (light chain) and 1IQW (heavy chain). Fv IL4 The dimer is IL13 / anti-IL13 Fab IL13 The complex was reassembled and optimized on the internal crystal structure. Fv IL4 To obtain an estimate of the capacity required by IL4 when bound to Fv, the crystal structure of IL4 (1RCB.pdb) was IL4 The homology model was then docked to identify complexes that merit further investigation. Twenty-two putative models of coalescence were produced.

[0130] In parallel, Fv IL4 The homology model for IL13 / Fab IL13 Internal crystal structure of the complex Fv derived from the structure IL13 One good solution was found that allows the construction of a relatively short linker while not presenting steric hindrance to the arrangement of the antigen-binding and constant domains as in the case of dual variable region immunoglobulins (see FIG. 3). In this arrangement, the Fv IL4 (V L1 ) is placed at the N-terminus of the light chain, followed by Fv IL13 (V L2 ) and Fc (C L1 On the heavy chain, Fv IL13 (V H2 ) at the N-terminus, followed by Fv IL4 (VH1 ) and the constant region (C H1 -C H2 -C H3 ) was placed.

[0131] As shown in Table 3, the light chain model is L1 and V L2 Interdomain linkers L1 and V L2 and C. L1 It was suggested that the interdomain linker L2 could be from one to three and zero to two glycine residues long, respectively. H2 and V H1 Interdomain linkers L3 and V H1 and C. H1 It was suggested that the interdomain linker L4 could be between 2 and 6 and 4 to 7 glycine residues long, respectively (see Table 3 and Figure 2). In this example, glycine was used as the prototype amino acid for the linker, but other amino acid residues could also serve as linkers. The structural stability of the proposed model was verified by linker conformation optimization, minimization, and molecular dynamics calculations. Systematic combinations between the four light chain and six heavy chain constructs resulted in 24 possible crossover dual variable region bispecific anti-IL4 and anti-IL13 antibody-like binding proteins (see Table 4).

[0132] [Table 3]

[0133] [Table 4]

[0134] In Table 4, the prefix "anti-" is not included, but IL13 is intended to mean anti-IL13 and IL4 to mean anti-IL4.

[0135] Example 3: Generation of CODV-Ig expression plasmid Nucleic acid molecules encoding the six heavy and four light variable chains listed in Table 4 were produced by gene synthesis at Geneart (Regensburg, Germany). Variable Light Chain The domain was fused to the constant light chain (IGKC, GenBank accession number Q502W4) by digestion with the restriction endonucleases ApaLI and BsiWI and subsequently ligated into the ApaLI / BsiWI sites of the episomal expression vector pFF, an analog of pTT described by Durocher et al. (2002, Nucl. Acids Res. 30(2): E9), to create a mammalian expression plasmid for expression of the light chain.

[0136] The variable heavy chain domain was either the "Ted" variant of the human constant heavy chain (IGHG1, GenBank accession no. 569F4) or a 6xHis-tagged C variant from human constant IGHG1 to create a bispecific Fab. H1 domain. Next, V H The domain is The IGHG1 or IGHG2 were digested with the restriction endonucleases ApaLI and ApaI, and then expressed by ligation into the ApaLI / ApaI sites of the episomal expression vector pFF, respectively. His-tagged C H1 The heavy chains were fused to the domains to create mammalian expression plasmids for expression of the heavy chains (IgG1 or Fab, respectively).

[0137] [Example 4] Expression of CODV-Ig Expression plasmids encoding the heavy and light chains of the corresponding constructs were propagated in E. coli DH5a cells and prepared from E. coli using the EndoFree Plasmid Mega Kit from Qiagen for transfection.

[0138] HEK293-FS cells grown in Freestyle medium (Invitrogen) were transfected with the indicated LC and HC plasmids encoding the heavy and light chains shown in Table 4, using 293fectin (Invitrogen) transfection reagent as described by the manufacturer. After 7 days, cells were removed by centrifugation and the supernatant was passed through a 0.22 μm filter to remove particulates.

[0139] The CODV-IgG1 construct was purified by affinity chromatography on a Protein A column (HiTrap Protein A HP column, GE Life Sciences). After elution from the column with 100 mM acetate buffer and 100 mM NaCl, pH 3.5, the CODV-IgG1 construct was desalted using a HiPrep 26 / 10 desalting column, formulated in PBS at a concentration of 1 mg / mL, and filtered using a 0.22 μm membrane.

[0140] The bispecific CODVFab construct was purified by IMAC on a HiTrap IMAC HP column (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 a HiPrep 26 / 10 desalting column, formulated in PBS at a concentration of 1 mg / mL, and filtered using a 0.22 μm membrane.

[0141] Protein concentrations were 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.

[0142] NuncF96-MaxiSorp-Immunoplates were coated with goat anti-human IgG (Fc specificity) [NatuTecA80-104A]. The antibody was diluted to 10 μg / ml in carbonate coating buffer (50 mM sodium carbonate, pH 9.6) and dispensed at 50 μL / well. The plate was sealed with adhesive tape and stored at 4°C overnight. 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 cover the plate. After 1 h at room temperature, the plate was washed three times with wash buffer. 100 μL of samples or standards (ranging from 1500 ng / ml to 120 ng / ml) were added and left at room temperature for 1 h. 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 1 hour incubation at room temperature, the plate was washed three times with wash buffer. 100 μL of ABTS substrate (10 mg ABTS tablets (Pierce 34026) in 0.1 M Na2HPO4, 0.05 M citric acid solution, pH 5.0). Before use, 10 μL of 30% H2O2 / 10 ml substrate buffer was dispensed into each well and color was allowed to develop. After color development (approximately 10-15 minutes), 50 μL of 1% SDS solution was added to stop the reaction. The plate was then incubated at 4°C for 1 hour ... 405 was read in.

[0143] Example 5: Characterization of CODV-Ig variants To determine whether the heavy and light chains of the CODV-Ig antibody-like protein were paired and properly folded, the aggregation level was measured by analytical size-exclusion chromatography (SEC). Analytical SEC was performed using an AKMS equipped with a TSK-gel G3000SWXL column (7.8 mm x 30 cm) and a TSK-gel SWXL guard column (Tosoh Bioscience). The analysis was performed in pairs using a TA Explorer 10 (GE Healthcare). The analysis was performed at 280 nm with 250 mM NaCl, 100 mM Na-phosphate, pH 6.7. 30 μL of protein sample (at 0.5-1 mg / ml) was applied to the column. For molecular size estimation, the column was calibrated with a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed with the UNICORN software v5. This was done using .11.

[0144] Table 5 shows the results of the first set of 24 different CODV-Ig molecules made with 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 each pair of light and heavy chains when proteins were produced, the aggregation level was measured using SEC. The most successful results are shown in Table 5, where LC4 (L1=1; L2=2) was paired with all six heavy chains. LC4 has the structure IL4V with linker L1 equal to 1. L -(Gly)-IL13V L -(Gly2)-C L1 Corresponding to, Here, a single amino acid residue is used to identify the two V L The domain was separated. LC4 is a L and C-terminal C H1 containing a Gly-Gly dipeptide linker between them, It had a new L2.

[0145] [Table 5]

[0146] When CODV-Ig molecules were produced, single concentration BIACORE experiments at intermediate IL13 and IL4 concentrations were performed to verify binding to the target antigen. CODV-Ig antibody-like molecules corresponding to the LC4:HC4 and LC4:HC6 combinations listed in Table 4 were selected for evaluation of full kinetic analysis using surface plasmon resonance.

[0147] As depicted in Table 5, most CODV-Ig molecules could be produced either not at all or only slightly as aggregates (up to 90%). The heavy / light chain combinations that resulted in acceptable aggregation levels (5-10%) after one chromatographic step were IL4V L -( Gly)-IL13V L -(Gly2)-C L1 The light chain was It was the finest chain among these CODV-Ig variants and acted as a platform for accepting different heavy chains of different linker composition.

[0148] 1.Dynamic 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 the purified antibodies was performed using surface plasmon resonance on a BIACORE3000 (GE Healthcare). For capture and orientation of the captured antibodies, a capture assay using a species-specific antibody (e.g., human-Fc specific MAB1302, Chemicon) was used. Capture antibodies were immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Life Sciences) using standard procedures. Analyte antibodies were captured at a flow rate of 10 μL / min with an adjusted RU value that could result in a maximum analyte binding of 30 RU. Binding kinetics were measured for recombinant human IL4 and IL13 over a concentration range between 0 and 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 surface was regenerated with 10 mM glycine, pH 2.5. Kinetic parameters were analyzed and calculated with the BIA evaluation program package v4.1 using a flow cell without capture antibody as a reference.

[0149] Table 6 below shows a comparison of the kinetics of each domain in the CODV-Ig format (Table 4, codes LC4:HC4 and LC4:HC6) with the parental BB13 (anti-IL13) and 8D4 (anti-IL4) antibodies (expressed as IgGs). As shown in Table 6, the CODV-Ig constructs did not show a decrease in binding properties to the corresponding antigens when compared to the parental anti-IL13 and anti-IL4 antibodies. The lack of on-rate observed in the DVD-Ig / TBTI format using the same Fv sequence did not occur in the CODV-Ig configuration. The oppositely oriented binding sites may allow for binding large antigens or cross-linking different cells in a bispecific antibody-like configuration and may also be suitable for a broad range of selection of parent antibodies. An additional advantage of CODV-Ig is that the linker residues do not protrude into the antigen binding site and reduce the accessibility of the antigen.

[0150] [Table 6]

[0151] 2. Co-injection of IL4 and IL13 to demonstrate additional antigen binding by CODV-Ig To study the additive binding of both antigens, a wizard-driven co-injection method was applied, where one antigen was injected immediately followed by the other antigen (IL4 then IL13 and vice versa) after a lag time. The resulting binding levels could be compared to those achieved with 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, BIACORE experiments were performed with the CODV-Ig combination [HC4:LC4] by co-injection of both antigens in three separate analytical cycles (see Figure 4). Co-injections were performed with 3.125 nM IL4 / 25 nM IL13 (and vice versa) and with 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 800 seconds, 6 Identical binding levels of 3 RU were achieved after injection of the antigen mixture or co-injection of the antigens, regardless of the order of co-injection. When the CODV-Ig protein was saturated with the first antigen (IL4), the second antigen (IL13) was injected and a secondary binding signal was observed. This observation was reproduced when the antigen injection order was reversed, demonstrating additive and non-inhibitory binding of both antigens by CODV-Ig. Thus, the CODV-Ig construct was able to simultaneously bind both antigens (i.e., demonstrating tetravalency) while saturating all binding sites (i.e., demonstrating dual specificity).

[0152] [Example 6] Permissible range of linker length for CODV-Ig The tolerance of linkers of different lengths was evaluated by constructing CODV-Ig molecules with various combinations of linker lengths for L1, L2 on the light chain and L3 and L4 on the heavy chain. CODV-Ig constructs were generated with heavy chain linkers L3 and L4 varying between 1 and 8 residues for L3 and 0 or 1 residue for L4. The heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the C-terminal binding domain, with the C H1-Fc. The light chain linkers L1 and L2 varied from 3 to 12 residues for L1 and from 3 to 14 residues for L2. The light chain contained anti-IL13 as the N-terminal binding domain and anti-IL4 as the C-terminal binding domain. L1 continued.

[0153] 1. Characterization of CODV-Ig variants Measurement of aggregation levels was by analytical size exclusion chromatography (SEC). Analytical SEC was performed using an AKTA Explorer 10 (GE Healthcare) equipped with a TSK gel G3000SWXL column (7.8 mm x 30 cm) and a TSK gel SWXL guard column (Tosoh Bioscience). The analysis was performed in 250 mM NaCl, 1 The column was run with 0.1 mM Na-phosphate pH 6.7 and detection at 280 nm. 30 μL of protein sample (at 0.5-1 mg / ml) was applied to the column. For molecular size estimation, the column was calibrated with a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using UNICORN software v5.11.

[0154] Recombinant human IL13 and IL4 were purchased from Chemicon (USA). Recombinant human TNF-α was purchased from Sigma Aldrich (H8916-10 μg), and recombinant human IL-1β (201 Recombinant human IL-23 (1290-IL / CF), recombinant human EGFR (344ER), and recombinant human HER2 (1129-ER-50) were purchased from R&D Systems. It was.

[0155] Kinetic binding analysis by BIACORE was performed as follows. Surface plasmon resonance (SPMR) analysis on the BIACORE3000 (GE Healthcare) was used to characterize the detailed kinetics of purified antibodies. For the capture and orientation of the investigated antibodies, a capture assay using species-specific antibodies (e.g., human-Fc specific MAB1302, Chemicon) was used. For the binding kinetics of IL4 and IL13, the corresponding CODV-IgFab was captured using an anti-human Fab capture kit (GE Healthcare) as in Example 10, Table 12. The capture antibodies were immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Life Sciences) using standard procedures. Analyte antibodies were captured at a flow rate of 10 μL / min with an adjusted RU value that resulted in a maximum analyte binding of 30 RU. Binding kinetics were measured for recombinant human IL4 and IL13 over a concentration range between 0 and 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 surface was regenerated with 10 mM glycine pH 2.5. Kinetic parameters were analyzed and calculated with the BIA evaluation program package v4.1 using a flow cell without capture antibody as a reference.

[0156] The binding affinities of CODV-Ig, CODV-Fab, and TBTI to EGFR and HER2 were measured using the ProteonXPR36 Protein Interaction Array System (Biorad). Antigens were immobilized by amine-reactive coupling on a GLC sensor chip (Biorad). Dilution series of bispecific antibody variants in PBSET buffer (Biorad) were analyzed in parallel in double-reference and one-shot kinetic modes. Data were analyzed using Proteon Manager software v3.0 (Biorad) by Langmuir's 1:1 model or by mass transfer or bivalent analyte models.

[0157] Table 7 summarizes the results of yield, aggregation (measured by size exclusion chromatography), and binding affinity of CODV-Ig with different size combinations of linkers. Near zero CODV-Ig molecules could not be produced, or if protein was produced, it showed high levels of aggregation (see batch ID numbers 101, 102, 106-111, and 132-137 in Table 7). Thus, in contrast to the molecular modeling predictions from Example 2, these results indicate that V L2 - C L It is shown that the transition (or L2) requires a linker of at least one residue (see Table 7).

[0158] [Table 7]

[0159] [Table 8]

[0160] In addition, the CODV-Ig linker length was found to be more sensitive to an increase of one amino acid residue than to an increase of two amino acid residues. For example, batch ID numbers 103 and 104 differ in L2 by only one amino acid residue, whereas batch ID number 103 has 6-fold more. Batch ID number 104 shows less than 2-fold aggregation. In contrast, in L2 Batch ID numbers 104 and 105, which differ by only two residues, showed similar profiles for yield, aggregation, and binding.

[0161] [Example 7] Heavy chain as a template for CODV-Ig In Examples 1-5, the optimal short linker size on the light chain suggested that the light chain was acting as a template by remaining in a linear sequence, and that a larger linker was required for the heavy chain so that it could properly fold into a cross-over configuration to fit the template light chain (see FIG. 5, panel A). It was next assessed whether the short linker specifically placed on the heavy chain to maintain the linear sequence on the heavy chain made it the "template" strand, and whether that pattern could repeat itself, and a larger linker was required to allow the non-template strand to properly fold and thus accommodate the template heavy chain (see FIG. 5, panel B).

[0162] Figure 6 illustrates these principles of CODV-Ig design based on having either the light chain or the heavy chain as a "template." To appreciate the general nature of this concept, CODV-Ig constructs were designed with heavy chain linker L3 varying between 1 and 8 residues for L3 and 0 or 1 residue for L4. The heavy chain was generated with anti-IL4 as the N-terminal binding domain and anti-IL4 as the C-terminal binding domain. Contains anti-IL13 as an inhibitor, H1 -Fc. The light chain linkers L1 and L2 are The light chains varied from 3 to 12 residues for L1 and from 3 to 14 residues for L2. , containing anti-IL13 as an N-terminal binding domain and anti-IL4 as a C-terminal binding domain, L1 continued.

[0163] Table 8 shows the yield, aggregation ( The results of the binding affinity and the size exclusion chromatography (measured by size exclusion chromatography) are summarized, in which the heavy chain is maintained in a linear arrangement as the template strand, and the light chain is allowed to fold into a cross-arrangement. The results show that CODV-Ig molecules with L4 near zero could not be produced or tagged. It was shown that when protein was produced, there was a high level of aggregation (L2 was equal to zero). (See Batch ID Nos. 207-209, 211-212, 219-224, 231-236, 243-252, and 263-266 in Table 8). The one exception was Batch ID No. 210, which had L1 of 7, L2 of 5, L3 of 2, and L4 of zero. This sequence produced sufficient protein and had acceptable levels of aggregation and binding, suggesting that some linker size combinations may be involved in L4 under some circumstances. It was suggested that a method to compensate for the zero-length linker may be found in

[0164] [Table 9]

[0165] [Table 10]

[0166] [Table 11]

[0167] The results in Tables 7 and 8 clearly show that a linker is required between the variable and constant domains to allow optimal folding. Only in rare sequences was a linker equal to zero tolerated (see batch ID numbers 103-105 where L1(LC) is zero, and batch number 210 where L4 is zero). However, in any case the corresponding transferred linker between the variable and constant regions on the other chain cannot be zero.

[0168] The above results showed that the combinations L1=7, L2=5, L3=1, and L4=2 were good starting points for optimizing new CODV-Igs with the heavy chain as a template. The ranges in Table 9 were shown to be reasonable ranges for successfully engineering new CODV-Igs from the two parent antibodies.

[0169] [Table 12]

[0170] Example 8: General applicability of the CODV-Ig format To evaluate the suitability of the CODV-Ig format for engineering new antibody-like binding proteins, variable regions from a number of existing human and humanized antibodies with specificity for insulin-like growth factor 1 receptor (IGF1R(1)), a secondary antibody against insulin-like growth factor 1 receptor (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 format (see Table 10).

[0171] [Table 13]

[0172] Antibody variable regions from known human and humanized antibodies were used to test the general applicability of the CODV-Ig format when designing bispecific antibody-like binding proteins. Also, possible position effects on the placement of the N-terminus or C-terminus of a particular antibody variable region on either the heavy or light chain were examined. Based on the design of CODV-Ig molecules with linker compositions of L1=7, L2=5, L3=1, and L4=2, different sequences of antibodies were introduced into the CODV-Ig format.

[0173] The activity of bispecific antibodies or derivatives against IL1β and TNFα was measured by using commercially available HEK-Blue TNFα / IL1β reporter cells (InvivoGen). To measure antibody activity against TNFα and IL1β, the antibodies were cycled at different concentrations. Cytokines were preincubated for 1 h and then added to 50,000 HEKBlue TNFα / IL1β cells. Cytokine-mediated induction of SEAP was measured in culture supernatants 24 h later by the QUANTI-Blue assay (InvivoGen).

[0174] As shown in Table 11, all constructs showed good to excellent yields and sufficient levels of aggregation (see in particular batch ID numbers 301 and 302 in Table 11). The measured affinity of each antibody variable domain was within the range of published or predicted affinities. No position effects were observed when affinity was assessed. In summary, no position effects were seen with any of the antibody variable domains used or with the use of these domains on either antibody chain, as shown in the table below.

[0175] [Table 14]

[0176] Example 9: Retention of parent antibody affinity in CODV-Ig format The same antibody sequences of anti-IL4 and anti-IL13 were incorporated into either the TBTI / DVD-Ig or CODV-Ig format for direct comparison of their configurations, linker positioning, and the affinities of the resulting molecules. As shown in FIG. 7, the affinity of each parent of the antibodies was maintained in the CODV format. As shown in the upper panel of FIG. 7, when the variable regions were placed in the TBTI / DVD-Ig format, the loss of affinity of the IL4 antibody located in the internal Fv2 position was manifested as a decrease in the on-rate of the antibody binding to antigen. In contrast, there was no loss of affinity of the CODV-Ig format compared to the parent antibody. (See Figure 7, lower panel).

[0177] [Example 10] Adaptability of CODV-Ig to Fab format The ability of the CODV-Ig format to yield fragments such as Fab fragments was next evaluated. Two different variable heavy chains were fused to each other via the linker L3, The V was then extended at the C-terminus by linker L4. H The complex then contains the C-terminal end of IGHG1 (GenBank Accession No. Q569F4), which contains the five amino acid sequence DKTHT (SEQ ID NO: 60) from the hinge region. H1 The two different variable light chains are fused to the corresponding heavy chains in a crossover configuration through the linker L1. The constant kappa chain (IGKC, GenBank accession no. Q502W4).

[0178] Fab fragments were expressed by transient transfection as previously described. Seven days after transfection, cells were removed by centrifugation, 10% v / v 1M Tris-HCl, pH 8.0 was added, and the supernatant was filtered through a 0.22 μm filter to remove particles. The Fab protein was purified by HisTrap high performance column (GE Healthcare). Proteins were captured using 500 mM MgCl2 and eluted via an imidazole gradient. Protein-containing fractions were pooled and desalted using a PD-10 or Sephadex column. The concentrated and sterile filtered (0.22 μm) protein solution was adjusted to 1 mg / ml and kept at 4°C until use.

[0179] The immediate advantage was observed in that the CODV-oriented Fab-like molecules showed no tendency to aggregate and retained the affinity of the parent antibody (see Table 12). For the binding protein constructs from batch ID numbers 401-421, antibody-like proteins in which the antibody variable regions were arranged as in the CODV-Ig molecule were directly compared 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 TBTI / DVD-Ig format (404, 409, 414, and 419), and CODV-Ig without a linker (405, 410, 415, and 420). As shown in Table 12, the results of this comparison indicated that a lack of affinity was more likely when the variable regions were incorporated into the TBTI or DVD-Ig format compared to the parent antibody. In contrast, both the CODV-Ig and CODV-IgFab-like formats were able to better maintain the parental affinity. The results further confirmed that the CODV-Ig molecule requires linkers between the variable regions and between the variable and constant domains (see Table 12).

[0180] [Table 15]

[0181] [Table 16]

[0182] Example 11: Replacement of variable domains in CODV-Ig and CODV-Fab To characterize the CODV format in a T cell engagement manner, a bispecific CODV Fab binding protein (CODV-Fab) with a TCR binding site (CD3ε) and a CD19 binding site was generated and compared to a bispecific Fab derived from the TBTI / DVD-Ig format (B-Fab). To investigate the importance of the orientation of the binding sites (TCRxCD19 vs. CD19xTCR), both orientations were evaluated for each binding protein.

[0183] The binding proteins were characterized in a cytotoxicity assay using NALM-6 (CD19 expressing) cells as target cells and primary human T cells as effector cells. CD3 positive cells were freshly prepared from human PBMCs. Effector and target cells were mixed at a 10:1 ratio and incubated for 20 h with the indicated concentrations of bispecific binding proteins. Apoptotic target cells were measured in a FACS-based assay using 7-aminoactinomycin staining.

[0184] The B-Fab format in the CD3-CD19(1060) configuration was shown to be active in inducing T cell-mediated cytotoxicity on NALM-6 cells with an EC50 of 3.7 ng / ml. Similar high activity was observed for the CD19-CD3 CODV-Fab(1109) with an EC50 of 3.2 ng / ml (see Figure 8).

[0185] Conformational exchange of the B-Fab molecules (Fabs of the TBTI / DVD-Ig format) to the CD19-CD3 orientation resulted in a marked lack of activity (see FIG. 8). The exchanged B-Fab molecules showed no activity at the highest concentration for both the orientation of the CODV-IgFab and the other orientation of the B-Fab. Maximal responses were observed with the CODV-IgFab and one orientation of the B-Fab (varying between 1 and 100 ng / ml). The CD19-CD3 orientation of the B-Fab did not achieve an optimal cytotoxic response, even at the highest concentration (30 μg / ml). In sharp contrast, the change in orientation of the domain in the CODV-Fab to the CD3-CD19 (1108) resulted in a molecule with significant activity in this assay (see FIG. 8). The domain swapping in CODV-Fab also reduced the induction of T cell-mediated cytotoxicity (EC50 increase of ~100), but this effect was much less pronounced than that seen with the B-Fab format, and the molecule was still able to induce maximal levels of cytotoxicity. Data are representative and obtained from three independent experiments.

[0186] [Example 12] Effect of amino acid sequence identity on CODV-Ig linker The optimized construct corresponding to batch ID 204 (see Example 7 and Table 8) was selected to investigate the effect of linker composition on linkers L1-L4. Linker lengths were set to 7, 5, 1, and 2 residues in length for L1, L2, L3, and L4, respectively (see Table 13). The test sequences were the native antibody V H and C. H1 Interdomain or κ or λ light chain antibody Fv and C L The candidate sequence was derived from a naturally occurring linker in the transition between domains. H and C H1 Domain ASTKGPS (SEQ ID NO: 48), which arises from endothelial cell transfer, and Fv and C of the κ and λ light chains, respectively L RTVAAPS (SEQ ID NO: 49) and GQPKAAP (SEQ ID NO: No. 50). Furthermore, one construct was generated with an arbitrary linker composition to show that any sequence could potentially be used in linkers L1-L4. This linker composition was obtained by randomly distributing the amino acids valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline at 15 positions of the four linkers. Phenylalanine, tyrosine, and tryptophan as aromatic amino acids, and methionine and cysteine ​​as amino acids were intentionally excluded to avoid possible increased aggregation.

[0187] A three-dimensional model of the construct for batch ID number 204 was generated to ensure suitability or refine the selection of the linker composition. Thus, serine was selected for linker L3 because positively and negatively charged residues were found in close proximity in the three-dimensional model. The residues in L4 are compatible with the solvent exposure of these positions as suggested by the model. Similarly, no issues with the linker composition of L1 and L2 were anticipated or predicted. A three-dimensional model of the proposed choices for linker composition was constructed.

[0188] As shown in Table 12, the linker composition can have a dramatic effect on yield. The sequences derived from the lambda chain on L1 (Batch ID Nos. 505-507 vs. Batch ID Nos. 501- 503) was a more productive generator (up to 8-fold increase). In fact, the linker based on random generation also gave good yields as shown in Table 13, batch ID number 508. Thus, linker composition may be one parameter considered during CODV-Ig optimization.

[0189] [Table 17]

[0190] The activity of bispecific antibodies and derivatives against the cytokines IL4 and IL13 was measured in commercially available HEK-Blue IL-4 / IL-13 reporter cells (InvivoGen). HEK-Blue IL-4 / IL-13 cells are a reporter cell line that detects STs induced by IL-4 or IL13. The assay was designed to monitor activation of the AT6 pathway. Stimulation of cells with either cytokine results in the production of the reporter gene secreted embryonic alkaline phosatase (SEAP), which can be measured in the culture supernatant by QUANTI-Blue assay. To test antibody activity against IL4 or IL13, cytokines were preincubated with different concentrations of antibody for 1 h and then added to 50,000 HEK-Blue IL-4 / IL-13 cells. Cytokine-mediated induction of SEAP was measured in the cell culture supernatant after 24 h incubation by QUANTI-Blue assay (InvivoGen). It was determined.

[0191] [Example 13] Introduction of cysteine ​​into CODV-Ig linker Published data suggest that the stability of antibodies and antibody-derived proteins is affected by the introduction of non-native disulfide bridges (see Wozniak-Knopp et al., 2012, "Stabilization of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds," PLoS ONE 7(1): e30083). To examine whether the equivalent Fc fragment generated from a human IgG1 antibody and engineered into a CODV-Ig molecule could be stabilized by the introduction of inter- and intrachain disulfide bridges, the equivalent Fc position of CODV-Ig construct batch ID number 204 (see Example 7) was mutated to a cysteine ​​residue and the mutant protein was overproduced, purified, and characterized (see Table 14).

[0192] As shown in Table 14, each of the mutant CODV-Ig molecules containing an additional cysteine ​​residue had a melting temperature that was the same as the melting temperature of CODV-Ig construct batch ID number 204.

[0193] Also, two simultaneous cysteines were found in Brinkmann et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7538-42, on each of the variable domains, Cysteine ​​substitutions were introduced into the heavy chain at position 100 and into the heavy chain at position 44. These positions are structurally conserved within the antibody fold and therefore have been shown to tolerate cysteine ​​substitutions without disrupting the integrity of the individual domains.

[0194] As shown in Table 15, CODV and CODV-Ig constructs in which cysteine ​​residues were introduced at Kabat position 100 in the light chain and 44 in the heavy chain onto each of the variable domains had higher melting temperatures than CODV and CODV-Ig constructs in which cysteine ​​residues were not introduced at these positions (see, e.g., batch ID numbers 704 and 706 and batch ID numbers 713 and 714).

[0195] 1. Thermostability measurements of CODV and TBTI variants The melting temperatures (Tm) of CODV and TBTI variants were measured using differential scanning fluorimetry (DSF). Samples were incubated at a final concentration of 0.2 μg / μl in D-PBS buffer (Invitrogen). The solution was diluted to 100% and added to 2 μl of a 40x concentrated solution of SYPRO-Orange dye (Invitrogen) in D-PBS in a white semi-skirted 96-well plate. All measurements were performed using MyiQ2 The PCR was performed in duplicate using a real-time PCR instrument (Biorad). Tm values ​​were calculated using iQ5 software. The melting curves were derived from the negative first derivatives using the ELISA software v2.1.

[0196] Next, the effect of introducing cysteine ​​residues into the linker or directly into the variable region was examined. In this example, batch ID number 204 (from Example 7 and Table 8) was used as a model CODV-Ig binding protein, and cysteines were introduced into L1, L3, and L4 based on the three-dimensional model. A glycine was substituted in the variable region. As shown in Table 16 below, the results show how the introduction of a cysteine ​​pair can affect yield and aggregation. All of the possible mutations were modeled to ensure that the disulfide bonds formed properly and that the correct geometry was maintained in the linker and environment in the model. In any case, batch ID number 808 showed good yield and low aggregation, thus suggesting that the proper cysteine ​​bridges can be formed.

[0197] While the present invention has been described with respect to various embodiments, it is to be understood that variations and modifications may occur to those skilled in the art. It is therefore intended by the appended claims to cover all such equivalent variations that fall within the scope of the invention as claimed. In addition, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0198] Each embodiment described herein may be combined with any other embodiment or embodiments unless expressly indicated otherwise. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or features or embodiment or embodiments indicated as being preferred or advantageous unless expressly indicated otherwise.

[0199] All references cited in this application are expressly incorporated herein by reference.

[0200] [Table 18]

[0201] [Table 19]

[0202] [Table 20]

[0203] [Table 21]

Claims

1. An antibody-like binding protein comprising four polypeptide chains forming four antigen binding sites, wherein two of the polypeptide chains have the formula: V L1 -L 1 -V L2 -L 2 -C L [I] The compound has a structure represented by and the two polypeptide chains have the formula: V H2 -L 3 -V H1 -L 4 -C H1 -Fc[II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; Fc is an immunoglobulin hinge region and C H2 , C H3 an immunoglobulin heavy chain constant domain; and L 1 , L 2 , L 3 and L 4 is an amino acid linker; Here, L 2 is one or more amino acid residues in length, and L 4 is 0 or more amino acid residues in length, and (a) L 3 is 0 to 8 amino acid residues in length, and L 4 is 0 to 3 amino acid residues in length, and L 2 The length of L 4 or is at least one amino acid residue longer and not greater than 14 amino acid residues in length; or (b) L 1 is 0 to 2 amino acid residues in length, and L 2 is 1 to 4 amino acid residues in length, and L 4 The length of L 2 at least one amino acid residue longer than the length of and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

2. It is an antibody-like binding protein that contains two polypeptide chains that form two antigen-binding sites. wherein the first polypeptide chain has the formula: V L1 -L 1 -V L2 -L 2 -C L [I] The compound has a structure represented by and the second polypeptide chain has the formula: V H2 -L 3 -V H1 -L 4 -C H1 [II] The compound has a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; and L 1 , L 2 , L 3 and L 4 is an amino acid linker; Here, L 2 is one or more amino acid residues in length, and L 4 is 0 or more amino acid residues in length, and (a) L 3 is 0 to 8 amino acid residues in length, and L 4 is 0 to 3 amino acid residues in length, and L 2 The length of L 4 or is at least one amino acid residue longer and not greater than 14 amino acid residues in length; or (b) L 1 is 0 to 2 amino acid residues in length, and L 2 is 1 to 4 amino acid residues in length, and L 4 The length of L 2 at least one amino acid residue longer than the length of and wherein the first and second polypeptides form a cross-light-heavy chain pair.

3. 2. The antibody-like binding protein of claim 1, wherein L 1 and L 3 are both at least one amino acid in length; and Here, L 2 The length of L 4 When the length of the 1 The length of is L 3 or L 4 The length of L 2 When the length of the 3 The length of is L 1 The antibody-like binding protein is at least twice as long as

4. 3. The antibody-like binding protein of claim 2, wherein L 1 and L 3 are both at least one amino acid in length; and Here, L 2 The length of L 4 When the length of the 1 The length of is L 3 or L 4 The length of L 2 When the length of the 3 The length of is L 1 The antibody-like binding protein is at least twice as long as

5. 2. The antibody-like binding protein of claim 1 , Here, L 1 is the length of 0 amino acid residues, and L 2 is 1-2 amino acid residues in length, and L 3 and L 4 are each 1 to 4 amino acid residues in length, or L 3 0 amino acid residues is the length of the group, L 1 is the length of one amino acid residue, and L 2 is 1-2 amino acid residues in length, and L 4 is 1 to 4 amino acid residues in length.

6. 3. The antibody-like binding protein of claim 2, Here, L 1 is the length of 0 amino acid residues, and L 2 is 1-2 amino acid residues in length, and L 3 and L 4 are each 1 to 4 amino acid residues in length, or L 3 0 amino acid residues is the length of the group, L 1 is the length of one amino acid residue, and L 2 is 1-2 amino acid residues in length, and L 4 is 1 to 4 amino acid residues in length.

7. 7. The antibody-like binding protein of any one of claims 1 to 6, capable of specifically binding to one or more antigen targets.

8. 8. The antibody-like binding protein of any one of claims 1 to 7, wherein one or more antigen targets 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 / Effector 1 (ESC1), and / or CCL25 (CCL26). 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), CSF 3 (GCSF), CTLA4, CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2, IGF1R, IL-1, IL-12, IL13, I L15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6, IL7, IL8, IL9, IL12 / 23, IL22, IL23, IL25, IL27, IL35, I The antibody-like binding protein is selected from the group consisting of TGB4 (b4 integrin), LEP (leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNFα, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1 (GPR5 / CCXCR1), DNGR-1 (CLEC91), and HMGB1.

9. 9. The antibody-like binding protein of any one of claims 1 to 8, which is bispecific and capable of binding to two different antigen targets.

10. 10. The antibody-like binding protein of claim 9, wherein the two different antigen targets are selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and IL12 / 23, TNFα and IL-12p40, TNFα and IL-1β, TNFα and IL-23, and IL17 and IL23.

11. The antibody-like binding protein of any one of claims 1 to 10, capable of inhibiting one or more functions of an antigen target.

12. The antibody-like binding protein of any one of claims 1 to 11, wherein L 1 , L 2 , L 3 and L 4 wherein at least one of the linkers selected from the group consisting of: contains at least one cysteine ​​residue.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody-like binding protein of any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid molecule of claim 13.

15. 14. An isolated host cell comprising the nucleic acid molecule of claim 13.

16. 15. An isolated host cell comprising the expression vector of claim 14.

17. 17. The host cell according to claim 15 or 16, which is a mammalian cell or an insect cell.

18. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of the antibody-like binding protein of any one of claims 1-12.

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