EA046243B1 - ANTIBODIES WITH FUNCTIONAL DOMAINS IN THE REGION OF THE BEND BETWEEN THE VARIABLE AND CONSTANT DOMAIN - Google Patents

Description

The present invention relates to the field of engineered antibodies with an additional functional domain, for example, multispecific antibody formats. In particular, the present invention relates to engineered antibodies in which an additional functional domain is inserted into the fold region of the antibody. Accordingly, for example, a multispecific antibody format is provided in which an additional binding site (specificity) is inserted into the fold region of the antibody.

In recent years, a variety of multispecific antibody formats have emerged, based on the molecular design of classic natural antibodies, such as IgG, that specifically bind to one type of antigen. Unlike classical natural antibodies such as IgG, multispecific antibodies are capable of binding to two or more different targets, thereby offering a wide range of applications.

Classic natural antibodies are Y-shaped molecules containing four polypeptide chains: two heavy chains and two light chains (Fig. 1A). Each light chain consists of two domains, the N-terminal domain is called the variable domain or VL domain, and the C-terminal domain is called the constant domain or CL. Each heavy chain consists of four or five domains, depending on the class of antibody. The N-terminal domain of the heavy chain is called the variable or VH domain. The next domain in the direction from N to the C-terminus is called the first constant domain or CH1. The next part of the heavy chain is called the hinge region, which is usually followed by the second, third and, in some cases, fourth constant domain or CH2, CH3 and CH4, respectively. The constant region, which contains constant domains, is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The γ, α, and δ heavy chains have a constant region consisting of three tandem (arranged in a line) Ig domains and a hinge region for additional flexibility; The μ and ε heavy chains have a constant region consisting of four immunoglobulin domains.

In addition to the hinge region, the bend region also provides flexibility for antigen binding. A kink is the junction between the variable domains and constant domains in the heavy and light chains of an antibody.

Typically, the C-terminus of the variable domain (VH or VL) is directly linked to the N-terminus of the outermost N-terminal constant domains (usually CH1 or CL), and the connection between the C-terminus of the variable domain and (VH or VL) and the N-terminus of the outermost The N-terminal constant domain (usually CH1 or CL) is called the bend or bend region. The bend region allows variable domains to bend and rotate relative to constant domains. The bending region is also called the molecular ball-and-socket joint because it provides ball-and-socket motion (Lesk A.M., Chothia C. Nature. 335(6186), 1988, 188-90).

In the assembled antibody, the VL and VH domains are combined together to form the antigen binding site. In addition, the CL and CH1 domains join together to form one heavy chain linked to one light chain. Each heavy chain hinge region includes at least one and often several cysteine residues. In the assembled antibody, the cysteine residues in the heavy chains are aligned such that disulfide bonds can form between the cysteine residues in the hinge regions and covalently link the two heavy and light chain heterodimers together. Thus, fully assembled antibodies are monospecific in that they bind one type of antigen and bivalent in that they have two independent antigen binding sites.

Based on the structure of such monospecific bivalent classical natural antibodies, multispecific antibody formats have been designed (see reviews by Spiess C, Zhai Q, Carter PJ Mol Immunol. 67(2A), 2015, 95-106 and Weidle UH et al., Cancer Genomics Proteomics. 10(1), 118). In general, prior art multispecific antibody formats can be classified into five different structural groups: (i) multispecific IgG, (ii) linked IgG, (iii) multispecific antibody fragments, (iv) multispecific fusion proteins, and (v) multispecific antibody conjugates ( Spiess S, Zhai Q, Carter PJ Mol Immunol 67(2A), 2015, 95106).

Multispecific IgG antibody formats are typically monovalent for each specificity (for each antigen). Initial attempts to combine the binding specificities of two whole antibodies against different target antigens for therapeutic purposes used chemically hybridized heteroconjugate molecules (Staerz et al., Nature 314, 1985, 628-631). Bispecific antibodies have been produced from hybridomas by heterohybridoma methods (Milstein and Cuello, Nature 305, 1983, 537-540). Multispecific IgG antibody formats include CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, conventional LC lug-to-valley, lug-to-valley assembly, charged pair , exchange of Fab fragments, SEEDbody, Triomab, LUZ-Y, Fcab, to λ-body and orthogonal Fab, for example, as described by Spiess C, Zhai Q., Carter P.J. Mol Immunol. 67(2A), 2015, 95-106.

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In the provided IgG antibody formats, classic monospecific IgG is created by attaching additional antigen-binding moieties to the N- and/or C-terminus of the heavy and/or light chain. Examples of such additional antigen binding fragments include single domain antibodies (unpaired VH or VL), paired antibody variable domains, such as Fv or scFv, or engineered scaffold proteins. Included IgG antibody formats include DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H) -V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgGscFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig and Zybody, for example, as described in Spiess C, Zhai Q, Carter PJ. Mol Immunol. 2015 67(2A), 95-106. DVI-IgG (four-in-one) is an oversized IgG antibody format combined with a multispecific IgG antibody format as described above. One of the important advantages of the provided multispecific IgG formats is that they can provide simultaneous antigen binding to all variable domains and therefore provide increased specific binding capacity.

Multispecific antibody fragments typically have lost one or more constant domains compared to natural antibodies.

Multispecific antibody fragments include nanobodies; nanobody-HAS, BiTE-antibodies; diabodies (diabodies); DART; TandAb; scDiabodies; sc-Diabody-CH3; Diabody-Offi; Triple Bodies (trivalent antibodies); Miniantibodies (miniantibodies); Miniantibodies; Minibodies; TriBi minibodies; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab')2; F(ab')2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb (tetravalent antibodies); scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and intracellular antibodies, as described, for example, in the article Spiess C, Zhai Q, Carter PJ, Mol Immunol. 2015 67(2A), 95-106. Multispecific fusion proteins include Dock and Lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin, as described, for example, in Spiess C, Zhai Q, Carter PJ, Mol Immunol. 2015 67(2A), 95-106, in particular in FIG. 1 and the corresponding description, for example, pages 95-101. Multispecific antibody conjugates include IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, as described, for example, in Spiess C, Zhai Q, Carter PJ, Mol Immunol. 2015 67(2A), 95-106, in particular in FIG. 1 and the corresponding description, for example, pages 95-101.

Typically, the antibody format is selected depending on the intended application. For example, a long pharmacological half-life is desirable for many applications. Typically this is achieved by including an Fc region - whereas for other applications a short half-life (and therefore lack of an Fc region) may be advantageous. Moreover, production yield may be important, particularly since the production of multispecific IgG antibody formats by coexpression of two different light and heavy chains in a single host cell is known to be very challenging and can result in abnormal antibody bindings that are difficult to remove. In addition, many antibody formats, such as added IgG antibody formats, typically require linkers to provide sufficient flexibility to bind different antigens simultaneously, but the flexible nature of linkers may make them more prone to proteolytic degradation, which may result in a failed antibody design. stability, aggregation and increased immunogenicity. It should be noted that for certain applications, antibody stability may be more important than binding ability, or vice versa, depending on the application. Additionally, the antibody format can be selected based on specificity, i.e. certain antigen binding sites achieve better production yields, stability and/or binding values in a certain antibody format - while other specificities achieve better performance in other antibody formats (e.g. due to to the size/structure/nature of the target antigen).

Thus, there is a need for additional antibody formats that provide different properties. Therefore, the purpose of the present invention is to develop a new antibody format with additional functionality, for example, multispecific antibodies or antibodies designed to provide additional functionality, for example, reporter functionality, translocation functionality or carrier functionality. A new antibody format may optionally be created by combining with known antibody formats, for example, to produce antibodies with multiple binding sites or bispecific antibodies with additional functionality. These objects are achieved by the spirit of the present invention as set forth in the text below and in the accompanying claims.

Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols and reagents described in the present invention, since they can be replaced. It should also be noted that the terminology used in the present invention does not limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as commonly accepted by those skilled in the art.

The elements of the present invention will be described below in the present invention. These elements are listed in certain embodiments of the present invention, however, it should be noted

- 2 046243 that they can be combined in any manner and in any quantity to create additional embodiments of the present invention. The various examples and preferred embodiments of the present invention described should not be construed as limiting the scope of the present invention. The present description should be considered as supporting and covering embodiments that combine the expressly described embodiments with any number of disclosed and/or preferred elements. In addition, any permutations and combinations of all described elements in this invention are to be considered as disclosed in the description of the present invention, unless the context indicates otherwise.

In this specification and in the claims that follow, unless the context otherwise requires, the term contents and variations such as contains and containing are intended to include the specified element, integer or step, but it is possible that also some other unnamed element, integer or stage. The term consists of is a specific embodiment of the term contains, in which any other unspecified element, integer, or step is excluded. In the context of the present invention, the concept includes includes the concept consists of. Accordingly, the concept including includes the concept consisting of, for example, a composition including X may consist solely of X or may include something additional, for example, X+Y.

In the context of the description of the present invention (especially in the context of the claims), references to the singular and plural are to be construed to cover both the singular and the plural, unless otherwise stated herein or unless the context clearly conflicts. The listing of value ranges in this document is simply intended to serve as a shortcut to individually refer to each individual value that falls within the range. Unless otherwise stated herein, each individual value is included in the description as if separately stated. No language in the specification should be construed as indicating any element not stated in the specification but essential to the practice of the invention.

The concept is not substantially exclusive of the concept entirely, for example, a composition that is substantially free of Y may be a composition completely free of Y. If necessary, the concept may be largely omitted from the description of the present invention.

The concept of approximately in relation to the numerical value of x means x ± 10%.

In the context of the present invention, the terms peptide, polypeptide, protein refer to peptides, oligopeptides or proteins, including fusion proteins, respectively, comprising at least two amino acids linked to each other, preferably by a common peptide bond, or, alternatively , modified by a peptide bond, for example in the case of isosteric peptides. In particular, the concepts of peptide, polypeptide, and protein also include peptidomimetics, which mean analogues of peptides containing non-peptide structural elements that can mimic or act as antagonists in relation to the biological action(s) of the natural parent peptide. A peptidomimetic has lost classical peptide characteristics, for example, peptide bonds that can be cleaved by enzymes.

A peptide, polypeptide or protein can consist of any 20 amino acids corresponding to the genetic code. In addition, the peptide, polypeptide or protein may also further comprise amino acids other than the 20 amino acids defined by the genetic code, or they may be composed of amino acids other than the 20 amino acids defined by the genetic code. In particular, a peptide, polypeptide or protein in the context of the present invention may equally consist of amino acids modified by natural processes, for example, post-translational maturation processes, or by chemical processes that are well known to the person skilled in the art. Such modifications are described in detail in the literature. These modifications can appear anywhere in the polypeptide: in the peptide backbone, in the amino acid chain, or even at the carboxy or amino termini. In particular, the peptide or polypeptide may be branched as a result of ubiquitination or be cyclically branched or not. This type of modification may be the result of natural or artificial post-translational processes that are well known to one of ordinary skill in the art. The terms peptide, polypeptide, protein in the context of the present invention, in particular, also include modified peptides, polypeptides and proteins. For example, modifications of a peptide, polypeptide or protein may include acetylation, acylation, ADPribosylation, amidation, covalent fixation of a nucleotide or nucleotide derivative, covalent fixation of a lipid or lipid derivative, covalent fixation of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation , including PEGylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids such as arginylation or ubiquitination. Such modifications are fully described in detail in the literature (Creighton

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I.E. Proteins Structure and Molecular Properties 1993, 2nd ed., New York; Post-translational Covalent Modifications of Proteins 1983, ed. Johnson V. S., ed. Academic Press, New York; Seifter et al. Meth. Enzymol. 182, 1990, 626-646; Rattan et al. Ann NY Acad Sci, 663, 1992, 48-62). Accordingly, the terms peptide, polypeptide, protein preferably include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins and others.

Preferably, however, the protein, polypeptide or peptide is a classical peptide, polypeptide or protein, the classical peptide, polypeptide or protein typically consisting of amino acids selected from the 20 amino acids determined by the genetic code linked to each other by a common peptide bond.

In the context of the present invention, the term first polypeptide chain refers to a polypeptide that is to be associated with a second polypeptide (second polypeptide chain). In particular, the first and second polypeptide chains are linked through a disulfide bond. The first polypeptide chain may contain one, two, or three antibody heavy constant domains. In a preferred embodiment of the present invention, it contains three antibody heavy chain constant domains: CH1, CH2 and CH3 and a hinge region between CH1 and CH2. These heavy chain constant domains may be derived from an antibody that is murine, chimeric, synthetic, humanized or human, and monoclonal or polyclonal. The first polypeptide chain may include one or more variable domains, preferably antibody heavy chain variable domains. In the context of the present invention, the term second polypeptide chain refers to a polypeptide that is linked to a first polypeptide (first polypeptide chain). In particular, the first and second polypeptide chains are linked through a disulfide bond. The second polypeptide chain may include a CL constant region of an antibody light chain. The light chain constant region may be derived from an antibody that is murine, chimeric, synthetic, humanized or human, and monoclonal or polyclonal. The second polypeptide chain may contain one or more variable domains, preferably antibody light chain variable domains.

In general, an antibody is a protein that specifically binds to an antigen. Typically, an antibody has a unique structure that allows it to specifically bind to a corresponding antigen, but antibodies typically have a similar structure and, in particular, are also known as immunoglobulins (Ig). In the context of the present invention, the term antibody covers various forms of antibodies, including, but not limited to, whole antibodies, antibody fragments, in particular antigen binding fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant antibodies or mutant antibodies ) as long as the specific properties of the present invention are maintained. Although the description of the present invention, including the claims of the present invention, may in some places clearly refer to antigen binding fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of an antibody, the term antibody is intended to include all categories antibodies, namely antigen binding fragment(s), antibody fragment(s), variant(s) and antibody derivative(s).

In the context of the present invention, the terms antigen binding fragment, fragment and antibody fragment are used interchangeably to refer to any fragment of an antibody of the present invention that retains (i) the antigen binding activity of the antibody, as well as (ii) additional functionality provided by an (additional) functional domain, e.g. , binding activity mediated by a (independent) binding site. In the antigen binding fragments of the present invention, the specific properties are retained according to the present invention. Typically, examples of antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab' or F(ab') ₂ . Antibody fragments can be prepared from antibodies by methods that include enzymatic cleavage, for example with pepsin or papain, and/or cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be produced by cloning and expressing portions of the heavy or light chain sequences. In addition, the term antibody as used herein includes both antibodies and antigen-binding fragments thereof.

In the present invention, the term human antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A. and van de Winkel, 2001, 368-374). Human antibodies can also be generated in transgenic animals (eg mice) which, following immunization, can produce the full immune repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene template into such a mutant mouse germline results in the production of human antibodies in response to the antigen (see, for example, Jakobovits A. et al., Proc. Natl. Acad. Sci. USA 90, 1993, 2551- 2555; Jakobovits A. et al., Nature 362, 1993, 255-258; Bruggemann M. et al., 1993, 3340). Human antibodies can also be generated from phage display libraries (Hoogenboom H.R., Win

- 4 046243 ter G., 227, 1992, 381-388; Marks J.D. et al., 222, 1991, 581-597). Methods Cole et al. and Boerner et al. applicable for the production of human monoclonal antibodies (Cole et al., 1985, 77; and Boerner R. et al., 147, 1991, 86-95). Preferably, human monoclonal antibodies are produced using improved immortalization of EBV-B cells as described in Traggiai E. et al., Nat Med, 10(8), 2004, 871-875. The term human antibody in the context of the present invention also refers to antibodies that are modified to produce properties consistent with the present invention.

Antibodies of the present invention can be obtained in purified form. Accordingly, the antibody of the present invention or an antigen binding fragment thereof may be a purified antibody or antigen binding fragment. Typically, the antibody may be presented in a composition that is substantially free of other polypeptides, for example, where less than 90% (by weight), typically less than 60%, and more typically less than 50% of the composition consists of other polypeptides.

In the context of the present invention, the term variable domain (or variable region, variable light chain domain (VL), variable domain heavy chain (VH)) refers to the domain of an antibody or antigen binding fragment thereof, which is the N-terminal domain in classical natural antibodies, typically the domain that provides the greatest variability in classical natural antibodies and is directly involved in the binding of the antibody to the antigen. Typically, the human variable light and heavy chain domains have the same general structure, and each domain contains framework regions (FRs) whose sequences are widely conserved (specifically four framework regions (FRs)) and three hypervariable or complementarity determining regions CDR regions (in particular, the three hypervariable regions'/CDRs). Framework regions typically adopt a β-sheet conformation, and CDRs can form loops connecting the β-sheet structure. CDRs on each strand are typically held in their three-dimensional structure by framework regions and combine with CDRs from another strand of the antigen binding site.

In the context of the present invention, the term hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains complementarity determining regions (CDRs). Framework regions (FR) are those regions of variable domains that are not hypervariable region residues as defined herein. The CDR and FR regions can be defined in accordance with the standard definition in Kabat et al., 1991. Typically, particularly in native monospecific IgG antibodies, the three CDRs (CDR1, CDR2 and CDR3) are located inconsistently in the variable domain. In other words, the CDRs on the heavy and/or light chain may be separated, for example, by framework regions, the framework region (FR) being a region in the variable domain that is less variable than the CDRs. For example, in an antibody, the variable domain (or each variable domain, respectively) may preferably comprise four framework regions separated by three CDRs. In particular, the variable domain of an antibody (variable domain VH or VL of the light or heavy chain) contains, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs on each chain are separated by the amino acids of such a framework region. Typically, the three CDRs of the heavy chain and the three CDRs of the attached light chain together form an antigen binding site (active site or paratope). In other words, since, in particular in native monospecific IgG antibodies, the antigen binding sites typically consist of two variable domains, there are six CDRs for each antigen binding site (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3 ). One antibody, in particular one native monospecific IgG antibody, typically has two (identical) antigen binding sites and therefore contains twelve CDRs (ie 2 x six CDRs).

Due to multispecificity, that is, different antigen binding sites, the heavy chain and/or light chain of multispecific antibodies, or antigen binding fragments thereof, may (each) include more than three CDRs, in particular, more than three different CDRs. For example, a multispecific antibody or antigen binding fragments thereof may comprise at least two different variable domains, each of the at least two different variable domains being derived from a different monospecific antibody, for example of the IgG type. Since such a monospecific antibody typically contains three CDRs in the heavy chain and three CDRs in the light chain forming an antigen binding site, the multispecific antibody of the present invention may particularly contain three CDRs of the heavy chain of the first antibody and three CDRs of the light chain of the first antibody, three CDRs the heavy chain of the second antibody and three CDRs of the light chain of the second antibody, optionally three CDRs of the heavy chain of the third antibody and three CDRs of the light chain of the third antibody, etc. Thus, the number of CDRs consisting of the heavy chain and/or light chain of a multispecific antibody is preferably a multiple of three, such as three, six, nine, twelve, etc. Thus, it is also preferable that the sum of CDRs consisting of both the heavy chain , and from the light chain of a multispecific antibody, was a multiple of six, for example six, twelve, eighteen, etc. Since the antigen binding site is usually characterized by a CDR, that is, CDRH1, CDRH2, and CDRH3, as well as CDRL1,

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CDRL2 and CDRL3, in the multispecific antibodies of the present invention, it is preferred that the CDRs are arranged such that the order (e.g., CDRH1, CDRH2 and CDRH3 and/or CDRL1, CDRL2 and CDRL3 derived from the same monospecific antibody) is maintained to preserve the site antigen binding, that is, to maintain the ability to specifically bind to a specific site in the antigen. This means that, for example, the order of CDRH1, CDRH2 and CDRH3 derived from the first monospecific antibody in the amino acid stretch is preferably not interrupted by any CDR derived from the second monospecific antibody. Importantly, if the multispecific antibody contains antigen binding sites derived from at least two different monospecific antibodies, the CDRs or variable domains of such monospecific antibodies are located in the multispecific antibody of the present invention such that the antigen receptor of each monospecific antibody from which the CDRs are derived ( or variable regions), is preserved, i.e. its ability to specifically bind to a specific site in the antigen is retained.

In the context of the present invention, the variable domain may be any variable domain (in particular, VH and/or VL) of a natural antibody, or the variable domain may be a modified/engineered variable domain. Modified/engineered variable domains are known in the art. Typically, variable domains are modified/engineered to delete or add one or more functions, for example by modification at the germline gene level (removal of somatic mutations) or by humanization.

In the context of the present invention, the term constant domains refers to antibody domains that are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions. Typically the heavy chain includes three or four constant domains, depending on the immunoglobulin class: CH1, CH2, CH3 and optionally CH4 (in the N- to C-terminal direction). Accordingly, the heavy chain constant region is usually formed as follows (in the direction from N- to C-terminus): CH1 - hinge (flexible polypeptide containing amino acids between the first and second heavy chain constant domains) - CH2 - CH3 (- CH4). The light chain usually contains only one constant domain, called CL, which usually also forms the light chain constant region. In the context of the present invention, the constant domain may be any constant domain (in particular, CL, CH1, CH2, CH3 and/or CH4) of a natural antibody, or the constant domain may be a modified/engineered constant domain.

Modified/designed constant domains are known in the art. Typically, constant domains are modified/designed to remove or add one or more functions, for example in the context of Fc region functionality. Depending on the amino acid sequence of the constant region of the heavy chains, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses, for example, IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called α, ε, γ and μ, respectively. Antibodies of the present invention are preferably of the IgG type.

In general, antibodies can be of any isotype (eg, IgA, IgG, IgM, i.e., α, γ, or μ heavy chain), but IgG is preferred. Within the IgG isotype, antibodies may be of the IgG1, IgG2, IgG3, or IgG4 subclasses, with IgG1 being preferred. Antibodies may have a k or λ light chain.

As used herein, the term recombinant antibody includes all antibodies that do not occur naturally, such as antibodies expressed using a recombinant expression vector (eg, providing one or more constant domains) transfected into a host cell.

The term multispecific in the context of the present invention in relation to an antibody or an antigen-binding fragment thereof means the ability of an antibody or an antigen-binding fragment thereof to bind to at least two different epitopes, for example, on different antigens or on the same antigen. Thus, the terms bispecific, trispecific, tetraspecific, etc. refer to the number of different epitopes to which an antibody can bind. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and thus can only bind to the same epitopes (but not to different epitopes). In contrast, a multispecific antibody has at least two different types of paratopes/binding sites and can thus bind to at least two different epitopes. In the present invention, the term paratope refers to the antigen binding site of an antibody. Moreover, one specificity can refer to one, two, three or more identical paratopes in a single antibody (the actual number of paratopes/binding sites in one individual antibody molecule is called valence). For example, one native IgG antibody is

- 6 046243 is monospecific and divalent, since it has two identical paratopes. Accordingly, a multispecific antibody contains at least two (different) paratopes/binding sites. Thus, the concept of multispecific antibodies refers to antibodies that have more than one paratope and are capable of binding to two or more different epitopes. The term multispecific antibodies includes, in particular, bispecific antibodies such as those described above, and also typically a protein, such as an antibody, a scaffold molecule that binds, in particular, to three or more different epitopes, that is, antibodies with three or more paratopes/binding sites .

In particular, a multispecific antibody or antigen-binding fragment thereof may contain two or more paratopes/binding sites, and some of the paratopes/binding sites may be identical, such that all paratopes/binding sites of the antibody belong to at least two different types of paratopes /binding sites and, therefore, the antibody has at least two specificities. For example, a multispecific antibody or antigen-binding fragment thereof may contain four paratopes/binding sites, with each two paratopes/binding sites being identical (i.e., having the same specificity), and thus the antibody or fragment thereof being bispecific and tetravalent (two identical paratope/binding site for each of the two specificities). Thus, one specificity refers in particular to one or more paratopes/binding sites exhibiting the same specificity (which generally means that such one or more paratopes/binding sites are identical), and thus two specificities can be realized by two , three, four, five, six or more paratopes/binding sites, provided they relate to only two specificities. In another embodiment of the present invention, the multispecific antibody may contain a single paratope/binding site for each (at least two) specificity, that is, the multispecific antibody contains a total of at least two paratope/binding sites. For example, a bispecific antibody contains a single paratope/binding site for each of two specificities, that is, the antibody contains a total of two paratope/binding sites. It is also preferred that the antibody contains two (identical) paratopes/binding sites for each of the two specificities, that is, the antibody contains a total of four paratopes/binding sites. Preferably, the antibody contains three (identical) paratopes/binding sites for each of the two specificities, that is, the antibody contains a total of six paratopes/binding sites.

In the context of the present invention, the term antigen refers to any structural substance that serves as a target for receptors of the adaptive immune response, in particular as a target for antibodies, T cell receptors and/or B cell receptors. An epitope, also called an antigenic determinant, is a part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T-cell receptors and/or B-cell receptors. Thus, one antigen contains at least one epitope, that is, one antigen contains one or more epitopes. An antigen may be (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or toxin. Thus, an antigen may be a peptide, protein, polysaccharide, lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a small molecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid) or any combination thereof. Preferably, the antigen is selected from (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide and (v) a glycolipid; more preferably, the antigen is a peptide, polypeptide or protein.

In the context of the present invention, the term antigen binding site means a portion of an antibody that includes a region that specifically binds and is complementary to part or all of an antigen. If the antigen is large, the antibody can only bind to a specific part of the antigen, called an epitope. Typically, two variable domains, particularly the VH heavy chain variable domain and the VL light chain variable domain, combine to form a single antigen binding site. Specifically, the antigen binding site is formed by three heavy chain variable domain CDRs and three light chain variable domain CDRs, that is, six CDRs as described above.

The concept of specifically binding and similar concepts do not imply non-specific adhesion.

The term linker (or spacer) in the present invention refers to a peptide adapted to connect different domains of a polypeptide or protein, for example, an antibody or an antigen-binding fragment thereof. Linkers are known in the art and are described in detail, for example, Reddy Chichili V.P., Kumar V. and Sivaraman J., 2013). Typically, linkers are designed in such a way that they do not affect functionality. In particular, the linker does not bind specifically to the target. The linker may contain any amino acids, and the amino acids glycine (G) and serine (S) may be

- 7 046243 preferred. Preferably, the linker consists of the amino acids glycine (G) and serine (S) (GSlinker). If two or more linkers are included in a single polypeptide or protein, the linkers may be the same or different from each other. In addition, the linker may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

In the context of the present invention, the term nucleic acid molecule or nucleic acid means DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded.

In the context of the present invention, the terms cell, cell line and cell culture are used interchangeably, and all such designations mean progeny. Thus, the words transformants and transformed cells include the primary subject cell and the cultures derived from it, without regard to the number of subcultures. Obviously, all offspring may not be completely identical in DNA due to intentional or accidental mutations. Included are progeny variants that have the same function or biological activity that were selected in the originally transformed cell. Where different designations are implied, this will be clear from the context.

In the context of the present invention, the term sequence variant (or simply variant) means any change to a specific sequence such that the changed sequence is any of the sequences listed in Sequence Tables and SEQ ID Numbers (Sequence Listings), i.e. sequences from SEQ ID NO: 1 to SEQ ID NO: 133. Thus, the term sequence variant includes nucleotide sequence variants and amino acid sequence variants. It should be noted that the sequence variants of the present invention are, in particular, functional sequence variants, that is, sequence variants that support a biological function, such as an antibody sequence. Preferred supported biological functions in the context of the present invention include (i) binding of the antibody to its antigens and (ii) the function provided by the (additional) functional domain, for example, binding of the (independent) binding site to its target.

Thus, preferred sequence variants are functional sequence variants having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, with at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the comparison sequence. In the context of the present invention, the phrase a functional sequence variant having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92 %, at least 95%, with at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the compared functional sequence means that the functional sequence variant has at least 70% sequence identity, preferably at least 75% sequence identity, preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 88% sequence identity , even more preferably at least 90% sequence identity, even more preferably at least 92% sequence identity, and even more preferably at least 95% sequence identity, even more preferably at least 96 % sequence identity, particularly preferably at least 97% sequence identity, particularly preferably at least 98% sequence identity, and most preferably at least 99% sequence identity to the corresponding comparison functional sequence.

The term sequence variant includes, in particular, those variants that contain mutations and/or substitutions compared to the compared sequence. Fc fragment sequence variants include, but are not limited to, those sequences that have an L to A substitution at the CH2 4 position, CH 2 5 position, or both.

Sequence identity is usually calculated relative to the total length of the sequence being compared (ie the sequence specified in the invention). In the present invention, the percent sequence identity can be determined, for example, using BLAST using default NCBI parameters (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; penalty for introducing a deletion into an alignment = 11 and penalty for continuing a deletion = 1].

In the context of the present invention, the term nucleotide sequence variant means a variant of an altered sequence in which one or more nucleotides relative to the compared sequence are deleted or replaced, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides in

- 8 046243 in the present invention is designated by the generally accepted one-letter designation (A, C, G or T). Due to the degeneracy of the genetic code, a variant of a nucleotide sequence can either lead to a change in the corresponding reference amino acid sequence, that is, a variant amino acid sequence, or not. Preferred sequence variants are those nucleotide sequence variants that do not result in amino acid sequence variants (silent mutations), but other mutations that are not silent mutations are also contemplated by the present invention, in particular mutant nucleotide sequences that correspond to amino acid sequences having identity with the compared sequence is at least 80%, preferably at least 90%, more preferably at least 95%.

The term amino acid sequence variant has an altered sequence in which one or more amino acids in the compared sequence are deleted or replaced, or one or more amino acids are inserted into the sequence of the compared amino acid sequence. As a result of the changes, the amino acid sequence variant is at least 80% identical to the compared sequence, preferably at least 90% identical, more preferably at least 95% identical, most preferably at least 99% identical amino acid sequence is identical to the compared sequence. Sequence variants that are at least 90% identical contain no more than 10 changes, i.e. any combination of deletions, insertions or substitutions per 100 amino acids of the compared sequence.

While non-conservative amino acid substitutions are acceptable, they are preferably conservative amino acid substitutions in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the sequence being compared. Examples are conservative amino acid substitutions, which represent the substitution of one aliphatic or hydrophobic amino acid, for example, alanine, valine, leucine or isoleucine, with another hydrophobic amino acid; substitution of one hydroxyl-containing amino acid, for example, serine and threonine, with another hydroxyl-containing amino acid; replacing one acid residue, for example, glutamic acid or aspartic acid, with another acid residue; replacing one amide-containing residue, for example, asparagine and glutamine, with another amide-containing residue; replacing one aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue; substitution of one basic residue, such as lysine, arginine and histidine, with another basic residue; and replacing one low molecular weight amino acid, such as alanine, serine, threonine, methionine or glycine, with another low molecular weight amino acid.

Amino acid sequence insertions include amino- and/or carboxy-terminal hybrids, varying in length from a single residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of one or many amino acid residues. Examples of terminal insertions include hybrids from the N- or C-terminus of the amino acid sequence of a reporter molecule or enzyme.

It is important to note that changes in sequence variants do not negate the functionality of the corresponding control sequence, in this case, for example, the functionality of the sequence of an antibody or antigen-binding fragment thereof to bind to its antigens, and/or additional functionality provided by a functional domain, e.g. binding to the target (independent) binding site. Approaches to identifying nucleotides and amino acid residues that can be substituted, inserted or deleted without affecting such functionality rely on the use of computer programs well known in the art.

In the context of the present invention, a nucleic acid sequence or an amino acid sequence derived from the designated nucleic acid, peptide, polypeptide or protein refers to the origin of the nucleic acid, peptide, polypeptide or protein. Preferably, the nucleic acid sequence or amino acid sequence that is derived from a particular sequence has an amino acid sequence that is substantially identical to the sequence or portion thereof from which it is derived and, while substantially identical, includes sequence variants as described above. Preferably, the nucleic acid sequence or amino acid sequence that is derived from a particular peptide or protein is derived from a corresponding domain in the particular peptide or protein. Thus, the concept corresponding refers in particular to the same functionality. For example, the extracellular domain corresponds to another extracellular domain (of another protein), or the transmembrane domain corresponds to another transmembrane domain (of another protein). The corresponding parts of the peptides, proteins and nucleic acids can be easily determined by one skilled in the art. Likewise, sequences derived from another sequence are usually easily identified by those skilled in the art because they are derived from that other sequence.

- 9 046243

Preferably, the nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the original nucleic acid, peptide, polypeptide or protein (of which it is a derivative). However, a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the original nucleic acid, peptide, polypeptide or protein (of which it is a derivative), in particular the nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a variant of the functional sequence, as described above, of the parent nucleic acid, peptide, polypeptide or protein (of which it is a derivative). For example, in a peptide/protein, one or more amino acid residues may be replaced by other amino acid residues, or one or more amino acid residues may be inserted or deleted.

In the context of the present invention, the term mutation refers to a change in the nucleic acid sequence and/or amino acid sequence compared to a reference sequence, for example, a corresponding genomic sequence. A mutation, for example, compared to a genomic sequence can be, for example, a (natural) somatic mutation, a spontaneous mutation, an induced mutation, for example caused by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology techniques for introducing specific and targeted changes in the nucleic acid sequence and/or amino acid sequence). Thus, it should also be taken into account that the concepts of mutation or mutation are associated with the production of mutations by a physical method, for example, in a nucleic acid sequence or in an amino acid sequence. Mutations include substitutions, deletions or insertions of one or more nucleotides or amino acids, as well as inversions of several (two or more) consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, it is preferred that a mutation may be made in a nucleotide sequence encoding said amino acid sequence to express the (recombinant) mutated polypeptide. The mutation can be achieved, for example, by changing, by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to produce a codon encoding another amino acid, or by synthesizing a sequence variant, for example, knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and creating the synthesis of a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide variant without the need to mutate one or more nucleotides of the nucleic acid molecule.

In the context of the present invention, the concept of disease as a whole is synonymous and is used interchangeably with the concepts of disorder and condition (state of health), since they all reflect a deviation from the norm in the state of the human or animal body or one of its parts, which disrupts the normal functioning, usually manifested in a difference signs and symptoms and leads to a decrease in the length or quality of life of a person or animal.

Several documents are cited in the text of the present invention. The substance of each of them (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions and other documents) mentioned above or below in the text is incorporated herein in their entirety by reference to their substance. Nothing herein should be construed as an admission that the invention is not entitled to precede such disclosure by virtue of a prior invention.

It should be noted that the present invention is not limited to the specific methodology, protocols and reagents specified in the present description, as they may vary. It should also be understood that the terminology used in the present invention is intended only to describe specific embodiments of the present invention and is not intended to limit its scope beyond the scope of the appended claims. Unless otherwise specified, all technical and scientific terms used in this description have the meaning known to those skilled in the art.

Antibodies and antigen-binding fragments with functional domains in the bend region

A first aspect of the present invention provides an antibody, or an antigen binding fragment thereof, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes, in an N- to C-terminal direction, (i) one or more variable domains, (ii) (optional) a functional domain, and (iii) one or more constant domains, the second polypeptide chain including, in an N- to C-terminal direction (iv) one or more variable domains forming antigen binding sites, with one or more variable domains (i) first polypeptide chain

- 10 046243 (v) an optional (additional) functional domain, and (vi) one or more constant domains, characterized in that the (additional) functional domain of (ii) the first polypeptide chain does not include a fragment of the second polypeptide chain and the optional (additional) functional domain (v) of the second polypeptide chain does not include a fragment of the first polypeptide chain.

The present invention is based on the surprising discovery that an (additional) functional domain can be inserted into the fold region of an antibody, that is, between the variable domain and the N-most constant domain, in particular CH1 or CL. In fig. 1A shows a classic natural antibody with the bend region indicated by arrows. The present inventors have unexpectedly discovered that antibodies modified in the fold region to impart such an additional functional domain can, for example, simultaneously bind (1) the antigen targeted by their variable domains and (2) the additional target targeted a binding site introduced into the fold region of the antibody, as shown in the examples herein. This is in contrast to prior art in which multispecific antibody formats are provided with additional binding sites either by replacing the heavy/light chain pair with another heavy/light chain pair with distinct specificity (as in the multispecific IgG antibody formats described above, which are often monovalent for each specificity) or by attaching additional binding sites to the N- or C-termini of the heavy or light chains (as in the IgG antibody formats described above), in the antibodies of the present invention an additional functional domain, for example an additional binding site (specificity) is inserted into the heavy and/or light chain, namely in the bend area. Surprisingly, it has been found that the N-terminus of a variable domain (eg, VH) of an inserted functional domain, such as a binding site, still forms a fully functional antigen binding site with the corresponding variable domain of another polypeptide chain (eg, VL) - even if the other polypeptide chain the chain is unmodified in the bend area. The same applies to multispecific antibodies: despite the built-in functional domain, the N-terminus of the variable domain(s) of the built-in functional domain still associates with corresponding variable domains in another polypeptide chain to form functional antigen binding sites (e.g. VH1-VH2 in one polypeptide chain with VL1 - VL2 in another polypeptide chain, etc.).

Thus, antibodies and antigen-binding fragments thereof according to the present invention are referred to as In-Elbow-Insert (IEI-Ig) Ig molecules. The significance of the results obtained by the authors of the present invention lies in the fact that it allows the production of many different new antibody formats, such as those shown in FIG. 1. IEIIg antibodies and antigen binding fragments thereof according to the present invention can either be based on classical natural antibodies, as shown in FIG. 1B, or they can be combined with other (multispecific) antibody formats to produce new antibody formats with even greater specificity, as illustrated in FIG. 1B.

For example, to prepare an antibody of the present invention, a classical monospecific antibody, preferably a human monoclonal antibody, can serve as the backbone of the antibody, and an additional functional domain, such as a binding site, is inserted into the fold region (i.e., between the variable and the extreme N- terminal constant domain, in particular the C-terminus of the variable domain and the N-terminus of CH1 and/or CL, respectively) of the heavy chain and/or light chain of the framework antibody. In other examples, classical monospecific antibody fragments, preferably human monoclonal antibody fragments, which contain (i) at least two polypeptide chains, such as a heavy and a light chain, and (ii) in each polypeptide chain, at least a variable domain and a constant domain domain may serve as a framework antibody, and a functional domain, such as a binding site, is inserted into a fold region of the first and/or second polypeptide chain of the framework antibody fragment. Preferred examples of such antibody fragments that can be used as a scaffold for preparing an antibody of the present invention by inserting a functional domain, such as a binding site, into a fold region include Fab, Fab' and F(ab') ₂ . Moreover, any recombinant antibody or fragment thereof, for example a multispecific, in particular bispecific, antibody or fragment thereof, which contains (i) at least two polypeptide chains, such as a heavy and a light chain, and (ii) in each polypeptide chains, at least a variable domain and a constant domain, may also serve as a framework antibody, and a functional domain, such as a binding site, is inserted into the fold region of the first and/or second polypeptide chain of the recombinant framework antibody (fragment).

Examples of antibodies that can act as scaffold antibodies include antibodies GCE536 (VH: SEQ ID NO: 1, VL: SEQ ID NO: 2, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4; Piccoli L. et al., Nature communications 6, 7375 (2015)); C1 (see Example 1; VH: SEQ ID NO: 5, VL: SEQ ID NO: 6, heavy chain constant region: SEQ ID NO: 3,

- 11 046243 light chain constant region: SEQ ID NO: 7); C1b (VH: SEQ ID NO: 8, VL: SEQ ID NO: 9, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) and FI174 (VH: SEQ ID NO: 10 , VL: SEQ ID NO: 11, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4; Pappas L. et al., Nature 516, 418-422 (2014)).

An antibody of the present invention or an antigen-binding fragment thereof can also be produced by cloning methods, for example, commercially available IgG vectors or sets of vectors that include constant domains/regions, for example, heavy and/or light chain. For example, the corresponding variable domains, for example, the variable domain of the heavy and light chains VH and VL, of the first antibody can be cloned into the corresponding polypeptide chains, which is provided by the vector; and, in addition, the additional functional domain can be cloned into the first and/or second polypeptide chain such that it is located between the (most C-terminal) variable domain and the (most N-terminal) constant domain, that is, in the fold region.

The first and/or second polypeptide chain of an antibody or antigen-binding fragment thereof according to the present invention contains an (additional) functional domain. In particular, a functional domain is a peptide or polypeptide that is linked to adjacent domains of an antibody polypeptide chain or antigen binding fragment by a (conventional) peptide bond. Specifically, the N-terminus of the (additional) functional domain is linked to the C-terminus (extreme C-terminus) of the variable domain, and the C-terminus of the (additional) functional domain is linked to the N-terminus (extreme N-terminus) of the constant domain by a peptide bond.

Typically, the term functional domain refers to a functional unit, for example, an antibody or an antigen-binding fragment. Typically, a functional domain provides a protein, such as an antibody or antigen-binding fragment, with (additional) functionality. Accordingly, the (additional) functional domain typically contains all the amino acids/sequences necessary to provide the (additional) function. In particular, the (optional) functional domain (ii) of the first polypeptide chain does not contain a fragment of the second polypeptide chain, and the optional (optional) functional domain (v) of the second polypeptide chain does not contain a fragment of the first polypeptide chain. That is, the second polypeptide chain (in particular, any fragment of it, for example, even one amino acid) is not required and does not participate in the (functionality of) the (additional) functional domain of the first polypeptide chain. In addition, if the second polypeptide chain also contains an (additional) functional domain, the first polypeptide chain (in particular, any fragment thereof, for example, even a single amino acid) is not required and does not participate in the (functionality) of the (additional) functional domain of the second polypeptide chain chains. All amino acids (amino acid sequences) required or involved in participating in the functionality of the (additional) functional domain of the first and/or second polypeptide chain are contained in the (additional) functional domain of that polypeptide chain itself - fragments or amino acids of another polypeptide chain are not required/involved . Accordingly, the (additional) functional domain is different from, for example, the antigen binding site formed by the variable domain of the first polypeptide chain together with the variable domain of the second polypeptide chain. That is, the (additional) functional domain is different from, for example, an antigen-binding site formed by the variable domains of two different polypeptide chains (however, the (additional) functional domain may still contain an antigen-binding site - if the included variable domains are located on the same polypeptide chain, as described in more detail below).

Particularly preferably, the (additional) functional domain of the first and/or second polypeptide chain contains or consists of an Ig-like domain; for example, the Ig-like domain of a protein or (poly)peptide, such as in the example below. The basic structure of immunoglobulin (Ig) molecules is a tetramer of two light chains and two heavy chains linked by disulfide bonds. There are two types of light chains: kappa and lambda, each of which consists of a constant domain (CL) and a variable domain (VL). There are five types of heavy chains: alpha, delta, epsilon, gamma and mu, all of which consist of a variable domain (VH) and three (in alpha, delta and gamma) or four (in epsilon and mu) constant domains (CH1 to CH4 ). Ig molecules are highly modular proteins in which the variable and constant domains have distinct fragments of conserved sequences. Domains in Ig and Ig-like molecules are grouped into four types: V-set (variable), C1-set (constant-1), C2-set (constant-2) and I-set (intermediate). Structural studies have shown that these domains share a basic Greek key beta sandwich structure, with different types differing in the number of strands in the beta sheets as well as in sequence. Immunoglobulin-like domains, which are related in both sequence and structure, can be found in several different protein families. Ig-like domains are involved in a variety of functions, including cell-to-cell recognition, cell surface receptors, muscle structure, and the immune system.

Preferred examples of Ig-like domains include the Ig-like domains of any of

- 12 046243 of the following proteins or (poly)peptides: A1BG (alpha-1B glycoprotein), ACAM, ADAMTSL1 (ADAMTS-like 1), ADAMTSL3 (ADAMTS-like 3), AGER (advanced glycosylation end products specific receptor), ALCAM (activated leukocyte cell adhesion molecule), ALPK3 (alpha kinase 3), AMIG01 (adhesion molecule with Ig-like domain 1), AMIGO2 (adhesion molecule with Ig-like domain 2), AMIGO3 (adhesion molecule with Ig-like domain 2), Ig-like domain 3), AXL (AXL receptor tyrosine kinase), BCAM (basal cell adhesion molecule (Lutheran blood group)), BOC (BOC associated cell adhesion regulated by oncogene), BSG (basijin (Ok blood group)), BTLA ( associated with B and T lymphocytes), C10orf72, C20orf102, CADM1 (cell adhesion molecule 1), CADM3 (cell adhesion molecule 3), CADM4 (cell adhesion molecule 4), CCDC141 (coil-coil domain containing 141), CD2, CD3, CD4 , CD8, CD19, CD22, CD33, CD47, CD48, CD80, CD84, CD86, CD96, CD101, CD160, CD200, CD244, CD276, CDON (associated cell adhesion regulated by oncogene), CEACAM1 (carcinoembryonic associated cell adhesion molecule antigen 1), CEACAM5 (cell adhesion molecule associated with carcinoembryonic antigen 5), CEACAM6 (cell adhesion molecule associated with carcinoembryonic antigen 6), CEACAM7 (cell adhesion molecule associated with carcinoembryonic antigen 7), CEACAM8 (cell adhesion molecule associated with with carcinembreal antigen 8), CEASM16 (cellular adhesion molecule associated with carcinoembreal antigen 16), CEASM18 (cellular adhesion molecule associated with carcinoembreal antigen 18), CEASEM20 (cellular adhesion molecule associated with carcinoembrilic antigen 20), Sea, sees Sam21 (molecule of cellular adhesion , associated with carcinoembryonic antigen 21), CH (N-like cell adhesion molecule), CILP (cartilage intermediate layer protein), CILP2 (cartilage intermediate layer protein 2), CLMP (CXADR-like membrane protein), CNTFR ((ciliary neurotrophic receptor factor), CNTN1 (contactin 1), CNTN2 (contactin 2), CNTN3 (contactin 3, CNTN4 (contactin 4), CNTN5 (contactin 5), CNTN6 (contactin 6), CSF1R (colony stimulating factor receptor 1), CXADR (CXADR, Ig-like cell adhesion molecule), DSCAM (DS cell adhesion molecule), DSCAML1 (DS cell adhesion molecule like 1 - molecule similar to DS cell adhesion molecule, 1), EMB (embigin), ESAM (endothelial cell adhesion molecule), FUR (F11 receptor), FAIM3, FCMR (Fc fragment of IgM receptor), HMCN1 (hemicentin 1), HMCN2 (hemicentin 2), FCAR (Fc fragment of IgA receptor), FCER1A (Fc fragment of IgE receptor Ia), FCGR1A (Fc fragment of IgG receptor Ia), FCGR1B (Fc fragment of IgG receptor Ib), FCGR1CP (Fc fragment of IgG receptor Ic, pseudogene), FCGR2A (Fc fragment of IgG receptor IIa), FCGR2B (Fc fragment of IgG receptor IIb), FCGR2C (Fc fragment of IgG receptor IIc), FCGR3A (Fc fragment of IgG receptor IIIa), FCGR3B (Fc fragment of IgG receptor IIIb), FCRH1, FCRH3, FCRH4, FCRL1 (Fc receptor-like protein 1), FCRL2 (Fc receptor-like protein 2), FCRL3 (Fc receptor-like protein protein 3), FCRL4 (Fc receptor-like protein 4), FCRL5 (Fc receptor-like protein 5), FCRL6 (Fc receptor-like protein 6), FCRLA (Fc receptor-like protein A), FCRLB (Fc receptor like B ), FGFR1, FGFR2, FGFR3, FGFR4, FGFRL1, FLT1 (fms-like tyrosine kinase 1), FLT3 (fms-like tyrosine kinase 3), FLT4 (fms-like tyrosine kinase 4), FSTL4 (follistatin-like 4), FSTL5 (follistatin -like 5), GP6 (platelet glycoprotein VI), GPA33 (glycoprotein A33), GPR116, GPR125, ADGRF5 (G protein adhesion coupled receptor F5), ADGRA2 (G protein adhesion receptor A2), hEMMPRIN, NEPACAM (adhesion molecule liver and glial cells), NEPACAM2 (member of the NEPACAM family 2), HLA-DMA, HLA-DMB, HLA-DQB, HLA-DQB1, HNT, HSPG2 (heparan sulfate proteoglycan 2), HYST2477, ICAM1 (intercellular adhesion molecule 1), ICAM2 (Intercellular adhesion molecule 2), ICAM3 (Intercellular adhesion molecule 3), ICAM4 (Intercellular adhesion molecule 4 (Landsteiner-Wiener blood group)), ICAM5 (Intercellular adhesion molecule 5), DCC (DCC netrin-1 receptor), NEO1 ( neogenin1), IGHA1, IGHD, IGHE, IGDCC4 (immunoglobulin superfamily member DCC 4), IGLON5 (IgLON family member 5), IGSF1 (immunoglobulin superfamily member 1), IGSF2 (immunoglobulin superfamily member 2), IGSF3 (immunoglobulin superfamily member 3), IGSF5 (Immunoglobulin Superfamily Member 5), IGSF9 (Immunoglobulin Superfamily Member 9), IGSF9B (Immunoglobulin Superfamily Member 9B), IGSF10 (Immunoglobulin Superfamily Member 10), IGSF11 (Immunoglobulin Superfamily Member 11), IGSF21 (Immunoglobulin Superfamily Member 21), IG SF23 ( member of the immunoglobulin 23 superfamily), IL1R1 (interleukin 1 receptor type 1), IL1R2 (interleukin 1 receptor type 2), IL1RAP (interleukin 1 receptor-associated protein), IL1RAPL1 (interleukin 1 receptor-associated protein-like protein 1), IL1RAPL2 ( interleukin 1 receptor-like protein 2), IL1RL1 (interleukin 1 receptor-like protein 1), IL1RL2 (interleukin 1 receptor-like protein 2), IL6R (interleukin 6 receptor), IL11RA (interleukin 11 receptor alpha subunit), IL12B (interleukin 12B), IL18BP (interleukin 18 binding protein), IL18R1 (interleukin 18 receptor 1), IL18RAP (interleukin 18 receptor associated protein), ISLR2 (immunoglobulin superfamily leucine-rich repeat 2), JAM2 (binding adhesion molecule 2), JAM3 (binding adhesion molecule 3), KDR (kinase receptor insertion domain), KIR-123FM, KIR2DL1 (killer cell immunoglobulin-like receptor, two Ig domains and

- 13 046243 long cytoplasmic tail 1), KIR2DL2 (immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2), KIR2DL3 (immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 3), KIR2DL4 (immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 4), KIR2DL5A (immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 5A), KIR2DL5B (immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 5B), KIR2DLX, KIR2DS1 (immunoglobulin-like killer cell receptor, two Ig domains and short cytoplasmic tail 1), KIR2DS2 (immunoglobulin-like killer cell receptor, two Ig domains and short cytoplasmic tail 2), KIR2DS3 (immunoglobulin -like killer cell receptor, two Ig domains and short cytoplasmic tail 3), KIR2DS4 (immunoglobulin-like killer cell receptor, two Ig domains and short cytoplasmic tail 4), KIR2DS5 (immunoglobulin-like killer cell receptor, two Ig domains and short cytoplasmic tail 5), kir3d, KIR3DL1 (immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1), KIR3DL2 (immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 2), KIR3DL3 (immunoglobulin -like killer cell receptor, three Ig domains and long cytoplasmic tail 3), KIR3DP1 (immunoglobulin-like killer cell receptor, three Ig domains of pseudogene 1), KIR3DS1 (immunoglobulin-like killer cell receptor, three Ig domains and short cytoplasmic tail 1), KIR3DX1 (immunoglobulin-like killer cell receptor, three Ig X1 domains), KIRREL1 (kirre-like nephrin family adhesion molecule 1), KIRREL2 (kirre-like nephrin family adhesion molecule 2), KIRREL3 (kirre nephrin family-like adhesion molecule 3), KIT (KIT proto-oncogene receptor tyrosine kinase), L1CAM, LAG3 (leukocyte activator 3), LAIR1 (leukocyte-associated immunoglobulin-like receptor 1), LAIR2 (leukocyte-associated immunoglobulin-like receptor 2) , LEPR (leptin receptor), LILRA1 (leukocyte immunoglobulin-like receptor A1), LILRA2 (leukocyte immunoglobulin-like receptor A2), LILRA3 (leukocyte immunoglobulin-like receptor A3), LILRA4 (leukocyte immunoglobulin-like receptor A4), LILRA5 (leukocyte immunoglobulin-like receptor A5), LILRA6 (leukocyte immunoglobulin-like receptor A6), LILRB1 (leukocyte immunoglobulin-like receptor B1), LILRB2 (leukocyte immunoglobulin-like receptor B2), LILRB3 (leukocyte immunoglobulin-like receptor B3), LILRB4 (leukocyte immunoglobulin-like receptor B4), LILRB5 (leukocyte immunoglobulin-like receptor B5), LILRP2, LRIG1, LRIG2, LRIG3, LRIT1, LRRC4, LSAMP, LSR (lipolysis-stimulated lipoprotein receptor), LY9 (lymphocyte antigen 9), MADCAM1 (addressin-type cell adhesion molecule in the vascular mucosa 1) , MAG (myelin-associated glycoprotein), MALT1 (MALT1 paracaspase), MCAM (melanoma cell adhesion molecule), MDGA1 (MAM domain containing glycosylphosphatidylinositol anchor 1), MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2), MERTK (MER proto-oncogene, tyrosine kinase), MFAP3, MIR, MILR1 ((mast cell immunoglobulin-like receptor 1), MMP23A (matrix metallopeptidase 23A (pseudogen)), MMP23B (matrix metallopeptidase 23B), MUSK (muscle-associated receptor tyrosine kinase), MXRA5 (associated with matrix remodeling 5), MYBPC3, MY0M1 (myomesin 1), MY0M2 (myomesin 2), MY0M3 (myomesin 3), NCA, NCAM1, NCAM2, NCR1 (natural cytotoxicity triggering receptor 1, NEGR1, NEO1, NFASC, NOPE, NPHS1 ( NPHS1, nephrin), NPTN (neuroplastin), NRCAM (neuronal cell adhesion molecule), NTRK1 (neurotrophic receptor tyrosine kinase 1), NRG1, NT, NTRK3, ObScN, OBSL1 (obscurin-like protein 1), OPCML, OsCaR (osteoclast-associated immunoglobulin-like receptor), PAPLN, PDCD1LG2 (programmed cell death protein ligand 2 1), PDGFRA (platelet-derived growth factor receptor alpha), PDGFRB (platelet-derived growth factor receptor beta), PDGFRL (platelet-derived growth factor receptor-like receptor), PECAM1 ( platelet-endothelial cell adhesion molecule 1), PRODH2, PSG1 (pregnancy beta-1 glycoprotein 1), PSG2 (pregnancy beta-1 glycoprotein 2), PSG3 (pregnancy beta 1 glycoprotein 3), PSG4 (pregnancy beta 1 glycoprotein 4), PSG5 (pregnancy beta-1-glycoprotein 5), PSG6 (pregnancy beta-1-glycoprotein 6), PSG7 (pregnancy beta-1-glycoprotein 7 (gene/pseudogene)), PSG8 (pregnancy beta-1-glycoprotein 8), PSG9 (pregnancy beta-1-glycoprotein 9), PSG10 (pregnancy beta-1-glycoprotein 10), PSG11 (pregnancy beta-1-glycoprotein 11), PSG11s' (pregnancy beta-1-glycoprotein 11s'), PTGFRN (postoglandin receptor F2 inhibitor), PTK7 (protein tyrosine kinase 7 (inactive)), PTPRD (protein tyrosine phosphatase, receptor type D), PTPRK (protein tyrosine phosphatase, receptor type K), PTPRM (protein tyrosine phosphatase, receptor type M), PTPRS (protein tyrosine phosphatase, receptor type S ), PTPRT (protein tyrosine phosphatase, receptor type T), PTPsigma, PUNC, PVR (poliomyelitis virus receptor), PVRL1, PVRL2, PVRL4, NECTIN1 (cell adhesion molecule nectin 1), NECTIN2 (cell adhesion molecule nectin 2), NECTIN3 (cell adhesion molecule nectin adhesion 3), RAGE, ROBO3 (bypass receptor 3), SCN1B (sodium voltage channel beta subunit 1), SDK1 (companion cell adhesion molecule 1), SDK2 (companion cell adhesion molecule 2), SEMA3A (semaphorin 3A), SEMA3B (semaphorin 3B),

- 14 046243

SEMA3E (Semaphorin 3E), SEMA3F (Semaphorin 3F), SEMA3G (Semaphorin 3G), SEMA4C (Semaphorin 4C), SEMA4D (Semaphorin 4D), SEMA4G (Semaphorin 4G), SEMA7A (Semaphorin 7A), (John Milton Hagen blood type)) , SIGIRR (single Ig and TIR domain containing - containing one Ig and TIR domain), SIGLEC1 (sialic acid-binding Ig-like lectin 1), SIGLEC5 (sialic acid-binding Ig-like lectin 5), SIGLEC6 (sialic acid-binding Ig- lectin-like 6), SIGLEC7 (sialic acid-binding Ig-like lectin 7), SIGLEC8 (sialic acid-binding Ig-like lectin 8), SIGLEC9 (sialic acid-binding Ig-like lectin 9), SIGLEC10 (sialic acid-binding Ig- lectin-like 10), SIGLEC11 (sialic acid-binding Ig-like lectin 11), SIGLEC12 (sialic acid-binding Ig-like lectin 12 (gene/pseudogene)), SIGLEC14 (sialic acid-binding Ig-like lectin 14), SIGLEC15 (sialic acid-binding Ig-like lectin 15), SLAMF1 (signaling lymphocytic activation molecule family member 1 - representative of the family of signaling molecules of lymphocyte activation 1), SLAMF6 (representative of the SLAM family 6), SLAMF8 (representative of the SLAM family 8), SIRPG; TARM1 (Tcell-interacting, activating receptor on myeloid cells 1), TEK (TEK receptor tyrosine kinase), THY1 (Thy-1 cell surface antigen), TIE1 (tyrosine kinase with immunoglobulin-like and EGF-like domains 1), TMEM81 (transmembrane protein 81 ), TMIGD1 (transmembrane and domain-containing immunoglobulin 1), TMIGD2 (transmembrane and domain-containing immunoglobulin 2), TTN (titin), TYRO3 (TYRO3 protein tyrosine kinase), UNC5D, VCAM1 (vascular cell adhesion molecule 1), VSIG1 (V-set and immunoglobulin domain-containing protein 1), VSIG2 (V-set and immunoglobulin domain-containing protein 2), VSIG4 (V-set and immunoglobulin domain-containing protein 4), VSIG10 (V-set and immunoglobulin domain-containing protein 10), VSIG10L (V-set-like and immunoglobulin domain-containing protein 10), VSTM1 (V-set and transmembrane domain-containing protein 1), VTCN1 (V-set domain-containing inhibitor of T-cell activation protein 1), ZPBP (transparent protein-binding protein zona) and ZPBP2 (zona pellucida binding protein 2).

More preferably, the Ig-like domain is the Ig-like domain of any of the following proteins: CD2, CD3, CD4, CD8, CD19, CD22, CD33, CD80, CD86, especially CD4.

Other preferred examples of Ig-like domains are described below in the present invention.

It is also preferred that the (additional) functional domain of the first and/or second polypeptide chain includes or consists of an extracellular and/or intracellular domain of a (known) protein. In addition, the (additional) functional domain of the first and/or second polypeptide chain may preferably include or consist of a (known) soluble globular protein domain. More preferably, the (additional) functional domain of the first and/or second polypeptide chain comprises or consists of an extracellular domain of a (known) protein or a domain of a (known) soluble globular protein.

Preferably, the (additional) functional domain of the first and/or second polypeptide chain has a length of up to 1000 amino acids, more preferably up to 750 amino acids, even more preferably up to 500 amino acids, even more preferably up to 400 amino acids, especially preferably up to 300 amino acids and most preferably up to 275 or 250 amino acids. It is further preferred that the (additional) functional domain of the first and/or second polypeptide chain has a length of from 5 to 1000 amino acids, more preferably from 10 to 750 amino acids, even more preferably from 20 to 500 amino acids, even more preferably from 50 to 400 amino acids , especially preferably from 70 to 300 amino acids and most preferably from 75 to 275 or from 100 to 250 amino acids.

It is also preferred that the (additional) functional domain of the first and/or second polypeptide chain has a size of up to 150 kDa, more preferably up to 100 kDa, even more preferably up to 80 kDa, even more preferably up to 70 kDa, especially preferably up to 50 kDa, and most preferably up to 30 or 25 kDa. It is further preferred that the (additional) functional domain of the first and/or second polypeptide chain has a size of from 0.5 kDa to 150 kDa, more preferably from 1 kDa to 100 kDa, even more preferably from 2.5 kDa to 80 kDa. more preferably 5 to 70 kDa, particularly preferably 7.5 to 50 kDa and most preferably 10 to 30 or 25 kDa.

The (additional) functional domain of the first and/or second polypeptide chain may comprise a monomeric domain or multimeric domains. A monomeric domain is a domain that mediates functionality without the participation of any other (additional) domain. Multimeric domains, eg two domains forming a dimer or three domains forming a trimer, jointly mediate functionality, particularly as a multimer, eg a dimer or trimer. In the case of multimeric domains, the (additional) functional domain of the first and/or second polypeptide chain may contain linkers, such as those described in the present invention, to provide sufficient flexibility for multimer formation, in particular, the linker may be located (immediately) adjacent to one or more multimeric domain(s), for example, between two multimeric domains or on each side of all multimeric domains. Prefer

- 15 046243 specifically, the (additional) functional domain of the first and/or second polypeptide chain contains or consists of one or more monomeric domains.

Typically, the (additional) functional domain of the first and/or second polypeptide chain may comprise or consist of a single protein domain or multiple protein domains. The term multiple protein domains can mean multimeric domains as described above and/or one or more monomeric domains as described above. For example, the (additional) functional domain of the first and/or second polypeptide chain may contain or consist of two or three monomeric domains, which may mediate the same or different functionality and/or which may optionally be linked by a linker. For example, the (additional) functional domain of the first and/or second polypeptide chain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (separate) protein domains.

Preferably, the (additional) functional domain is a human protein, peptide or polypeptide, or a fragment (in particular a domain) or derivative thereof.

In particular, the (additional) functional domain is not a linker (peptide), for example a GS linker. Even though the (additional) functional domain may optionally contain a linker (peptide) such as a GS linker, it preferably does not consist of a linker (peptide) such as a GS linker. In other words, even if the (additional) functional domain contains a linker (peptide), such as a GS linker, it preferably contains an additional amino acid sequence mediating a function other than (solely) the function of linking two peptides to each other. Accordingly, the (additional) functional domain is preferably different from the linker (peptide), such as the GS linker. In particular, the (additional) functional domain may not contain a linker (peptide), such as a GS linker. Typically, the functionality provided by the (additional) functional domain is preferably not a simple linkage of two (poly)peptides. Even though the (additional) functional domain can link two (poly)peptides, such as an adjacent variable and constant domain, and optionally provides flexibility, the (additional) functional domain preferably provides an (additional) function other than the (poly)peptide bond and flexibility.

Preferably, the second polypeptide chain contains an (additional) functional domain (v). In this case, the first and second polypeptide chains both contain an (additional) functional domain, resulting in the formation of an antibody or antigen-binding fragment thereof with two different (separate) (additional) functional domains. The (additional) functional domains of the first polypeptide chain and the second polypeptide chain may be the same or different, that is, the same or different functional domains. That is, even if the (additional) functional domains of the first and second polypeptide chains have identical amino acid sequence, these (additional) functional domains are independent, that is, they mediate their functions independently of each other. For example, the (additional) functional domains of the first and second polypeptide chain bind to distinct targets (e.g., distinct antigens or distinct epitopes of the same antigen, distinct molecules, or distinct portions of the same molecule), and no independent binding sites requires the participation of another to communicate with the target.

However, more preferably, the second polypeptide chain does not contain an (additional) functional domain (v) and the C-terminus of the C-terminal variable domain of the second polypeptide chain is preferably directly linked to the N-terminus of the N-terminal terminal constant domain of the second polypeptide chain. For example, if the second polypeptide chain is a light chain, the C-terminus of the C-terminal VL domain (or single VL domain) is preferably directly linked to the N-terminus of the CL domain. For example, if the second polypeptide chain is a heavy chain, the C-terminus of the C-terminal VH domain (or single VH domain) is preferably directly linked to the N-terminus of the CH1 domain.

Thus, the present invention also provides an antibody, or an antigen binding fragment thereof, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes, in an N- to C-terminal direction, (i) one or more variable domains, (ii) ( an additional) functional domain, and (iii) one or more constant domains, the second polypeptide chain comprising, in an N- to C-terminal direction, (iv) one or more variable domains forming antigen binding sites, with one or more variable domains a first polypeptide chain, and (v) one or more constant domains, characterized in that the C-terminus of the C-terminal variable domain of the second polypeptide chain is directly connected to the N-terminus of the N-terminal constant domain of the second polypeptide chain.

Thus, the term functional domain refers to a functional unit, for example, an antibody or an antigen binding fragment, as described above. In short, functionality

- 16 046243 This domain typically represents a protein, for example an antibody or antigen binding fragment, with (additional) functionality as described above. Accordingly, the (additional) functional domain typically contains all the amino acids/sequences necessary to provide the (additional) function. In particular, the (additional) functional domain (ii) of the first polypeptide chain does not contain a fragment of the second polypeptide chain, as described in more detail above.

Particularly preferably, the (additional) functional domain of the first polypeptide chain contains or consists of an Ig-like domain as described above. Preferred examples of Ig-like domains are those described above and those described below in the present invention.

It is also preferred that the (additional) functional domain of the first polypeptide chain includes or consists of an extracellular and/or intracellular domain of a (known) protein. In addition, the (additional) functional domain of the first polypeptide chain may preferably include or consist of a (known) soluble globular protein domain. More preferably, the (additional) functional domain of the first polypeptide chain comprises or consists of an extracellular domain of a (known) protein or a domain of a (known) soluble globular protein.

Preferably, the (additional) functional domain of the first polypeptide chain is up to 1000 amino acids in length, more preferably up to 750 amino acids, even more preferably up to 500 amino acids, even more preferably up to 400 amino acids, especially preferably up to 300 amino acids, and most preferably up to 275 or 250 amino acids amino acids. It is further preferred that the (additional) functional domain of the first polypeptide chain has a length of from 5 to 1000 amino acids, more preferably from 10 to 750 amino acids, even more preferably from 20 to 500 amino acids, even more preferably from 50 to 400 amino acids, especially preferably 70 to 300 amino acids, and most preferably 75 to 275 or 100 to 250 amino acids.

It is also preferred that the (additional) functional domain of the first polypeptide chain has a size of up to 150 kDa, more preferably up to 100 kDa, even more preferably up to 80 kDa, even more preferably up to 70 kDa, especially preferably up to 50 kDa, and most preferably up to 30 or 25 kYes. It is further preferred that the (additional) functional domain of the first polypeptide chain has a size of from 0.5 kDa to 150 kDa, more preferably from 1 kDa to 100 kDa, even more preferably from 2.5 kDa to 80 kDa, even more preferably from 5 to 70 kDa, particularly preferably 7.5 to 50 kDa and most preferably 10 to 30 or 25 kDa.

The (additional) functional domain of the first polypeptide chain may comprise a monomeric domain or multimeric domains as described above. In addition, the (additional) functional domain of the first polypeptide chain may contain or consist of a single protein domain or more than one protein domain, as described above.

In addition, the (additional) functional domain of the first polypeptide chain may contain or consist of a single protein domain or more than one protein domain, as described above.

Preferably, the (additional) functional domain is a human protein, peptide or polypeptide or a fragment (in particular a domain) or derivative thereof.

In particular, the (additional) functional domain is not a linker (peptide) such as a GS linker as described above. Even though the (additional) functional domain may optionally contain a linker (peptide) such as a GS linker, it preferably does not consist of a linker (peptide) such as a GS linker. That is, even if the (additional) functional domain contains a linker (peptide), such as a GS linker, it preferably contains an additional amino acid sequence mediating a function other than (exclusively) linking two peptides to each other. Accordingly, the (additional) functional domain is preferably different from the linker (peptide), such as the GS linker. In particular, the (additional) functional domain may not contain a linker (peptide), such as a GS linker. Typically, the functionality provided by the (additional) functional domain is preferably not a simple linkage of two (poly)peptides. Even though the (additional) functional domain can link two (poly)peptides, such as adjacent variable and constant domains, and optionally provide flexibility, the (additional) functional domain preferably provides an (additional) function other than the (poly)peptide bond and flexibility.

In addition, the C-terminus of the C-terminal variable domain of the second polypeptide chain is preferably directly linked to the N-terminus of the N-terminal constant domain of the second polypeptide chain. For example, if the second polypeptide chain is a light chain, the C-terminus of the larger C-terminal VL domain (or single VL domain) is preferably directly linked to the N-terminus of the CL domain. For example, if the second polypeptide chain is a heavy chain, the C-terminus of the C-terminal VH domain (or single VH domain) is preferably directly linked to the N-terminus of the CH1 domain.

In general, preferably in an antibody or antigen binding fragment thereof according to the present invention

- 17 046243 where one or more of the first polypeptide chain constant domains are heavy chain constant domains, preferably comprising at least a CH1 constant domain, and the second polypeptide chain constant domain is a light chain CL constant domain. More preferably, the one or more first polypeptide chain constant domains are heavy chain constant domains, preferably comprising at least a CH1 constant domain, the one or more first polypeptide chain variable domains are heavy chain VH variable domains, the second polypeptide chain constant domain is a CL constant domain light chain, and one or more variable domains of the second polypeptide chain are VL light chain variable domains. Thus, it is preferred that the first polypeptide chain is or is derived from a heavy chain and the second polypeptide chain is or is derived from a light chain. It is particularly preferred that the constant region of the first polypeptide chain comprises three constant domains, namely CH1, CH2 and CH3 (particularly in the N- to C-terminal direction CH1-CH2-CH3), most preferably with a hinge region between CH1 and CH2 .

In another embodiment of the present invention, it is also preferred that in the antibody or antigen binding fragment thereof, the first polypeptide chain constant domains are light chain CL constant domains and one or more of the second polypeptide chain constant domains are heavy chain constant domains, preferably including at least a CH1 constant domain. . More preferably, the first polypeptide chain constant domains are a light chain CL constant domain, one or more of the first polypeptide chain variable domains are light chain VL variable domains, one or more of the second polypeptide chain constant domains are heavy chain constant domains, preferably including at least a heavy chain constant domain the CH1 domain, and one or more variable domains of the second polypeptide chain are VH heavy chain variable domains. In other words, it is preferable that the second polypeptide chain is or is derived from a heavy chain and the first polypeptide chain is or is derived from a light chain. It is particularly preferred that the constant region of the second polypeptide chain comprises three constant domains, namely CH1, CH2 and CH3 (particularly in the N- to C-terminal direction CH1-CH2-CH3), most preferably with a hinge region between CH1 and CH2 .

It is further preferred that in the first and/or second polypeptide chain, the constant domains immediately adjacent to the (additional) functional domain (and/or variable region if the second polypeptide does not contain an (additional) functional domain) are not included in the Fc region. In native antibodies, the constant region following the variable domains, CH1 and CL, is usually not included in the Fc region. Accordingly, it is preferred that one polypeptide chain (first or second) contains a CH1 constant domain and the other polypeptide chain (different from the first or second) contains a CL constant domain.

Preferably the heavy chain constant domain, in particular CH1 or some other heavy chain constant domain, is selected from the following classes: γ, α, μ, ε and δ, preferably γ, for example from IgG1, IgG2, IgG3 or IgG4.

It is further preferred that the light chain constant domain, in particular CL, is selected from the following classes: k and λ.

Preferably, the antibody or antigen binding fragment thereof according to the present invention is a recombinant antibody or antigen binding fragment. In the context of the present invention, the term recombinant means that the recombinant antibody or antibody fragment does not occur in nature.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains or consists of a carrier domain, a reporter domain, a tag, a localization domain, a (independent) binding site, an enzyme or an enzymatic domain, a receptor or a functional fragment thereof, or a ligand or a functional fragment thereof.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention contains or consists of an enzyme or an enzymatic domain thereof. An enzyme is a polypeptide or protein catalyst, that is, an enzyme that typically speeds up a chemical reaction. The molecules that enzymes can act on are called substrates, and the enzyme converts the substrates into various molecules called products. Almost all metabolic processes in a cell require enzymes to proceed at a rate fast enough to support life. Preferred enzymes include oxidoreductases, transferases, hydrolases, lysases, isomerases and ligases. For enzymes that form a dimer, the functional domain of the first and/or second polypeptide chain may contain two identical domains linked by a linker. For example, enzymes may be useful for activating a prodrug at a specific site, at

- 18 046243 example, in a tumor. Examples of preferred enzymes and the use of antibodies representing such enzymes are described in the articles of Andrady S. et al., Immunotherapy. 2011, 3(2): 193-211, and Boado R.J. et al., Biotechnol Bioeng. 2008, 99(2):475-484.

Preferred enzymes are selected from the group consisting of: dehydrogenase, luciferase, DMSO reductase, alcohol dehydrogenase (NAD), alcohol dehydrogenase (NADP), homoserine dehydrogenase, aminopropanol oxidoreductase, diacetyl reductase, glycerol dehydrogenase, propanediol phosphate dehydrogenase, propanediol phosphate dehydrogenase, glycerol -3-phosphate dehydrogenase ( NAD+), Dxylulose reductase, L-xylulose reductase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, HMG-CoA reductase, glucose oxidase, L-gulonolactone oxidase, thiamine oxidase, xanthine oxidase, acetaldehyde dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyr uvate dehydrogenase, oxoglutarate dehydrogenase, biliverdin reductase, monoamine oxidase, dihydrofolate reductase, methylenetetrahydrofolate reductase , sarcosine oxidase, dihydrobenzophenanthridine oxidase, NADH dehydrogenase, urate oxidase, nitrite reductase, nitrate reductase, glutathione reductase, thioredoxin reductase, sulfite oxidase, cytochrome c oxidase, coenzyme Q - cytochrome c reductase, catechol oxidase, laccase, cytochrome c peroxidase, catalase, myeloperoxidase, thyroid peroxidase, glutathione peroxidase, 4oxyphenylpyruvate dioxygenase, ReniUa-luciferin-2-monooxygenase, Cypridina-luciferin-2-monooxygenase, firefly luciferase, Watasenia-luciferin-2-monooxygenase, Oplophorus-luciferin-2-monooxygenase, aromatase, CYP2D6, CYP2E1, CYP3A4, cytochrome P450 oxidase, dioxygenase nitric oxide , nitric oxide synthase, aromatase, phenylalanine hydroxylase, tyrosinase, superoxide dismutase, ceruloplasmin, nitrogenase, deiodinase, glutathione^-transferase, catechol-o-methyl transferase, DNA methyltransferase, histone methyltransferase, aspartate carbamoyltransferase (ATCase), ornithine transcarbamoyl for, aminolevulinic acid synthase, choline acetyltransferase, factor XIII, gammaglutamyl transpeptidase, transglutaminase, hypoxanthine guanine phosphoribosyltransferase, thiaminase, alanine transaminase, aspartate transaminase, butyrate kinase, nuclease, endonuclease, exonuclease, acid hydrolase, phospholipase A, phospholipase C, acetylcholinesterase, choline sterase, lipoprotein lipase, ubiquitin carboxy-terminal hydrolase L1, phosphatase, alkaline phosphatase, fructose bisphosphatase, CGMP specific phosphodiester esterase type 5, phospholipase D, restriction enzyme type 1, restriction enzyme type 2, restriction enzyme type 3, restriction enzyme type 4, deoxyribonuclease I, RNase H, ribonuclease, amylase, sucrase, chitinase, lysozyme, maltase , lactase, beta-galactosidase, hyaluronidase, adenosine methitonine hydrolase, S-adenosyl-L-homoserine hydrolase, alkenylglycerophosphocholine hydrolase, alkenylglycerophosphoethanolamine hydrolase, cholesterol-5,6-oxide hydrolase, hepoxyline epoxide hydrolase, isochorosematase, leukotriene A4 hydrolase, limonene-1,2 -epoxide hydrolase, microsomal epoxide hydrolase, trans-epoxysuccinate hydrolase, alanine aminopeptidase, angiotensin converting enzyme, serine protease, chemotrypsin, trypsin, thrombin, factor X, plasmin, acrosin, factor VII, factor IX, prolyl oligopeptidase, factor XI, elastase, factor XII, proteinase K, activator tissue plasminogen, protein C, separase, pepsin, rennet, renin, trypsinogen, plasmepsin, matrix metalloproteinase, metalloendopeptidase, urease, beta-lactamase, arginase, adenosine deaminase, GTP-cyclohydrolase I, nitrilase, helicase, DnaB helicase, RecQ helicase, ATPase , NaK-ATPase, ATP synthase, kynureninase, haloacetate dehalogenase, lyase, ornithine decarboxylase, uridine monophosphate synthetase, Aromatic L-amino acid decarboxylase, RubisCO (ribulose-1,5bisphosphate carboxylase/oxygenase), carbonic anhydrase, tryptophan synthase, phenylalanine ammonia lyase, cystathionine gamma lyase, cystathionine beta-lyase, leukotrian C4 synthase, dichloromethane dehalogenase, halohydrin dehalogenase, adenylate cyclase, guanylate cyclase, amino acid racemase, serine racemase, mandel racemase, UDP-glucose-4-epimerase, methylmalonyl-CoA epimerase, FKBP: FKBP1A, FK BP1B, FKBP2, FKBP3 , FKBP5, FKBP6, FKBP8, FKBP9, FKBP10, FKBP52, FKBPL, cyclophilin, parvulin, prolyl isomerase, 2-chloro-4-carboxymethylbut-2-ene-1,4-olid isomerase, beta-carotene isomerase, farnesol 2 isomerase , Furilfuramid-Isomerase, linoleat-ameraza, maleA-amerase, maleilacetathizomez, maleilpiruvat-amenomerase, parvulin, photosomerase, prolikopen-ismerase, sulfate, izmeraza, retinol-amerase, zeta-karotin-ammeraza, zoil-sosa-sosa-sosa-sosa Zomeraza, proteindimulfide -isomerase, phosphoglucomutase, muconate cycloisomerase, 3-carboxy-^,g-muconate cycloisomerase, tetrahydroxypteridine cycloisomerase, inositol-3-phosphate synthase, 3-carboxycis.cis-muconate cyclase. chalcone isomerase, chloromuconate cycloisomerase, (+)-bornyl diphosphate synthase, cycloeucalenol cycloisomerase, alpha-pinene oxide decyclase, dichloromuconate cycloisomerase, copalyl diphosphate synthase, ent-copalyl diphosphate synthase, syn-copalyl diphosphate synthase , terpentedienyl diphosphate synthase, halimadienyl diphosphate synthase, ^)-beta-macrocarpene synthase, lycopene epsilon cyclase, lycopene beta cyclase, prosolanapyrone-III cycloisomerase, D-ribose pyranase, steroid delta isomerase, topoisomerase, 6-carboxytetrahydropterin synthetase , FARSB, glutamine synthetase, CTP synthetase, arginate succinate synthase, pyruvate carboxylase, acetyl-CoA carboxylase and DNA ligase.

More preferred enzymes may be selected from the group consisting of carboxypeptidase, β-lactamase, cytosine deaminase, β-glucuronidase, purine nucleoside phosphorylase, granzyme B, caspase and RNase, for example, HPR (human pancreatic RNase, barnase, seminal

- 19 046243

Bovine RNase, onconase) RapLRl, angiogenin, diker, DIS3-like exonuclease 2, phosphodiesterase ELAC 2, RNase HIII, RNase T2 and tRNA splicing ribonuclease.

A functional enzyme fragment may be any fragment of an enzyme that has the ability to mediate functionality. Typically such fragments are called domains. Accordingly, the functional fragment of the enzyme may be any domain of the enzyme. Preferred examples include functional fragments (eg, domains) of the (exemplified) enzymes described above. Preferably, the functional enzyme fragment that includes the (additional) functional domain is the catalytic domain of the enzyme. The catalytic domain of an enzyme is the region of the enzyme that interacts with its substrate and causes the enzymatic reaction. For example, the (additional) functional domain of the first polypeptide chain and/or the second polypeptide chain may be the catalytic domain of any of the following enzymes: carboxypeptidase, β-lactamase, cytosine deaminase, β-glucuronidase, purine nucleoside phosphorylase, granzyme B, caspase and RNase, e.g. HPR (human pancreatic RNase), barnase, bovine seminal RNase, onconase, RapLR1, angiogenin, Dicer, DIS3-like exonuclease 2, ELAC phosphodiesterase 2, RNase HIII, RNase T2, and tRNA splicing ribonuclease.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains or consists of a carrier domain. In the context of the present invention, the term carrier domain refers to an amino acid sequence that mediates the conjugation of an antibody to another molecule. In a preferred embodiment of the present invention, the carrier domain enables conjugation of an antibody or an antigen-binding fragment thereof, for example, to a drug, an imaging agent, or a nanoparticle. In general, preferred examples of conjugates that may be useful in the context of the present invention are described in Wu A.M. and Senter P. D., Nat. Biotechnol. 2005, 23, 1137-1146.

For example, drugs that can be conjugated to an antibody include anti-cancer drugs, such as those described in Thomas A. et al., Lancet Oncol. 2016 Jun;17(6):e254-62. doi: 10.1016/S1470-2045(16)30030-4. For example, imaging agents that can be conjugated to an antibody are described in Steve Knutson et al., PLoS One 2016; 11(6): e0157762. Such drugs are preferably cytotoxic agents. Preferred examples of drugs that can be conjugated to the antibody or antigen binding fragment of the present invention include doxorubicin, truncated exotoxin A of Pseudomonas aeruginosa, maytansinoid DM1.

Examples of imaging agents that can be conjugated to an antibody or antigen-binding moiety of the present invention include radioisotopes such as those described in Schubert M. et al., Bioconjug Chem. 2017 Apr 19;28(4):1176-1188, and Bhusari P et al., Int J Cancer. 140(4), 2017, 938-947. Preferred examples of radioisotopes include ⁹⁰ Y, ¹³¹ I and ¹⁷⁷ Lu.

Additional examples of imaging agents that can be conjugated to an antibody or antigen binding moiety of the present invention include fluorescent dyes, quantum dots, and iron oxide. Examples of fluorescent dyes include those described below as reporter domains. An example of iron oxide nanoparticles is described by Hengyi Xu et al. in Biomaterials. 32(36), 2011, 9758-9765.

Antibody conjugates (ie antibodies conjugated to other molecules) are known in the art. In particular, the antibody-conjugated molecule may be linked to the antibody by a cleavable or non-cleavable linker (for example, as described by Thomas H. Pillow in Pharmaceutical Patent Analyst Vol. 6, No. 1, February 3rd, 2017, https://doi. org/10.4155/ppa-2016-0032 or Beck A et al in Nat Rev Drug Discov 2017 May;16(5):315-337). Examples of such linkers that can be used to link a molecule to an antibody or antigen binding fragment are described, for example, in EP 2927227 and Thomas H. Pillow, Pharmaceutical Patent Analyst Vol. 6, No. 1, February 3rd, 2017, https://doi.org/10.4155/ppa-2016-0032. However, in the prior art, linkers are attached directly to the Ig domains of the antibody (namely, the variable and/or constant domains of the antibody), which may interfere with the functioning of the Ig domains of the antibody. Therefore, the (additional) functional domain of the antibody or antigen binding fragment of the present invention can be used to attach a linker to the antibody. Preferred linkers differ from classical linkers in that they are designed to contain additional cysteines or lysines. Preferably, the carrier domain contains one or more non-canonical amino acids suitable for site-specific conjugation, for example, as described in Link A.J. et al., Curr Opin Biotechnol. 2003 Dec;14(6):603-9. In addition, the carrier domain can be designed to be recognized by specific enzymes (eg, formylglycine generating enzyme, sortase and/or transglutaminase) that modify specific amino acids, which can then be used for conjugation, as described in section 6 of Dennler R. et al., Antibodies. 2015, 4:197-224.

- 20 046243

Other preferred carrier domains are those for conjugation, for example, genetically modified cross-reacting material (CRM) diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxin (D) and H. influenzae protein D (HiD), for example, as described by Pichichero M.E. in Hum Vaccin Immunother. 2013 Dec; 9 (12): 2505-23.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains or consists of a reporter domain. The reporter domain is usually encoded by a reporter gene. Reporter domains are those domains whose presence (for example, in a cell, an organism) can be easily established. Reporter domains include, for example, fluorescent proteins such as GFP/EGFP (green fluorescent protein/enhanced green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein) and CFP (cyan fluorescent protein), luciferases and enzymes, for example, beta-galactosidase and peroxidase. Reporter domains may be useful for in vivo and ex vivo applications. For example, fluorescent proteins cause the cell to fluoresce when excited by light of a specific wavelength, luciferases cause a reaction in the cell that results in the emission of light, and enzymes, such as beta-galactosidase, transform the substrate into a colored product. There are several different ways to measure or quantify a reporter, depending on the specific reporter and what properties are desired to be determined. In general, microscopy is useful for obtaining both spatial and temporal information about reporter activity, especially at the single cell level. Liquid-based cytometers are best suited for measuring the distribution of reporter activity across a large population of cells. Plate readers are usually best suited for measuring a large number of different samples over time. An enzyme such as beta-galactosidase and peroxidase that can react with a given substrate may be useful, for example, for ex-vivo staining of human samples, for example in tumor diagnosis.

Preferably, the reporter domain contains or consists of an amino acid sequence encoding GFP/EGFP, YFP, RFP, CFP, luciferase, beta-galactosidase or peroxidase. In addition, the fluorescent tags described below are also useful as reporter domains.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains or consists of a localization domain. Typically, a localization domain directs a protein to a specific target, such as at the organismal or cellular level. A localization domain may direct an antibody or antigen binding fragment of the present invention to a specific location in a cell, such as the nucleus, membrane, periplasm, extracellular secretion, a specific part of the body, or other location.

For example, to target an antibody or antigen binding fragment of the present invention to a cell, the (additional) functional domain may contain or consist of a cell penetrating peptide. The term cell penetrating peptides (CPPs, also called protein transduction domains (PTDs)) is commonly used to refer to short peptides that are capable of transporting various types of cargo molecules across the plasma membrane and thus facilitating cellular uptake various molecular cargo (from nano-sized particles to small chemical molecules and large DNA fragments). Cell-penetrating peptides typically have an amino acid composition that either contains a relatively high content of positively charged amino acids, such as lysine or arginine, or has a sequence that contains an alternating set of polar/charged amino acids and non-polar hydrophobic amino acids. These two types of structures are called polycationic or amphipathic, respectively. Typically, cell penetrating peptides (CPPs) are peptides of 8-50 residues that are able to cross the cell membrane and penetrate most types of cells. Alternatively, they are also called a protein transduction domain (PTD), reflecting their origin in natural proteins. Frankel and Pabo, together with Green and Lowenstein, described the ability of transactivating transcription activator of human immunodeficiency virus 1 (HIV-TAT) to enter cells (Frankel A.D., Pabo CO., Cell, 55(6), 1988, 1189-1193). In 1991, transduction of the Antennapedia homeodomain (DNA-binding domain) from Drosophila melanogaster into nerve cells was described (Joliot A. et al., Proc Natl Acad Sci USA, 88(5), 1991, 1864-1868). In 1994, it was established that the first 16-membered CPP peptide, named penetratin, was different from the third helix of the Antennapedia homeodomain (Derossi D. et al., J Biol Chem, 269(14), 1994, 10444-10450), described in 1998 by identifying the minimal TAT domain required for protein transduction (Vives E., Brodin, P., Lebleu, B. J Biol Chem, 272(25), 1997, 16010-16017). Over the past two decades, dozens of peptides of various origins have been described, including viral proteins, for example, VP22 (Elliott G., O'Hare P., Cell, 1997, 88(2), 223233), or from venoms, for example, melittin (Dempsey , C.E., Biochim Biophys Acta, 1990. 1031 (2), 143-161), mastoporan (Konno K. et al., Toxicon, 2000. 38(11), 1505-1515), maurocalcin (Esteve E., et al. ., J Biol

- 21 046243

Chem, 2005, 280(13), 12833-12839), crotamine (Nascimento F.D. et al., J Biol Chem, 2007, 282(29), 2134921360) or buforin (Kobayashi S. et al., Biochemistry, 2004, 43 (49), 15610-15616). Synthetic CPPs have also been developed to include poly-arginine (R8, R9, R10 and R12) (Futaki S. et al., J Biol Chem, 2001. 276(8): p. 5836-40) or transportan (Pooga M. with al., FASEB J, 1998, 12(1), 67-77). Any of the described CPPs may be used as a cell penetrating peptide in an antibody or antigen binding fragment of the present invention. Various CPPs that can be used as a cell penetrating peptide in an antibody or antigen binding fragment of the present invention are also reviewed in: Milletti F., Drug Discov Today, 2012, 17(15-16), 850-860.

Another example of a localization domain that can be used in an antibody or antigen binding fragment thereof of the present invention is a domain for crossing the blood-brain barrier, for example, as described in Farrington G.K. et al., FASEB J, 2014, 28(11), 4764-4778.

Another example of a localization domain is a nuclear localization domain. The nuclear localization domain directs a protein, in particular an antibody or an antigen binding fragment thereof, according to the present invention, to the cell nucleus. A nuclear localization domain may be useful for an antibody or antigen binding fragment to block transcription factor activity and modulate gene expression. Preferred examples of nuclear localization domains are described in Kalderon D. et al., Cell 1984, 39(3, part 2), 499-509, and Lusk C.P., Blobel G., King M.S., Nature Reviews Molecular Cell Biology 2007 , 8(5), 414-420.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment thereof according to the present invention contains or consists of a label. More preferably, the tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescent tag.

The tag represents the peptide sequence grafted into the recombinant protein. Examples of tags include affinity tags, solubilization tags, chromatographic tags, epitope tags, fluorescent tags, and protein tags. Affinity tags can be useful for purifying proteins from raw biological sources using affinity technology. Examples of affinity tags include chitin binding protein (CBP), maltose binding protein (MBP), and glutathione B-transferase (GST). Another example is the semi(His) tag, which binds to metal matrices. Solubilization tags can be used, especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, to promote proper protein folding and not interfere with sedimentation. Examples of solubilization tags include thyredoxin (TRX) and uo.rn(NANP). Chromatographic tags can be applied to modify the chromatographic properties of a protein to provide different resolution depending on the separation method. Chromatographic tags often consist of polyanionic amino acids, such as the FLAG tag.

Epitope tags are short peptide sequences that are chosen because high-affinity antibodies can be reliably produced in many different species. They are usually derived from viral genes, which explains their high immunoreactivity.

Epitope tags include V5 tag, Myc tag, HA tag and NE tag. These labels are particularly useful for Western blotting, immunofluorescence, and immunoprecipitation experiments, although they also find use in antibody purification. Fluorescent tags can be used to visually read a protein. GFP and its variants are the most commonly used fluorescent tags. GFP can be used as a foldable reporter (fluorescing when folded and not fluorescent when not). Protein tags can enable specific enzymatic modification (eg, biotinylation with biotin ligase) or chemical modification (eg, reaction with FlAsH-EDT2 for fluorescence imaging). Tags can be combined, for example, to link proteins to many other components. The tag can be removed by chemical agents or enzymes, such as proteolysis or intein splicing.

Preferred examples of tags include, but are not limited to, the following tags: twin-Strep-tag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK; SEQ ID NO: 20); AviTag is a peptide that is biotinylated by the enzyme BirA and the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE; SEQ ID NO: 21); Calmodulin-tag is a peptide bound by the calmodulin protein (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 22); a polyglutamate tag is a peptide that binds efficiently to an anion exchange resin such as Mono-Q (EEEEEE; SEQ ID NO: 23); Etag is an antibody recognition peptide (GAPVPYPDPLEPR; SEQ ID NO: 24); FLAG-tag is a peptide recognized by an antibody (DYKDDDDK; SEQ ID NO: 25); HA-tag is a hemagglutinin-derived peptide recognized by an antibody (YPYDVPDYA; SEQ ID NO: 26); His-tag is a peptide of 5-10 histidines linked with a nickel or cobalt chelate (HHHHHH; SEQ ID NO: 27); Myc-tag is a c-myc-derived peptide recognized by an antibody (EQKLISEEDL; SEQ ID NO: 28); NE-tag, a synthetic 22 046243 peptide of 18 amino acids (TKENPRSNQEESYDDNES; SEQ ID NO: 29), recognized by an IgG1 monoclonal antibody, which is useful in a variety of methods, including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation and affinity purification recombinant proteins; S-tag, ribonuclease A-derived peptide (KETAAAKFERQHMDS; SEQ ID NO: 30); SBPtag, a peptide that binds to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 31); Softag 1, mammalian expression tag (SLAELLNAGLGGS; SEQ ID NO: 32); Softag 3, prokaryotic expression tag (TQDPSRVG; SEQ ID NO: 33); Strep-tag, a peptide that binds to streptavidin or a modified streptavidin called streptactin (Strep-tag II: WSHPQFEK; SEQ ID NO: 34); TC tag, a tetracysteine tag that is recognized by the biarsenic compounds FlAsH and ReAsH (CCPGCC; SEQ ID NO: 35); V5 tag, antibody recognition peptide (GKPIPNPLLGLDST; SEQ ID NO: 36); VSV-tag, antibody recognition peptide (YTDIEMNRLGK; SEQ ID NO: 37); Xpress tag (DLYDDDDK; SEQ ID NO: 38); Isopeptag, a peptide that covalently binds pilin C protein (TDKDMTITFTNKKDAE; SEQ ID NO: 39); SpyTag, a peptide that covalently binds to the SpyCatcher protein (AHIVMVDAYKPTK; SEQ ID NO: 40); SnoopTag, a peptide that covalently binds to the SnoopCatcher protein (KLGDIEFIKVNK; SEQ ID NO: 41); Ty1 tag (EVHTNQDPLD; SEQ ID NO: 42); BCCP (carboxybiotin carrier protein), a protein domain biotinylated by BirA, recognized by streptavidin; glutathione^-transferase (GST)-tag, a protein that binds to immobilized glutathione; green fluorescent protein tag, a spontaneously fluorescent protein that can bind to nanobodies; HaloTag, a mutant bacterial haloalkane dehalogenase that covalently attaches to a reactive haloalkane substrate, allowing attachment to a wide range of substrates; maltose binding protein (MBP)-tag, a protein that binds to amylose agarose; Nus (N-utilization substance)-tag; thioredoxin (Trx)-tag; Fasciola hepatica 8-kDa antigen (Fh8)-tag; small ubiquitin modified tag (SUMO)-tag; solubility enhancing peptide sequences (SET)-tag; IgG domain B1 of protein G (GB1)-tag; Repeat of the IgG ZZ domain of protein A (ZZ)-tag; solubility enhancement ubiquitous tag (SNUT)-tag; 17 kDa protein (Skp)-tag; phage T7 protein kinase (T7PK)-tag; E. coli secreted protein A (EspA) tag; monomeric protein of bacteriophage T7, encoded by the 0.3(Orc) gene (Orc protein)/Mocr tag; E. coli trypsin inhibitor (Ecotin)-tag; calcium binding protein (CaBP) tag; stress response arsenate reductase (ArsC)-tag; N-terminal fragment of translation initiation factor IF2 (GB2 domain C-tag; expression tag (N-terminal fragment of translation initiation factor IF2); stress response proteins RpoA, SlyD, Tsf, RpoS, PotD, Crr-tags; acidic proteins E . coli tags msyB, yjgD, rpoD (see, for example, Costa S. et al., Frontiers in Microbiology 2014, (5), 63; especially Table 1 in Costa et al., 2014).

Accordingly, it is preferred that the label includes or consists of any amino acid sequence from SEQ ID NO: 20-42 or a variant of such sequence. Most preferably, the tag is a Strep tag, in particular according to SEQ ID NO: 20 or 34.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of an antibody, or an antigen binding fragment thereof, in accordance with the present invention contains or consists of a receptor or a functional fragment thereof. The term receptor means a polypeptide or protein that binds a specific (signaling) molecule, its ligand, and which can initiate a response, for example, in a cell. In nature, receptors are particularly located on/in the cell membrane (cell surface receptors) or intracellularly (intracellular receptors). Preferred receptors include ion channel coupled (ionotropic) receptors, G protein coupled (metabotropic) hormone receptors, enzyme coupled hormone receptors, cytoplasmic receptors and nuclear receptors. For receptors that form a dimer, the functional domain of the first and/or second polypeptide chain may comprise two identical domains connected by a linker.

Preferred are receptors containing an Ig-like domain. In particular, the receptor may be an inhibitory receptor comprising an Ig-like domain or an activating receptor comprising an Ig-like domain. Preferred examples of inhibitory receptors containing an Ig-like domain include: programmed cell death protein 1 (PD-1 or PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), B and T lymphocyte attenuator (BTLA), T -cell immunoglobulin and mucin domain-containing receptor-3 (TIM-3; also called hepatitis A virus cellular receptor 2 (HAVCR2)), T-cell receptor with Ig and ITIM domains (TIGIT), cell surface glycoprotein receptor 1 CD200 ( CD200R1), 2B4 (CD244; SLAMF4), Trem/trigger receptor expressed on myeloid cells)-like transcript 2 (TLT2), leukocyte immunoglobulin-like receptor superfamily B representative 4 (LILRB4), and killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2). Preferred examples of activated receptors containing an Ig-like domain include inducible T-cell COStimulator (ICOS) and CD28. Specifically, the receptor is programmed cell death protein 1 (pro

- 23 046243 grammed cell death protein 1 - PD-1 or PD1) or Signaling lymphocytic activation molecule (SLAM).

Other preferred receptors are soluble receptors, such as those described in Heaney M.L., Golde D.W. J Leukoc Biol. 1998, 64(2), 135-146. Examples include TNFR (tumor necrosis factor receptor), p55, p75, Fas (CD95), nerve growth factor receptor, CD27, CD30, growth hormone receptor, GM-CSF receptor, erythropoietin receptor (EpoR), thrombopoietin receptor, G-CSF receptor, IL-1RI (interleukin 1 receptor), IL-1RII (interleukin 1 receptor), IL-2Ra (interleukin 2 receptor, Tac, CD25), IL-4R (interleukin 4 receptor), IL-5Ra (interleukin 5 receptor a ), IL-7R (interleukin 7 receptor), IL-6Ra (interleukin 6 receptor), gp130, CNTFR (ciliary neurotrophic factor receptor), LIFR (leukemia inhibitory factor receptor), leptin receptor, IL-11R (interleukin 11 receptor) , IL-12 p40 (p40 protein capable of binding to the interleukin 12 receptor), stem cell factor receptor (c-kit), interferon receptor, lipopolysaccharide receptor (CD14), complement receptor type I (CD35), hyaluronate receptor (CD44), CD58, IgE receptor (FcεRII, CD23), IgG receptor (FcyRII), ICAM-1 (CD54), ICAM-3 (CD50), transforming growth factor β receptor type III, epidermal growth factor receptor (c-erb B), receptor Vascular endothelial growth factor receptor, platelet-derived growth factor receptor, fibroblast growth factor, colony stimulating factor receptor-1 (MCFR, c-fms), ARK (adrenergic receptor kinase), Tie (angiopoietin receptor), insulin receptor, insulin-like growth factor II receptor and mannose-6-phosphate.

More preferably, the soluble receptor is a soluble cytokine receptor, for example, a class I cytokine receptor superfamily receptor, a class II cytokine receptor superfamily receptor, an IL-1/TLR family receptor, a TGF-β family receptor, a TNFR superfamily receptor, or an IL-17R. Preferred receptors of the class I cytokine receptor superfamily include IL-4Ra, IL-5Ra, IL-6Ra, IL-7Ra, IL-9Ra, EpoR, G-CSFR, GM-CSFRa, gp130, and LIFRa. Preferred receptors of the class II cytokine receptor superfamily include type I IFNRs, for example, IFNAR1 and IFNAR2a. Preferred IL-1/TLR family receptors include IL-1RII and IL1RacP. Preferred receptors of the TGF-β receptor family include TeR-I and activin receptor-like kinase 7. Preferred TNFR superfamily receptors include TNFRSF6/Fas/CD95 and TNFRSF9/4-1BB/CD137. Accordingly, preferred examples of cytokine receptors include IL-4Ra, IL-5Ra, IL-6Ra, IL-7Ra, IL-9Ra, EpoR, G-CSFR, GM-CSFRa, gp130, LIFRa, IFNAR1, IFNAR2a, IL-1RII, IL-1RacP, TeR-I, activin receptor-like kinase 7, TNFRSF6/Fas/CD95, TNFRSF9/4-1BB/CD137 and IL-17R. An antibody or antigen-binding fragment containing a functional domain comprising such a receptor, or a functional fragment thereof, can modulate the inflammatory response when the antibody reaches its target. For example, soluble IL-1 receptor type II (sIL-1RII), which is produced primarily by proteolytic cleavage in response to various stimuli, can attenuate excess IL-1 bioactivity, primarily by binding ΓΒ-1β. For example, soluble IL-1RAcP, which is produced by alternative splicing rather than ectodomain cleavage. For example, soluble IL-6 receptors bind IL-6 with an affinity similar to membrane IL-6R, thereby prolonging the half-life of IL-6.

A functional receptor fragment may be any receptor fragment that has the ability to mediate functionality. Typically such fragments are called domains. Accordingly, the functional fragment of the receptor may be any domain of the receptor. Preferred examples include functional fragments (eg, domains) of the receptors described above as examples. Preferably, the functional receptor fragment, which consists of an (additional) functional domain, represents the extracellular domain of the receptor. For example, the (additional) functional domain of the first polypeptide chain and/or the second polypeptide chain may be an extracellular domain of any of the following receptors: IL-4Ra, IL5Ra, IL-6Ra, IL-7Ra, IL-9Ra, EpoR, G-CSFR , GM-CSFRa, gp130, LIFRa, IFNAR1, IFNAR2a, IL-1RII, IL-1RacP, TβR-I, activin receptor-like kinase 7, TNFRSF6/Fas/CD95, TNFRSF9/4-1BB/CD137, IL-17R, p55, p75, nerve growth factor receptor, CD27, CD30, growth hormone receptor, thrombopoietin receptor, IL-1RI (interleukin 1 receptor I), IL-2Ra (interleukin 2 receptor a, Tac, CD25), CNTFR (ciliary neurotrophic factor receptor ), leptin receptor, IL-11R (interleukin 11 receptor), IL-12 p40 (p40 protein capable of binding to the interleukin 12 receptor), stem cell factor receptor (c-kit), interferon receptor, lipopolysaccharide receptor (CD14), receptor complement type I (CD35), hyaluronate receptor (CD44), CD58, IgE receptor (FcεRII, CD23), IgG receptor (FcyRII), ICAM-1 (CD54), ICAM-3 (CD50), transforming growth factor receptor type III P , epidermal growth factor receptor (c-erb B), vascular endothelial growth factor receptor, platelet-derived growth factor receptor, fibroblast growth factor, colony stimulating factor receptor-1 (MCFR, c-fms), ARK (adrenergic receptor kinase), Tie (receptor angiopo

- 24 046243 ethin), insulin receptor, insulin-like growth factor II receptor and mannose-6phosphate receptor.

Preferably, the functional receptor fragment that consists of the (additional) functional domain is an Ig-like domain. For example, the (additional) functional domain of the first polypeptide chain and/or the second polypeptide chain may be an Ig-like domain of any of the following receptors: PD1, SLAM, LAIR1, CTLA4, BTLA, TIM-3, TIGIT, CD200R1, 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28. Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody, or antigen binding fragment thereof, in accordance with the present invention does not contain a transmembrane domain. Most preferably, the receptor includes or consists of the amino acid sequence set forth below, which is any of the sequences of SEQ ID NO: 1315 or a functional variant of such a sequence.

In addition, it is particularly preferred that the functional domain of the first polypeptide chain and/or the second polypeptide chain includes or consists of a mutant fragment of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), as described in WO 2016/207402 A1. The LAIR1 mutant fragment shown as SEQ ID NO: 13, or a sequence variant thereof, which is at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably 90%, particularly preferably 95%, and most preferably at least 98% identical in sequence.

Particularly preferably, the functional domain of the first polypeptide chain and/or the second polypeptide chain includes or consists of a PD1 Ig-like fragment or SLAM, for example, the amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15; or a sequence variant thereof which is at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably 90%, particularly preferably 95%, and most preferably at least 98% sequence identical.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention includes or consists of a ligand or a functional fragment thereof. The term ligand means a molecule that specifically binds to a specific site on a protein or some other molecule. In the context of the present invention, a ligand means a peptide, polypeptide or protein as it is contained in a polypeptide chain. Ligand binding occurs in particular through intermolecular forces, such as ionic bonds, hydrogen bonds and van der Wals forces. Preferred examples of ligands are cytokines and ligands of any of the receptors described above, in particular the receptors PD1, SLAM, LAIR1, CTLA4, BTLA, TIM-3, TIGIT, CD200R1, 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28 , for example, PD-L1, PD-L2, B7-1, B7-2, B7-H4 (B7 homolog), galectin-9, polio virus receptor (PVR), OX-2 membrane glycoprotein, CD48, B7-H3 ( homolog B7), MHCI and ICOS-L.

Preferably the ligand is a cytokine or a functional fragment thereof. Cytokines are typically small proteins (~5-20 kDa) that are important in cell signaling. They are released by cells and influence the behavior of other cells and sometimes the behavior of the releasing cell itself. The cytokine may be selected from chemokines, for example, the SIS cytokine family, the SIG cytokine family, the SCY cytokine family, the platelet factor 4 and intercrine superfamily, CC chemokine ligands (CCL-1 to CCL-28, particularly CCL12), CXCL1 CXCL17, XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β), fractalkine (or CX3CL1); interferons, e.g. IFN type I, IFN type II and IFN type III, in particular IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-SSS, IFN-ω, IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12); interleukins, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, Il-13, Il-14, Il-15, Il-16, Il-17, Il-18, Il-19, Il-20, Il-21, Il-22, Il-23, Il-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL33, IL-34, IL-35 and IL-36; lymphokines, for example, IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF and interferon-gamma; tumor necrosis factors, for example, CD40LG (TNFSF5); CD70 (TNFSF7); EDA; FASLG (TNFSF6); LTA (TNFSF1); LTB (TNFSF3); TNF, TNFa, TNFSF4 (OX40L); TNFSF8 (CD153); TNFSF9; TNFSF10 (TRAIL); TNFSF11 (RANKL); TNFSF12 (TWEAK); TNFSF13; TNFSF13B; TNFSF14; TNFSF15; and TNFSF18; and colony-stimulating factors, for example, CSF1 (also known as macrophage colony-stimulating factor), CSF2 (also known as granulocyte macrophage colony-stimulating factor; GM-CSF and sargramostim), CSF3 (also known as granulocyte colony-stimulating factor (G-CSF and filgrastim), and also from synthetic CSF, for example, promegapoietin. Accordingly, preferred examples of cytokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 , IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL -22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34 , IL-35, IL-36,

- 25 046243

CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, CCL-6, CCL-7, CCL-8, CCL-9, CCL-10, CCL-11, CCL-12, CCL- 13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-20, CCL-21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-26, CCL-27, CCL-28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, fractalkine, IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-κ, IFN-ω, IL10R2, IFNLR1, CD40LG, CD70, EDA, FASLG (TNFSF6), LTA (TNFSF1), LTB (TNFSF3) , TNFa, TNFSF4 (OX40L), TNFSF8 (CD153), TNFSF9, TNFSF1O (TRAIL), TNFSF11 (RANKL), TNFSF12 (TWEAK), TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, CSF1, CSF2 (GM-CSF) and CSF3 (G-CSF). More preferred examples of cytokines include IL-2, IL6, IL10, IL-12, IL-15, IL-17, interferons, GM-CSF and TNF. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts and various stromal cells, resulting in a given cytokine being produced by multiple cell types. An antibody or antigen-binding fragment comprising a functional domain containing such a cytokine or a functional fragment thereof may induce a pro-inflammatory immunostimulatory response or an anti-inflammatory immunosuppressive or cytotoxic response depending on the cytokine selected.

Other preferred ligands include, for example, hormones that are peptides, polypeptides or proteins. Hormones are signaling molecules that are transported by the circulatory system to distant target organs, particularly to regulate physiology and behavior. Hormones are usually produced by the glands of multicellular organisms. A particularly preferred hormone is (human) growth hormone. Additional examples of hormones include TRH, vasopressin, insulin, prolactin, ACTH, oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, leptin, angiotensin II, basic fibroblast growth factor-2, and parathyroid hormone-related protein.

A functional ligand moiety may be any ligand moiety that has the ability to mediate functionality. Typically such fragments are called domains. Accordingly, the functional ligand moiety may be any domain of the ligand. Preferred examples include functional fragments (eg, domains) of the (exemplified) ligands described above. Preferably, the functional ligand moiety that consists of the (additional) functional domain is an Ig-like domain.

Preferably, the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains or consists of an (independent) binding site. Accordingly, it is preferred that the first and/or second polypeptide chain of an antibody or antigen-binding fragment thereof according to the present invention contains an (independent) binding site.

In general, the term (independent) binding site refers to a region of a polypeptide chain to which a specific target (eg, molecule and/or ion) can bind, particularly through the formation of a chemical bond, eg, a non-covalent bond. A noncovalent bond is a relatively weak chemical bond that does not involve a tight exchange of electrons. Multiple noncovalent bonds often stabilize the conformation of macromolecules and provide highly specific interactions between molecules. Accordingly, the binding site represents a functional domain of the first and/or second polypeptide chain that provides binding functionality. In particular, the binding site is not a linker, such as a GS linker. Even though the binding site may not necessarily contain a linker (peptide), such as a GS linker, it preferably does not consist of a linker (peptide), such as a GS linker. That is, even if the binding site contains a linker (peptide), for example a GS linker, it preferably contains an additional amino acid sequence providing a function other than (exclusively) linking two peptides to each other. Accordingly, the binding site is preferably different from the linker (peptide), for example, a GS linker. In particular, the binding site may not contain a linker (peptide), for example a GS linker. A linker typically does not provide linking functionality.

It is important that the (independent) binding site (ii) of the first polypeptide chain does not contain a fragment of the second polypeptide chain. Accordingly, the optional independent binding site (v) of the second polypeptide chain does not contain a fragment of the first polypeptide chain. That is, the second polypeptide chain (in particular, any fragment of it, even one amino acid) is not required or does not participate in the independent binding site of the first polypeptide chain. Moreover, if the second polypeptide chain also contains an independent binding site, the first polypeptide chain (in particular, any fragment thereof, even a single amino acid) is not required or does not participate in the independent binding site of the second polypeptide chain. Accordingly, the independent binding site is different from the antigen binding site formed by the variable domain of the first polypeptide chain together with the variable domain of the second polypeptide chain. That is, the independent binding site is different from the antigen binding site formed by the variable domains of two different polypeptide chains (however, the independent binding site may still contain an antigen binding site, provided

- 26 046243 that the variable domains are located in the same polypeptide chain, which is described in more detail below).

Preferably, the (independent) binding site is selected from the group consisting of receptors and functional fragments thereof, ligands and functional fragments thereof, CD molecules and functional fragments thereof, single chain antibodies and antigen binding fragments thereof, antigens and functional fragments thereof, and tags.

More preferably, the (independent) binding site contains or consists of a receptor or a functional fragment thereof. Receptors are usually capable of binding to a (specific) ligand. Accordingly, receptors can also be referred to as (independent) binding sites. Various receptors are described above, as well as preferred embodiments and examples of their use, respectively.

In the context of a binding site, a functional receptor fragment is characterized by retaining the ability of the receptor to bind its ligand. Since the binding site may contain a receptor or a functional fragment thereof, the term functional refers to the binding function of the receptor in the context of the binding site. Other receptor fragments/domains may preferably not be included in the (independent) binding site. For example, a receptor may include one or more transmembrane domains that are not normally involved in the binding function of the receptor and that are thus preferably not included in the (independent) binding site. Accordingly, it is most preferred that the receptor fragment that consists of an (independent) binding site is simply a receptor binding site (especially without any additional receptor domains).

It is also more preferable that the (independent) binding site contains or consists of a ligand or a functional fragment thereof. Ligands are usually able to bind to a (specific) receptor. Accordingly, ligands may also be referred to as (independent) binding sites. Various ligands, preferred embodiments thereof and, accordingly, examples of their use are described above.

In the context of a binding site, a functional ligand moiety is that moiety of the ligand that retains its binding ability. Since the binding site may contain a ligand or a functional fragment thereof, the term functional refers to the binding function of the ligand in the context of the binding site. Other ligand fragments/domains may preferably not be included in the (independent) binding site. Accordingly, it is most preferred that the ligand moiety that consists of an (independent) binding site is simply a ligand binding site (especially without any additional ligand domains).

Preferably, the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is a CD (cluster of differentiation) molecule or a functional fragment thereof. The CD (cluster of differentiation) molecule is a marker on the cell surface. CD molecules often act as receptors or ligands or are involved in cell adhesion. The CD nomenclature was developed and maintained within the HLDA (Human Leukocyte Differentiation Antigens) working group established in 1982. Examples of CD molecules that can serve as binding sites in the context of the present invention can be obtained, for example, from various sources known to one skilled in the art, for example, at http://www.ebioscience.com/resources/human-cd- chart.htm, in the BD Bioscience Human and Mouse CD Marker Handbook (available at https://www.bdbiosciences.com/documents/cd_marker_handbook.pdf) or www.hcdm.org. Accordingly, the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain may be a CD marker or a functional fragment thereof, for example a CD (human) marker described in the BD Bioscience Human and Mouse CD Marker Handbook (available at https:/ /www.bdbiosciences.com/documents/cd_marker_handbook.pdf) or other sources of CD marker charts, which usually also indicate binding partners so that the appropriate binding site can be selected.

A functional CD molecule fragment is a fragment of the CD molecule that retains the binding ability of the CD molecule. In the context of the present invention, the binding site may comprise a CD molecule or a functional fragment thereof, and accordingly it is the binding function of the CD molecule to which the term functional refers. Other fragments/domains of the CD molecule may preferably not contain an (independent) binding site. Accordingly, it is most preferred that the fragment of the CD molecule that consists of an (independent) binding site is simply a binding site for the CD molecule (especially without any additional domains of the CD molecule). Preferably, the functional fragment of the CD molecule that consists of the (independent) binding site is an Ig-like domain.

Preferably, the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is a single chain antibody (eg scFv or VHH) or an antigen binding fragment thereof. It is also preferable that the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is an antigen or a functional thereof

- 27 046243 ny fragment, for example, epitope.

Preferably, the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is a single chain antibody or an antigen binding fragment thereof. A single chain antibody is a recombinant antibody consisting of only a single polypeptide chain. Preferred examples of single-chain antibodies include single-chain antibodies without constant domains, such as single-chain antibodies, single-chain variable fragment (scFv) antibodies and single-chain diabodies (scDb), as well as single-chain antibodies with constant domains, such as single-chain Fab fragments (scFab) ; Hust M. et al., BMC Biotechnol 2007, 8(7), 14).

Preferred examples of single chain antibodies based on single chain variable fragments (scFv) include scFv (single VH domain and single VL domain) and tandem scFvs, such as tandem di-scFv (BiTE), tandem tri-scFv and tandem tetra-scFv. ScFv.

A single domain antibody (also called a nanobody) is an antibody fragment containing/consisting of only one (monomeric) variable domain. Like a whole antibody, a single domain antibody is capable of selectively binding to a specific antigen. The first single-domain antibodies were constructed from heavy chain antibodies found in camels; these antibodies designate VHH or VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR, immunoglobulin new antigen receptor - a new immunoglobulin antigen receptor), from which single-domain antibodies called V _NAR or VNAR fragments can be obtained. Another approach is to separate dimeric variable domains from common immunoglobulin G ( IgG) human or mouse into monomers. Accordingly, single domain antibodies can be derived from heavy or light chain variable domains (VH or VL). Preferred examples of single domain antibodies include VHH, VNAR, as well as IgG-derived VH and IgG-derived VL.

Most preferably, the functional domain of the first polypeptide chain and/or the second polypeptide chain is VHH or scFv. The most preferred example of VHH is T3VHH or F4-VHH. For example, the single domain antibody preferably contains or consists of the amino acid sequence of SEQ ID NO: 16 or 18, or a variant thereof that is at least 70%, at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably 90%, especially preferably 95%, and most preferably at least 98% sequence identical. The most preferred example of scFv is TT39.7-scFv or MPE8-scFv. For example, a single domain antibody contains or consists of the amino acid sequence of SEQ ID NO: 17 or 19, or a variant thereof that is at least 70%, at least 75%, more preferably at least 80% %, even more preferably at least 85%, even more preferably 90%, especially preferably 95%, and most preferably at least 98% identical in sequence.

Preferably, the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is an antigen or a functional fragment thereof, in particular an epitope. An antigen is a molecule or part of a molecule that can be bound by an antibody. Since an antigen or a functional fragment thereof consists of a polypeptide chain, it should be noted that in the context of the present invention, if the binding site is an antigen or a functional fragment thereof, said antigen or a functional fragment thereof is a peptide or polypeptide. An antigen usually contains one or more epitopes. An epitope is the part of an antigen that is bound by an antibody (recognized by the antibody). Preferred examples of antigens include, but are not limited to, serum proteins such as cytokines such as IL4, IL5, IL9 and IL13, bioactive peptides, cell surface molecules such as receptors, transporters, ion channels, viral and bacterial proteins, RAGE (Receptor for Advanced Glycosylation End Products - receptor for advanced glycosylation of end products), GPVI and collagen.

A functional antigen fragment is a fragment of an antigen that retains antigen binding capacity. Accordingly, the antigen fragment is preferably an epitope or contains one or more epitopes. Other antigen fragments/domains may preferably not be included in the (independent) binding site. Accordingly, it is most preferred that the antigen fragment that is included in the (independent) binding site is an epitope or includes more than one epitope (particularly without any additional antigen domains).

It is also preferred that the (independent) binding site of the first polypeptide chain and/or the second polypeptide chain is a tag containing the binding site. Most tags can bind, such as affinity tags. Accordingly, those tags that have the ability to bind to another molecule can also be called (independent) binding sites. Various labels, including those labels that contain a binding site, are described above, and preferred embodiments and examples thereof are used accordingly.

In most cases, the (additional) functional domain of the first polypeptide chain and/or the second polypeptide chain is an Ig-like domain, scFv, VHH or Strep-tag. IN

- 28 046243 in particular, the (additional) functional domain of the first polypeptide chain and/or the second polypeptide chain preferably includes or consists of any amino acid sequence SEQ ID NO: 13-20 from the list below, or a functional variant of such a sequence that, at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity.

Preferably, the first polypeptide chain contains a single variable domain of (i) an N-terminal (optional) functional domain (ii), and the second polypeptide chain contains a single variable domain of (iv) an N-terminal optional (optional) functional domain (v) or an N-terminal one or several constant domains (vi), wherein the single variable domain (iv) of the second polypeptide chain forms an antigen-binding site with the variable domain (i) of the first polypeptide chain. Preferred examples of such antibodies and antigen binding fragments according to the present invention are shown in FIG. 1B and 1B (all formats in FIGS. 1B and 1C except DVD-Ig antibodies are shown in the bottom-most panel of FIG. 1B).

In another embodiment, the first polypeptide chain may comprise two or more variable domains of (i) an N-terminal (optional) functional domain (ii), and the second polypeptide chain may comprise two or more variable domains of (iv) an N-terminal optional (optional) domain. functional domain (v) or the N-terminus of one or more constant domains (vi), two or more variable domains (iv) of the second polypeptide chain forming antigen binding sites with two or more variable domains (i) of the first polypeptide chain. For example, a scaffold antibody having an (additional) functional domain inserted into its fold region may be a bispecific antibody in DVD-Ig format, for example as shown in FIG. 1B, bottommost panel of DVDIg.

It is also preferred that the first polypeptide chain comprises a single chain antibody, eg scFv, or a single domain antibody, eg VHH, the N-terminus of an N-terminal variable domain (i) and/or the C-terminus of a C-terminal constant domain (iii). Alternatively or additionally, the second polypeptide chain may comprise a single chain antibody, eg scFv, or a single domain antibody, eg VHH, the N-terminus of the N-terminal variable domain (i) or the C-terminus of the C-terminal constant domain (vi). Preferred examples thereof are shown in FIGS. 1B and include scFv- (H) IgG, IgG (H) -scFv, scFv- (L) IgG, IgG (L) -scFv, V-(H) IgG, IgG (H) -V, V- (L) IgG and IgG(L)-V antibodies with one or more (additional) functional domains inserted into the bend region of the heavy and/or light chain(s).

Preferably, each first polypeptide chain and second polypeptide chain comprise a single constant domain, in particular a CL domain and a CH1 domain, respectively. Preferred examples thereof are shown in FIGS. 1B and include F(ab) ₂ and Fab antibody fragments with one or more (additional) functional domains inserted into the bend region of the heavy and/or light chain(s).

In another embodiment of the present invention, it is also preferred that the first polypeptide chain or the second polypeptide chain comprise a single constant domain, in particular a CL domain; and the other, the first polypeptide chain or the second polypeptide chain, contains a CH1 domain and one or more additional constant domains, for example, a CH2 domain and/or a CH3 domain. Preferred examples of such antibodies and antigen binding fragments according to the present invention are shown in FIG. 1B and 1C (all formats of FIGS. 1B and 1B except F(ab)2 and Fab based antibody fragments shown in the top panels of FIGURE 1B).

Preferably, the antibody or antigen binding fragment thereof according to the present invention contains an Fc fragment, in particular an Fc region. More preferably, the Fc fragment is derived from a human antibody, such as human IgG1, IgG2, IgG3 and/or IgG4, with human IgG1 being particularly preferred.

In the context of the present invention, the term Fc fragment refers to a sequence derived from a portion of the heavy chain of an immunoglobulin starting in the hinge region immediately upstream of the papain cleavage site (for example, residue 216 in native IgG, with the first residue of the heavy chain constant region being at position 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, the Fc fragment may be a complete Fc fragment or a portion thereof (eg, a domain). A complete Fc fragment contains at least a hinge domain, a CH2 domain, and a CH3 domain (eg, amino acid positions EU 216-446). An additional lysine residue (K) is sometimes present at the extreme C-terminus of the Fc fragment, but is often cleaved from the mature antibody. Each of the amino acid positions in the Fc fragment was numbered in accordance with the Kabat numbering system adopted in the EU, see, for example, the publication by Kabat et al., in the book: Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1983 and 1987.

Preferably, in the context of the present invention, the Fc fragment includes at least one of the following sequences: a hinge (eg, upper, middle and/or lower hinge region) domain, CH2 domain, CH3 domain, or a variant, part, or fragment thereof. In prev

- 29046243 In some embodiments of the present invention, the Fc moiety comprises at least a hinge domain, a CH2 domain, or a CH3 domain. More preferably, the Fc fragment is a complete Fc fragment. The Fc fragment may also contain one or more amino acid insertions, deletions or substitutions relative to the natural Fc fragment. For example, at least one of the hinge domains of the domain, the CH2 domain or the CH3 domain (or portions thereof), may be removed. For example, an Fc fragment may contain or consist of: (i) a hinge domain (or part thereof) hybridized with a CH2 domain (or part thereof), (ii) a hinge domain (or part thereof) hybridized with a CH3 domain (or part thereof) ), (iii) a CH2 domain (or part thereof) hybridized with a CH3 domain (or part thereof), (iv) a hinge domain (or part thereof), (v) a CH2 domain (or part thereof), or (vi) a CH3 domain or parts thereof.

One skilled in the art will appreciate that the Fc fragment can be modified to differ in amino acid sequence from the complete Fc fragment of a naturally occurring immunoglobulin molecule while retaining at least one desired function of the naturally occurring Fc fragment. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement fixation. Portions of naturally occurring Fc fragments that are responsible and/or necessary for such functions are well known to those skilled in the art.

For example, to activate complement, the C1q cascade binds to at least two IgG1 molecules or one IgM molecule attached to the target antigen (Ward E.S., Ghetie V. Ther. Immunol. 2, 1995, 77-94). Burton D.R. (Mol. Immunol. 22, 1985, 161-206) described that the heavy chain region containing amino acid residues 318 to 337 is involved in complement fixation. Duncan A.R., Winter G. (Nature 332, 1988, 738-740) reported that using site-directed mutagenesis Glu318, Lys320 and Lys322 form a binding site for Clq. The role of residues Glu318, Lys320, and Lys 322 in C1q binding was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement-mediated lysis.

For example, FcR binding may be mediated by the interaction of an Fc moiety (antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and various other cellular targets (eg, tumor cells) coated with the corresponding antibody through antibody-dependent cellular cytotoxicity (ADCC; Van de Winkel J.G., Anderson C.L., J. Leukoc. Biol. 49, 1991, 511-524). FcR receptors are defined by their immunoglobulin class specificity; Fc receptors for IgG antibodies are designated FcyR, for IgE as FcεR, for IgA as FcaR, and so on, and neonatal Fc receptors are designated as FcRn. Binding to the Fc receptor is described, for example, in the publications of Ravetch J.V., Kinet J.P., Annu. Rev. Immunol. 9, 1991, 457-492; Capel P. J. et al., Immunomethods, 4, 1994, 25-34; de Haas M. et al. J Lab. Clin. Med. 126, 1995, 330-341; Gessner J. E. et al. Ann. Hematol. 76, 1998, 231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a wide range of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity and release of inflammatory mediators, as well as clearance of immune complexes and regulation of antibody production. Therefore, receptor cross-linking Fc fragments (FcyR) are preferred. In humans, three classes of FcyRs have been characterized, namely: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complex IgG with medium to low affinity, is widely expressed particularly on leukocytes, is a central player in antibody-mediated immunity and can be divided into FcyRIIA, FcyRIIB and FcyRIIC, which have different functions in the immune system. system, but bind with similar low affinity to IgG-Fc, and the ectodomains of these receptors are highly homologous; and (iii) FcyRIII (CD16), which binds IgG with moderate to low affinity and exists as two types: FcyRIIIA, found on NK cells, macrophages, eosinophils and some monocytes and T cells, and mediating ADCC and FcyRIIIB, which is highly expressed on neutrophils, FcyRIIA is found in many cells involved in killing (eg, macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcyRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, mast cells and eosinophils.

With regard to FcyRI binding, modification of the native IgG with at least one of E233G236, P238, D265, N297, A327 and P329 reduces FcyRI binding. IgG2 residues at positions 233-236, replaced by IgG1 and IgG4, reduce binding to FcyRI by 10 times and eliminate the response of human monocytes to antibody-sensitized erythrocytes (Armor K.L. et al. Eur. J. Immunol. 29, 1999, 26132624). Regarding FcyRII binding, reduced binding to FcyRIIA was found, for example, for the IgG mutation at at least one of positions E233-G236, P238, D265, N297, A327, P329, D270,

- 30 046243

Q295, A327, R292 or K414. Regarding FcyRIII binding, reduced binding to FcyRIIIA was found, for example for mutation at at least one of positions E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303 , A327, K338 and D376. Mapping of binding sites on human IgG1 for Fc receptors, the aforementioned mutation sites, and methods for measuring binding to FcyRI and FcyRIIA are described in Shields R.L. et al. J Biol. Chem 276, 2001, 6591-6604.

With regard to binding to the critical FcyRII, two regions of the native IgG Fc appear to be critical for FcyRII and IgG interactions, namely: (i) the lower hinge site of the IgG Fc, in particular the amino acid residues L, L, G, G (234 - 237, numbering EU) and (ii) the adjacent region of the CH2 domain in the IgG Fc, in particular the loop and strands in the upper CH2 domain adjacent to the lower hinge region, for example in the P331 region (Wines, B.D. et al. J. Immunol. 164 , 2000, 5313-5318). In addition, FcyRI appears to bind to the same site on the IgG Fc, whereas FcRn and protein A bind to a different site on the IgG Fc, which appears to be at the CH2-CH3 interface (Wines, BD with J Immunol 164, 2000, 5313-5318).

For example, the Fc fragment may contain or consist of at least a portion of the Fc fragment that is known in the art to be necessary for FcRn binding or increased half-life. Alternatively or additionally, the Fc fragment of an antibody of the present invention contains at least a portion known in the art that is required for binding to protein A, and/or the Fc fragment of an antibody of the present invention contains at least a portion of the molecule An Fc known in the art that is required for protein G binding. Accordingly, a preferred Fc fragment contains at least a portion known in the art that is required for FcyR binding. As stated above, a preferred Fc fragment may thus comprise at least (i) the lower hinge site of the native IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numbering) and (ii ) an adjacent region of the CH2 domain of a native IgG Fc, in particular the loop and strands in the upper CH2 domain adjacent to the lower loop region, for example in the P331 region, for example regions of at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids in the upper CH2 domain of the native IgG Fc around P331, for example between amino acids 320 and 340 (EU numbering) of the native IgG Fc.

Preferably, the antibody, or antigen binding fragment thereof, of the present invention includes an Fc region. In the context of the present invention, the term Fc region means the part of an immunoglobulin formed by two or more parts of the Fc molecule of the heavy chains of an antibody. For example, the Fc region may be a monomeric or single chain Fc region (ie, a scFc region). Single-stranded Fc regions consist of Fc fragments linked within a single polypeptide chain (eg, encoded in a single contiguous nucleic acid sequence). Examples of the scFc region are described in WO 2008/143954 A2. Preferably, the Fc region is a dimeric Fc region. The term dimeric Fc region or dcFc refers to a dimer formed by the Fc fragments of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc fragments (eg, the Fc region of a natural immunoglobulin) or a heterodimer of two non-identical Fc fragments.

The Fc fragments of the Fc region may be of the same or different classes and/or subclasses. For example, Fc fragments can be derived from an immunoglobulin (eg, human immunoglobulin) of the IgG1, IgG2, IgG3, or IgG4 subclass. Preferably, Fc fragments of the Fc region are of the same class and subclass. However, the Fc region (or one or more Fc portions of the Fc region) may also be chimeric, and the chimeric Fc region may contain Fc fragments derived from different classes and/or subclasses of immunoglobulins. However, the Fc region (or one or more Fc fragments of the Fc region) may also be chimeric, and the chimeric Fc region may contain Fc fragments derived from different classes and/or subclasses of immunoglobulins. For example, at least two of the Fc fragments of a dimeric or single chain Fc region may be from different immunoglobulin classes and/or subclasses. Additionally, or alternatively, the chimeric Fc regions may comprise one or more chimeric Fc fragments. For example, a chimeric Fc region or fragment may contain one or more portions derived from a first subclass immunoglobulin (eg, IgG1, IgG2, or IgG3 subclass), although the remainder of the Fc region or fragment is of a different subclass. For example, an Fc region or fragment of an Fc polypeptide may comprise a CH2 and/or CH3 domain derived from a first subclass immunoglobulin (eg, IgG1, IgG2, or IgG4 subclass) and a hinge region from a second subclass immunoglobulin (eg, IgG3 subclass). For example, the Fc region or fragment may comprise a hinge region and/or a CH2 domain derived from a first subclass immunoglobulin (eg, IgG4 subclass) and a CH3 domain derived from a second subclass immunoglobulin (eg, IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region may comprise an Fc fragment (eg, a complete portion of an Fc molecule) from an immunoglobulin for a first subclass (eg, the IgG4 subclass) and an Fc group from an immunoglobulin of a second subclass (eg, the IgG1, IgG2, or IgG3 subclass). For example, the Fc region or fragment may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, an Fc region or fragment may contain up to

- 31 046243 men CH1 and CH2 domain from the IgG4 molecule and CH3 domain from the IgG1 molecule. For example, the Fc region or fragment may comprise part of a CH2 domain from a particular antibody subclass, for example, positions EU 292-340 of the CH2 domain. For example, the Fc region or fragment may contain amino acids at positions 292-340 CH2 derived from the IgG4 fragment and a CH2 residue derived from the IgG1 fragment (in another case, 292-340 of the CH2 domain may be derived from the IgG1 fragment and the CH2 residue derived from an IgG4 fragment).

In addition, the Fc region or fragment may (additionally or alternatively), for example, comprise a chimeric hinge region. For example, a chimeric hinge may be derived, for example, in part from an IgG1, IgG2 or IgG4 molecule (eg, the upper and lower middle loop sequences) and in part from an IgG3 molecule (eg, the middle loop sequences). In another example, the Fc region or fragment may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge can be obtained, for example, by introducing a proline substitution (Ser228Pro) at position EC 228 in the middle hinge domain of the IgG4 hinge region. In another embodiment of the present invention, the chimeric hinge may contain amino acids at positions EU 233-236 from the IgG2 antibody and/or the Ser228Pro mutation, where the remaining amino acids of the hinge are derived from the IgG4 antibody. Chimeric loops that can be used in the Fc fragment of an antibody according to the present invention are described in US 2005/0163783 A1.

In the present invention, it is preferred that the Fc fragment or Fc region contains or consists of an amino acid sequence derived from a human immunoglobulin sequence (eg, an Fc region or Fc fragment from a human IgG molecule). However, the polypeptides may contain one or more amino acids from a mammal of another species. For example, a primate Fc fragment or a primate binding site may be included in the polypeptides of the present invention. In another embodiment, one or more mouse amino acids may be present in the Fc group or Fc region.

Preferably, the antibody of the present invention includes, in particular, in addition to the Fc fragment as described above, other parts derived from the constant region, in particular from the IgG constant region, from the IgG1 constant region, more preferably from the human IgG1 constant region. More preferably, the antibody of the present invention includes, in particular, in addition to the Fc fragment as described above, all other parts of the constant regions, in particular all other parts of the IgG constant regions, preferably all other parts of the IgG1 constant regions, more preferably all other parts human IgG1 constant regions.

Particularly preferred constant region sequences are the amino acid sequences of SEQ ID NO: 3, 4 or 7. Preferably, the heavy chain constant region comprises or consists of IgG1 CH1-CH2-CH3, in particular comprising or consisting of the amino acid sequence SEQ ID NO: 3 or its variant of the functional sequence according to the description of the present invention. Preferably, the light chain constant region contains or consists of an IgG1 CL, in particular, contains or consists of the amino acid sequence of SEQ ID NO: 4 or a functional sequence variant thereof as described herein.

It is also preferable that the antibody or antigen binding fragment thereof according to the present invention does not contain an Fc region or Fc fragment. In particular, the constant domains required for the bend region (CH1 and CL) are usually not involved in the Fc fragment or Fc region. Therefore, the antibody or antigen binding fragment thereof according to the present invention does not contain an Fc fragment or Fc region. Examples of such antibodies (or antibody fragments) include Fab or F(ab) ₂ -based antibodies (IEI-Fab or IEI-F(ab) ₂ ).

Preferably, the first polypeptide chain and/or the second polypeptide chain of the antibody or antigen binding fragment of the present invention includes one or more linkers. For example, the first polypeptide chain and/or the second polypeptide chain of an antibody or antigen binding fragment of the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 linkers. More preferably, the first polypeptide chain and/or the second polypeptide chain of the antibody or antigen binding fragment of the present invention includes one or two linkers. It is also preferred that the first polypeptide chain and/or the second polypeptide chain of the antibody or antigen binding fragment of the present invention contains three or four linkers. Generally, a linker can provide greater flexibility to the polypeptide chain. Preferably, the first polypeptide chain and/or the second polypeptide chain comprises a linker between the constant region and the (additional) functional domain. In another embodiment, the first polypeptide chain and/or the second polypeptide chain may not contain a linker between the constant region and the (additional) functional domain. If the second polypeptide does not contain an (additional) functional domain, the second polypeptide chain may contain a linker between a constant region and a variable region. Alternatively, the second polypeptide chain may not contain a linker between the constant

- 32 046243 region and variable region. Preferably, the first polypeptide chain and/or the second polypeptide chain comprises a linker between the (additional) functional domain and a variable domain, in particular the C-terminal most variable domain. In another embodiment, the first polypeptide chain and/or the second polypeptide chain may not contain a linker between the (additional) functional domain and the variable domain, in particular the C-terminal most variable domain. If the first polypeptide chain and/or the second polypeptide chain contains more than one variable domain, it is also preferred that the first polypeptide chain and/or the second polypeptide chain contain one or more linkers between the variable domains. In another embodiment, the first polypeptide chain and/or the second polypeptide chain may not contain one or more linkers between the variable domains. More preferably, the first polypeptide chain and/or the second polypeptide chain comprises a linker between a constant region and a (additional) functional domain and a linker between a (additional) functional domain and a variable domain, in particular the C-terminal most variable domain. In another embodiment, the first polypeptide chain and/or the second polypeptide chain may contain neither a linker between the constant region and the (additional) functional domain, nor a linker between the (additional) functional domain and the variable domain, in particular the C-terminal most variable domain. In addition, the first polypeptide chain may contain a linker between the constant region and the (additional) functional domain and a linker between the (additional) functional domain and the variable domain, in particular the C-terminal most variable domain, although the second polypeptide chain may not contain either (additional) functional domain. functional domain, nor a linker between the constant region and the variable region. It is also preferred that the first polypeptide chain and/or the second polypeptide chain comprise more than one variable domain, such that the first polypeptide chain and/or the second polypeptide chain comprise a linker between a constant region and an (additional) functional domain; a linker between the (additional) functional domain and the variable domain, in particular the C-terminal most variable domain; and one or more linkers between the variable domains.

If the first polypeptide chain and/or the second polypeptide chain contains more than one linker, the linkers may be the same or different.

Preferably the linker comprises up to 20 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, more preferably up to 15 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids, even more preferably up to 10 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, and even more preferably up to 5 amino acids, for example 1, 2, 3, 4 or 5 amino acids. The linker may be derived from a natural amino acid sequence, for example, from flanking regions, or from artificial amino acid sequences. Preferably, the (additional) functional domain corresponds to one exon or more than one exon, and the amino acid sequence of the linker is derived from intronic sequences flanking the single exon or more than one exon. For example, (i) the linker between the variable region and the (additional) functional domain (which is encoded by an exon or sequence variant thereof) may be encoded by a flanking intronic sequence immediately upstream (in the 5'-3' direction) of the exon or sequence variant thereof used as an (additional ) functional domain and/or (ii) a linker between the (additional) functional domain (which is encoded by the exon or sequence variant thereof) and the constant region may be encoded by a flanking intronic sequence immediately after (in the 5'-3' direction) of the exon or sequence variant thereof for use as an (additional) functional domain. Preferably, the linker does not contain any Cys (C) residues. Preferably, the linker contains or consists of one or more glycine residues (Gly) and/or one or more serine residues (Ser) (GS linker). Preferred examples of GS linkers are the amino acid sequences of SEQ ID NO: 43-48, most preferably the linker is the sequence of SEQ ID NO: 45. Preferred examples of intronic sequence linkers are the amino acid sequences of SEQ ID NO: 49-52. Most preferably, the linker contains or consists of an amino acid sequence, namely any of the following sequences SEQ ID NO: 43-52.

For example, the first polypeptide chain and/or the second polypeptide chain may comprise (in the N- to C-terminal direction) a linker with the sequence SEQ ID NO: 49, or a functional sequence variant thereof, immediately followed by an (additional) functional domain with the sequence SEQ ID NO: 14, or a functional sequence variant thereof. In addition, the first polypeptide chain and/or the second polypeptide chain may contain (in the N- to C-terminal direction) an (additional) functional domain of SEQ ID NO: 14, or a functional sequence variant thereof immediately followed by a linker with sequence SEQ ID NO: 50, or a functional variant of the sequence. Most preferably, the first polypeptide chain and/or the second polypeptide chain may comprise

- 33 046243 rule from N- to C-terminus) (variable domain immediately followed by) a linker of the sequence SEQ ID NO: 49, or a functional variant of the sequence thereof, immediately followed by an (additional) functional domain of the sequence SEQ ID NO: 14, or a functional sequence variant thereof, immediately followed by a linker having the sequence SEQ ID NO: 50, or a functional sequence variant thereof (immediately followed by a constant domain).

For example, the first polypeptide chain and/or the second polypeptide chain may contain (in the N- to C-terminal direction) a linker with the sequence SEQ ID NO: 51, or a functional variant of the sequence, immediately followed by an (additional) functional domain with the sequence SEQ ID NO: 15, or a functional sequence variant thereof. In addition, the first polypeptide chain and/or the second polypeptide chain may contain (in the N- to C-terminal direction) an (additional) functional domain of SEQ ID NO: 15, or a functional sequence variant thereof immediately followed by a linker with sequence SEQ ID NO: 52, or a functional variant of the sequence. Most preferably, the first polypeptide chain and/or the second polypeptide chain may contain (in the N- to C-terminal direction) (variable domain immediately followed by) a linker having the sequence SEQ ID NO: 51, or a functional sequence variant thereof, followed by immediately followed by an (additional) functional domain of SEQ ID NO: 15, or a functional sequence variant thereof, immediately followed by a linker of SEQ ID NO: 52, or a functional sequence variant thereof (immediately followed by a constant domain).

In another embodiment, it is also preferred that the first polypeptide chain and/or the second polypeptide do not contain any linkers.

Preferably, the first polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention comprises or consists of (in N- to C-terminal direction): V-DCH1, where

V denotes variable domain (i);

D denotes (additional) functional domain (ii); and CH1 indicates the CH1 constant domain (iii).

V and D and/or D and CH1 may be connected via a linker as described above, or may be directly connected to each other.

More preferably, the first polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention contains or consists of (in the N- to C-terminal direction): V-D-CH1-CH2-CH3, wherein the designation V-D-CH1 is the same as described above, and CH2 and CH3 are the CH2 constant domain and the CH3 constant domain, respectively.

It is also preferred that the first polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention contains or consists of (in the N- to C-terminal direction): (V) _a -D-CH1, wherein the designation VD-CH1 is the same as described above, and A is an integer from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, and even more preferably 1 or 2, and the V variable domains can be connected to each other directly or through a linker.

Preferably, the second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention comprises or consists of (in N- to C-terminal direction): VCL, where

V denotes variable domain (iv), and CL denotes constant domain (vi).

V and CL may be connected via a linker as described above, or may be connected to each other directly.

It is also preferred that the second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention comprises or consists of (in the N- to C-terminal direction): (V) _a -CL, wherein the designation V-CL is the same as described above, And

A is an integer from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, and even more preferably 1 or 2, and the V variable domains can be connected to each other directly or through a linker.

For example, the variable domain (i) of the first polypeptide chain of an antibody or antigen binding fragment thereof in accordance with the present invention may contain or consist of an amino acid sequence of SEQ ID NO: 1, 5, 8 or 10, or a functional variant of such a sequence that at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% identical to the sequence.

For example, the variable domain (iv) of the second polypeptide chain of an antibody or antigen binding fragment thereof in accordance with the present invention may contain or consist of amino

- 34 046243 an acid sequence from SEQ ID NOs 2, 6, 9 or 11, or from a functional variant of such a sequence that is at least 80%, preferably at least 85%, more preferably, according to at least 90%, even more preferably at least 95%, and most preferably at least 98% identical in sequence.

Preferably, an antibody or antigen binding fragment thereof according to the present invention comprises one or two first polypeptide chains and one or two second polypeptide chains. Examples of such antibodies or antigen binding fragments thereof are shown in FIG. 1B and 1B. It is also preferred that the first polypeptide chain and the second polypeptide chain are linked by a disulfide bond, thereby forming a pair. In particular, the heavy chain and the light chain of an antibody or an antigen binding fragment thereof according to the present invention may be linked by a disulfide bond, thereby forming a pair. In addition, it is also preferred that the antibody or antigen binding fragment thereof comprises two first polypeptide chains and two second polypeptide chains, and that the first two polypeptide chains and/or two second polypeptide chains are linked by one or more, for example two, disulfide bonds. In particular, the two heavy chains of an antibody or antigen binding fragment thereof may be linked by one or more bonds, such as two disulfide bonds.

Preferably, the antibody or antigen binding fragment thereof of the present invention is bivalent for each specificity/antigen. In particular, it is preferred that the (additional) functional domain(s) contains or consists of (independent) binding site(s) and (1) (independent) binding site(s) and (2) antigen binding site(s) formed by one or several variable domains of the first pair of the first and second polypeptide chains, corresponds to (1) (independent) binding site(s) and (2) antigen binding site(s) formed by one or more variable domains of the second pair of the first and second polypeptide chains. In another embodiment of the present invention, the antibody or antigen binding fragment thereof may be monovalent for each specificity (antigen).

Accordingly, it is preferred that the antigen binding site(s) formed by one or more variable domains of the first pair of the first and second polypeptide chains and the antigen binding site(s) formed by one or more variable domains of the second pair of the first and second polypeptide chains are the same or different .

In addition, it is preferable that the antibody or antigen binding fragment thereof according to the present invention contains at least two (additional) functional domains, which may be the same or different. For example, an antibody or antigen binding fragment thereof in accordance with the present invention may contain 2 or 4 (additional) functional domains, which may be the same or different.

If the antibody or antigen binding fragment of the present invention contains more than one (additional) functional domain, for example, on the same or different polypeptide chains, the (additional) functional domains may be the same or different. Preferably, they may belong to the same or a separate group of functional domains. For example, at least two or all of the (additional) functional domains may contain or consist of (independent) binding sites. For example, at least two or all of the (additional) functional domains may contain or be composed of carrier domains. For example, at least two or all of the (additional) functional domains may contain or consist of reporter domains. For example, at least two or all of the (additional) functional domains may contain or consist of labels. For example, at least two or all of the (additional) functional domains may contain or consist of localization domains. Even if the (additional) functional domains belong to the same group of functional domains, the subgroup, and in particular their amino acid sequence, may still be the same or different. For example, at least two or all of the (additional) functional domains may contain or consist of the same amino acid sequences. In another embodiment of the present invention, it is also preferred that the (additional) functional domains belong to a specific group of functional domains. For example, one (additional) functional domain may contain or consist of an (independent) binding site, while another (additional) functional domain of an antibody or antigen-binding fragment may contain or consist of a carrier domain. For example, one (additional) functional domain may contain or consist of an (independent) binding site, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a reporter domain. For example, one (additional) functional domain may contain or consist of a (independent) binding site, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a tag. For example, one (additional) functional domain may contain or consist of (independently

- 35 046243 other) binding site, while another (additional) functional domain of the antibody or antigen-binding fragment may contain or consist of a localization domain. For example, one (additional) functional domain may contain or consist of a carrier domain, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a reporter domain. For example, one (additional) functional domain may contain or consist of a carrier domain, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a tag. For example, one (additional) functional domain may contain or consist of a carrier domain, while another (additional) functional domain of an antibody or antigen-binding fragment may contain or consist of a localization domain. For example, one (additional) functional domain may include or consist of a reporter domain, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a tag. For example, one (additional) functional domain may contain or consist of a reporter domain, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a localization domain. For example, one (additional) functional domain may contain or consist of a localization domain, while another (additional) functional domain of an antibody or antigen binding fragment may contain or consist of a tag.

It is further preferred that an antibody or antigen binding fragment thereof according to the present invention comprises two first polypeptide chains and two second polypeptide chains forming a first and second pair of first and second polypeptide chains, and wherein the first pair of first and second polypeptide chains comprises, at least one (additional) functional domain(s) and/or a second pair of first and second polypeptide chains comprises at least one (additional) functional domain(s). For example, the first pair of first and second polypeptide chains may contain at least one (additional) functional domain(s), while the second pair of the first and second polypeptide chains may not contain (additional) functional domain. For example, the first pair of first and second polypeptide chains may contain no (additional) functional domain, whereas the second pair of first and second polypeptide chains may contain at least one (additional) functional domain(s). In most cases, the first pair of first and second polypeptide chains contain at least one (additional) functional domain(s), and the second pair of the first and second polypeptide chains contain at least one (additional) functional domain(s).

Preferably, the antibody or antigen binding fragment thereof according to the present invention is derived from an IgG-like antibody, Fab or F(ab)2. That is, the antibody or antigen-binding fragment thereof contains all the variable and constant regions of an IgG-like antibody, Fab or F(ab)2. In particular, an IgG-like antibody, Fab or F(ab)2, can be used as a scaffold antibody in which an (additional) functional domain is inserted into the fold region.

Preferably, two or more variable domains (i) and (iv) of an antibody or antigen binding fragment thereof according to the present invention are derived from a monoclonal antibody. In particular, two or more variable domains (i) and (iv) of an antibody or an antigen-binding fragment thereof of the present invention are (correspond to) two or more variable domains of a monoclonal antibody.

It is also preferred that the variable domains and/or constant domains of the antibody or antigen binding fragment thereof of the present invention are from a human antibody or are humanized. In particular, the variable domains and/or constant domains of an antibody or an antigen binding fragment thereof according to the present invention correspond to the variable domains and/or constant domains of a human antibody or a humanized antibody or an antigen binding fragment thereof.

Preferably, the (additional) functional domain(s) of an antibody or antigen binding fragment thereof according to the present invention comprises or consists of an amino acid sequence that is a human or humanized antibody sequence.

Preferably, the first polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention contains or consists of any amino acid sequence of SEQ ID NO: 53, 55, 56, 58, 59, 60, 61, 62, 64, 65 , 66, 68, 69 or 70, which is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95 % and most preferably at least 98% identical to the sequence, and/or the second polypeptide chain of an antibody or antigen binding fragment thereof in accordance with the present invention contains or consists of any amino acid sequence from among the sequences

- 36 046243

SEQ ID NOs 54 57, 63 or 67, or a functional sequence variant that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably, at least 95% and most preferably at least 98% sequence identical.

Preferably, the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention may not contain or consist of any amino acid sequence from SEQ ID NO: 75-92. It is also preferred that the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention may not contain or consist of any amino acid sequence of SEQ ID NOs 96-112. More preferably, the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof of the present invention may not contain or consist of any amino acid sequence of SEQ ID NOs: 75-95. Even more preferably, the (additional) functional domain consisting of the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention may not contain or consist of any amino acid sequence from SEQ ID NO: 113-130 . Even more preferably, the (additional) functional domain included in the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention may not contain or consist of any amino acid sequence from SEQ ID NO: 113 -133. Most preferably, the first and/or second polypeptide chain of an antibody or antigen binding fragment thereof of the present invention may not contain or consist of any amino acid sequence from SEQ ID NO: 75 to 133. Optionally, an (additional) functional domain consisting from the first and/or second polypeptide chain of an antibody or an antigen-binding fragment thereof, of the present invention may not contain or consist of a (mutated) LAIR1 fragment.

Nucleic acid molecule.

Another aspect of the present invention relates to a nucleic acid molecule comprising a first polynucleotide encoding a first polypeptide chain of an antibody or an antigen binding fragment thereof according to the present invention, and/or a second polypeptide encoding a second polypeptide chain of an antibody or antigen binding fragment thereof according to the present invention.

A nucleic acid molecule is a molecule, preferably composed of nucleic acid components. The term nucleic acid molecule preferably refers to DNA or RNA molecules. In particular, it is used as a synonym for the concept of polynucleotide. Preferably, the nucleic acid molecule is a polymer containing or consisting of nucleotide monomers that are covalently linked to each other by phosphodiester bonds of the sugar/phosphate backbone of the molecule. The term nucleic acid molecule also includes modified nucleic acid molecules, for example, with modified bases, modified sugars, modified scaffolds, etc., DNA or RNA molecules.

Preferably, the nucleic acid molecule is a DNA or RNA molecule. Examples of nucleic acid molecules and/or polynucleotides include, for example, a recombinant polynucleotide, vector, oligonucleotide, RNA molecules, such as rRNA, mRNA, microRNA, siRNA or tRNA, or DNA molecules, such as cDNA. Preferred are nucleic acid sequences encoding all or part of the first and/or second polypeptide chain. Thus, the present invention preferably provides nucleic acid sequences encoding all or part of the light and heavy chains, in particular VH and VL sequences and/or (additional) functional domains of exemplary antibodies or antigen binding fragments of the invention.

It is also preferred that the nucleic acid molecule of the present invention includes a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least , is 98% or at least 99% identical to the nucleic acid encoding the (additional) functional domain, the VH sequence used and/or the VL sequence used in the (typical) antibody or antigen binding fragment thereof in accordance with the present invention.

In general, a nucleic acid molecule can be altered by insertion, deletion, or substitution of certain nucleic acid sequences.

Changes resulting from such changes include, but are not limited to, changes to introduce restriction sites, codon changes, addition or optimization of transcription and/or translation control sequences, etc. It is also possible to change the nucleic acid to

- 37 046243 change the coded amino acids. For example, it may be useful to introduce one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the amino acid sequences of the antibodies. Such point mutations may modify effector functions, antigen binding affinity, post-translational modifications, immunogenicity, etc., may introduce amino acids for attachment of covalent groups (eg, tags), or may introduce tags (eg, for purification purposes). Mutations can be introduced at specific sites or can be introduced randomly followed by selection (eg molecular evolution). For example, one or more nucleic acids encoding any of the (additional) functional domains, the VH sequence and/or the VL sequence of a (typical) antibody of the present invention may be randomly or targetedly mutated to introduce different properties into the encoded amino acids. Such changes may be the result of an iterative process in which the original changes are maintained and new changes are introduced at other nucleotide positions. In addition, changes achieved in independent stages can be combined. Various properties introduced into the encoded amino acids can lead to increased affinity, but the properties are not limited to this.

Vector.

The present invention further provides vectors, for example expression vectors, containing a nucleic acid molecule in accordance with the present invention. Preferably, the vector contains a nucleic acid molecule as described above.

The term vector refers to a nucleic acid molecule, preferably a recombinant nucleic acid molecule, that is, a nucleic acid molecule that does not occur in nature. A vector in the context of the present invention is suitable for incorporating or storing a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that provides convenient storage of a nucleic acid molecule. For example, the vector may contain a sequence corresponding to, for example, a desired antibody or antibody fragment of the present invention. An expression vector can be used to produce expression products such as RNA, for example mRNA or peptides, polypeptides or proteins. For example, an expression vector may contain sequences necessary for transcription of a sequence of a region of the vector, such as a promoter sequence. A cloning vector is typically a vector including a cloning site that can be used to incorporate nucleic acid sequences into the vector. The cloning vector can be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector that is suitable for transferring nucleic acid molecules into cells or organisms, such as viral vectors. The vector in the context of the present invention may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the context of the present invention contains a cloning site, a selection marker, for example an antibiotic resistance factor, and a sequence suitable for propagation of the vector, for example an origin of replication. Preferably, the vector in the context of the present invention is a plasmid vector.

Cells.

In another embodiment, the present invention provides cells expressing an antibody or antigen binding fragment thereof in accordance with the present invention, and/or containing a nucleic acid molecule or vector in accordance with the present invention.

Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, or plant cells. Preferably, the cells are mammalian cells, more preferably a mammalian cell line. Preferred examples are CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human liver cells, myeloma cells or hybridoma cells.

In particular, the cell can be transfected with a vector of the present invention, preferably an expression vector. The term transfection refers to the introduction of nucleic acid molecules, eg DNA or RNA molecules (eg, mRNA), into cells, preferably eukaryotic cells. In the context of the present invention, the term transfection covers any method known to one skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells, for example mammalian cells. Such methods include, for example, electroporation, lipofection, e.g., cationic lipid and/or liposome-based methods, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, e.g., DEAE-dextran or polyethylenimine, and etc. Preferably, the introduction is not viral.

In addition, cells of the present invention can be stably or transiently transfected with a vector of the present invention, for example, to express an antibody or antigen binding fragment thereof in accordance with the present invention. Preferably, cells are stably transfected

- 38 046243 is used with a vector according to the present invention encoding an antibody or an antigen binding fragment thereof according to the present invention. In another embodiment of the present invention, it is also preferred that the cells are transiently transfected with a vector of the present invention encoding an antibody or antigen binding fragment thereof of the present invention.

Composition.

The present invention also provides a composition comprising one or more of the following components:

(i) an antibody or fragment thereof of the present invention, (ii) a nucleic acid molecule of the present invention, (iii) a vector comprising a nucleic acid of the present invention, and/or (iv) cells expressing an antibody of the present invention, and/or comprising a vector or nucleic acid molecule of the present invention.

Thus, the present invention also provides a composition containing an antibody or antigen binding fragment thereof, in accordance with the present invention, a nucleic acid, in accordance with the present invention, a vector, in accordance with the present invention, and/or a cell in accordance with the present invention.

Preferably, the composition is a pharmaceutical composition. The pharmaceutical composition may also preferably contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the excipient may facilitate administration, it should not, by itself, induce the production of antibodies harmful to the individual to whom the composition is administered. There should also be no toxicity from administration. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive viral particles. In general, the pharmaceutically acceptable carriers in the pharmaceutical composition of the present invention may be active ingredients or inactive ingredients.

Pharmaceutically acceptable salts may be used, for example salts of mineral acids such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in the pharmaceutical composition may further contain liquids, for example, water, saline, glycerin and ethanol. In addition, such compositions may contain auxiliary substances, for example, wetting or emulsifying agents, or pH buffering agents. Such excipients allow pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions and slurries for administration by the subject.

The pharmaceutical compositions of the present invention can be prepared in various forms. For example, the compositions may be formulated as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared (eg, lyophilized composition such as Synagis™ and Herceptin™, for reconstitution with sterile water containing a preservative). The composition may be prepared, for example, in the form of an ointment, cream or powder. The composition may be prepared, for example, in the form of a tablet or capsule, in the form of a spray or in the form of a syrup (optionally flavored). The composition can be prepared, for example, in the form of an inhaler using a finely divided powder or spray. The composition can be prepared, for example, in the form of drops. The composition may be in the form of a kit formulated such that the combination composition can be reconstituted, for example, immediately before administration. For example, the lyophilized antibody may be provided in a kit with sterile water or sterile buffer.

Preferably, the active ingredient in the composition is an antibody molecule, an antibody fragment, or variants and derivatives thereof, particularly that the active ingredient in the composition is an antibody, an antibody fragment, or variants and derivatives thereof, in accordance with the present invention. The composition may contain agents that protect the antibody from destruction in the gastrointestinal tract, but which release the antibody after it is absorbed from the gastrointestinal tract.

For a detailed discussion of pharmaceutically acceptable carriers, see Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th ed., ISBN: 0683306472.

The pharmaceutical compositions of the present invention typically have a pH of 5.5-8.5, in some embodiments the pH may be 6-8, and in other embodiments the pH may be about 7. The pH may be maintained through the use of a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic in relation to humans. In one embodiment of the present invention, the pharmaceutical compositions of the present invention are supplied in hermetically sealed containers.

The composition may be in the form of a suspension, solution or emulsion in oil or water.

- 39 046243 body and, in particular, it may contain auxiliary substances, for example suspending, preserving, stabilizing and/or dispersing agents. In another embodiment of the present invention, the antibody molecule may be in dry form for reconstitution with a suitable sterile liquid before use.

The composition may contain a diluent, such as water or saline. The term diluent generally means a material that is suitable for storing, transporting and/or administering a compound, for example, a pharmaceutically active compound, in particular an antibody of the present invention. For example, the diluent may be a physiologically acceptable liquid that is suitable for storing, transporting and/or administering a pharmaceutically active compound, in particular the antibodies of the present invention.

The composition may be an aqueous solution that is pyrogen-free and has a suitable pH, isotonic and stable. Those skilled in the art can prepare suitable solutions using, for example, isotonic diluents, eg, sodium chloride injection, Ringer's injection, lactated Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included. The composition of the present invention may be provided, for example, in a pre-filled syringe.

The composition as described above may also be in dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets, carriers such as lactose and corn starch are also commonly added. Lubricants such as magnesium stearate are also commonly added. For capsules, useful diluents include lactose and dried cornstarch.

When aqueous suspensions are required, the active ingredient may be combined with emulsifying and suspending agents. Certain sweeteners, flavors or colors may also be added if desired.

Additional examples of carriers included in the composition include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, a polyoxypropylene compound, emulsifying wax and water. Alternatively, the composition may be formulated into a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, wax cetyl esters, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

In one embodiment of the present invention, the composition of the present invention may include antibodies of the present invention, wherein the antibodies may constitute at least 50% by weight (e.g., 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more) of the total protein in the composition. In such a composition, the antibodies are preferably in purified form.

Pharmaceutical compositions may include an antimicrobial agent, especially if it is packaged in a multi-dose format. They may contain a detergent such as Tween (polysorbate), eg Tween-80. Detergents are typically included at low concentrations, such as less than 0.01%. The compositions may also include sodium salts (eg, sodium chloride) to provide tonicity. For example, a NaCl concentration of 10 ± 2 mg/ml is commonly used.

Additionally, the pharmaceutical compositions may contain a sugar alcohol (eg, mannitol) or a disaccharide (eg, sucrose or trehalose), for example, in an amount of about 15-30 mg/ml (eg, 25 mg/ml), especially if they are to be lyophilized. or if they include material that has been reconstituted from lyophilized material. The pH of the lyophilization composition may be adjusted to 5 to 8, or 5.5 to 7, or approximately 6.1 prior to lyophilization.

The compositions of the present invention may also contain one or more immunoregulatory agents. In one embodiment of the present invention, one or more immunoregulatory agents contain an adjuvant.

Antibody production.

The antibodies of the present invention can be prepared by any method known in the art. For example, a framework antibody may be provided and genetically engineered into the fold region(s). To obtain a framework antibody, for example, the general methodology for obtaining monoclonal antibodies using hybridoma technology is well known (Kohler G., Milstein C, 1975; Kozbar et al., 1983). In particular, another method can be used, namely the EBV immortalization method described in WO 2004/076677.

The preferred method is described in WO 2004/076677. In this method, B cells producing the antibody of the present invention are transformed with Epstein-Barr virus (EBV) and polyclonal B cell activator. Additional cellular growth and differentiation promoters may optionally be added during the transformation step to further enhance efficiency. These stimulators can be cytokines, such as IL-2 and IL-15. Another object is present

- 40 046243 of the invention provides for the addition of IL-2 during immortalization for an additional increase, but its use is not mandatory. Immortalized B cells obtained using these methods can then be cultured using methods known in the art, using antibodies isolated from them.

Another preferred method is described in WO 2010/046775. In this method, plasma cells are cultured in limited numbers or as individual plasma cells in microculture plates. Antibodies can be isolated from plasma cell cultures. In addition, RNA can be isolated from plasma cell cultures and PCR can be performed using methods known in the art. The VH and VL regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector, which is then transfected into HEK293T cells or other host cells. Cloning of nucleic acid into expression vectors, transfection of host cells, cultivation of transfected host cells, and isolation of the generated antibody can be accomplished using any methods known to those skilled in the art.

The antibody fragments of the present invention can be prepared from antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or cleavage of disulfide bonds by chemical reduction. In another embodiment of the present invention, antibody fragments can be obtained by cloning and expressing portions of the heavy or light chain sequences. The concept of antibody fragments includes Fab, Fab' and F(ab%) fragments.

For example, the present invention also provides a method for producing an antibody or antigen binding fragment thereof of the present invention, comprising:

transforming a eukaryotic host cell by a method (for example, as described above) of incorporating one or more nucleic acid molecules, for example, as described above, encoding a first polypeptide chain and a second polypeptide chain, for example, into a vector of the present invention as described above, culturing the host cell under suitable conditions allowing for expression of said nucleic acid molecules, causing or allowing said first and second polypeptide chains to combine to form an antibody or an antigen binding fragment thereof, and optionally purifying the antibody or antigen binding fragment thereof from the culture medium.

Standard molecular biology techniques can be used to obtain DNA sequences encoding the antibodies or antibody fragments of the present invention. The required DNA sequences can be synthesized in whole or in part by oligonucleotide synthesis methods. If necessary, site-directed mutagenesis and polymerase chain reaction (PCR) methods can be used.

Any suitable host cell/vector system may be useful for expressing DNA sequences encoding the antibody molecules of the present invention or fragments thereof. Bacterial, eg E. coli, and other microbial systems may be partially useful for the expression of antibody fragments, eg Fab and F(ab')2 fragments, and especially Fv fragments and single chain antibody fragments, eg single chain Fvs. Eukaryotic expression systems in host cells, such as mammals, can be used to produce larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells for this purpose include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma, or hybridoma cells.

For example, a cell line can be transfected with two vectors, a first vector encoding a first polypeptide chain, such as a heavy chain polypeptide, and a second vector encoding a second polypeptide chain, such as a light chain polypeptide. In another embodiment of the present invention, a single vector may be used comprising sequences encoding first and second polypeptide chains, eg, light chain and heavy chain polypeptides.

If desired, antibodies can be further purified using filtration, centrifugation and various chromatographic techniques, such as HPLC or affinity chromatography. Methods for purifying antibodies, such as monoclonal antibodies, including methods for producing pharmaceutical grade antibodies, are well known in the art.

Methods and applications.

Another aspect of the present invention provides an antibody or antigen binding fragment thereof of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention or a (pharmaceutical) composition of the present invention for use in medicine.

Accordingly, the present invention also provides a method of preventing or treating a disease or disorder in a subject, comprising the step of administering to the subject an antibody or antigen binding fragment thereof of the present invention, a nucleic acid of the present

- 41 046243 the invention, a vector of the present invention, a cell of the present invention or a (pharmaceutical) composition of the present invention.

It will be appreciated that one or more specificities/specificities of an antibody or antigen binding fragment thereof according to the present invention may be selected in accordance with the disease or disorder to be prevented and/or treated. For example, for the prevention and/or treatment of malaria, the antibody or antigen-binding fragment thereof may contain specificity for a malaria antigen, for example, an antigen binding site (formed by variable domains) and/or an (additional) functional domain in the fold region that specifically binds to a malaria antigen . An example is the mutated fragment of LAIR1 described in the present invention, in particular the mutated fragment of LAIR1 described in WO 2016/207402 A1, for example, containing the amino acid sequence SEQ ID NO: 13 or a sequence variant thereof, which is at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 88%, at least 90%, at least 92%, at least 95%, and even more preferably at least 98% sequence identical. An antibody having such specificity (such (antigenic) binding site) may be particularly useful in providing immunity to malarial parasites in the blood (particularly Plasmodium falciparum).

Diseases to be treated and/or prevented using an antibody/antigen binding fragment of the present invention, nucleic acid molecules of the present invention, a vector of the present invention, cells of the present invention, or a composition of the present invention include cancer, infectious diseases, and autoimmune disorders. Moreover, treatment and/or prevention of cancer and/or infectious diseases is preferable.

Preferably, an antibody/antigen binding fragment of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a composition of the present invention may be useful for (medication preparation) for the prevention, treatment and/or alleviation of cancer or tumor diseases. In general, the term cancer refers to solid tumors, in particular malignant solid tumors such as sarcomas, carcinomas and lymphomas, and blood cancers such as leukemia. Cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors and blastomas.

Preferably, an antibody/antigen binding fragment of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a composition of the present invention may be useful for (medication preparation) for the prevention, treatment and/or alleviation of infectious diseases. Infectious diseases include viral, retroviral, bacterial and protozoal infectious diseases.

Moreover, the antibody/antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the composition of the present invention may be useful for (medicine preparation) for the prevention, treatment and/or alleviation of a condition for autoimmune diseases. Typically, autoimmune diseases occur because the body has an abnormal immune response to substances and tissues normally found in the body (autoimmunity). This may be limited to certain organs or may involve specific tissue located in different locations. Autoimmune diseases can be classified according to the corresponding type of hypersensitivity: type I (i.e., urticaria caused by autologous serum), type II, type III, or type IV.

In addition, an antibody or antigen binding fragment thereof of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, or a (pharmaceutical) composition of the present invention may also be useful for (in vitro) diagnostics. Diagnostic methods may involve contacting an antibody or antibody fragment with a sample. Such samples may be isolated from a subject, for example, from an isolated tissue sample taken, for example, from the nasal passages, sinuses, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries , pituitary gland, adrenal gland, thyroid gland, brain, skin or blood, plasma or serum. Diagnostic methods may also include detection of the antigen/antibody complex, in particular after contact of the antibody or antibody fragment with the sample. This detection step is usually performed on a bench, that is, without any contact with the human or animal body. Examples of detection methods are well known to one skilled in the art and include, for example, the ELISA (enzyme-linked immunosorbent assay) method.

Also for diagnostic purposes, it should be appreciated that one or more specificities/specificities of an antibody or antigen binding fragment thereof in accordance with the present invention can be selected

- 42 046243 act in accordance with the disease or disorder that needs to be prevented and/or treated, essentially as described above for the prevention and/or treatment of diseases.

In addition, the present invention also provides an assay for detecting antigen or quantifying antigen binding, comprising:

incubating an antigen with an antibody or an antigen-binding fragment thereof in accordance with the present invention under conditions that allow binding of the antigen to a multivalent antibody, and detecting binding of the antigen to the antibody.

For example, such an analysis may be useful in a diagnostic context, as described above.

Brief description of the figures

A brief description of the accompanying figures will now be given. The figures are intended to illustrate the present invention in more detail. However, they are in no way intended to limit the scope of the present invention.

Fig. 1(A). A classic monospecific antibody containing two heavy chains (gray), each with one VH variable domain and three constant domains CH1, CH2 and CH3, and two light chains (white), each with one VL variable domain and one CL constant region. The bending area is indicated by arrows.

Fig. 1(B). Preferred examples of the antibodies of the present invention (Inserted Ig molecules; IEI-Ig) derived from the classic monospecific antibody shown in FIG. 1A): an antibody containing one (additional) functional domain in the bend region of each heavy chain, but does not contain an insertion in the bend region of the light chain; an antibody containing one (additional) functional domain in the bend region of each light chain, but does not contain an insertion in the heavy chain; an antibody containing one (additional) functional domain in the bend region of each heavy chain and each light chain; an antibody containing two (additional) functional domains in the bend region of each heavy chain, but without insertion in the bend region of the light chain; and an antibody containing two (additional) functional domains in the bend region of each light chain, but without insertion in the bend region of the heavy chain.

Fig. 1(B). Preferred examples of antigen-binding fragments and antibodies of the present invention (injection inserted Ig molecules; IEI-Ig) derived from the classic monospecific antibody shown in FIG. 1A): derivatives from antibody fragments or from bispecific antibodies: F(ab) ₂ fragment, including one (additional) functional domain in the bend region of each heavy chain, but without insertion into the bend region in the light chain; a Fab fragment containing one (additional) functional domain in the bend region of each light chain, but without insertion into the bend region of the heavy chain; antibodies with CrossMab/Knobs-in-holes/orthogonal Fab/Fab-arm substitutions, containing one (additional) functional domain in the bend region of one heavy chain, but without insertion into the bend region in the light chains and in the other heavy chain; scFv-(H)IgG, containing one (additional) functional domain in the bend region of each heavy chain, but without insertion into the bend region of the light chain; IgG(H)-scFv, containing one (additional) functional domain in the bend region of each heavy chain, but without insertion into the bend region of the light chain; scFv-(L)IgG, containing one (additional) functional domain in the bend region of each heavy chain, but without insertion into the bend region of the light chain; IgG(L)-scFv, containing one (additional) functional domain in the bend region of each heavy chain, but without insertion into the bend region of the light chain; and DVD-Ig, containing one (additional) functional domain in the bend region of each heavy chain, but without insertion in the bend region of the light chain.

Fig. 2. Schematic from Example 1 of the seven antibody constructs of the present invention (C2 to C8) and comparison with the scaffold antibodies (GCE536, C1).

Fig. 3. EC ₅₀ values from Example 2 determined by nonlinear regression analysis of binding (OD) values and relative antibody concentrations in ELISA tests performed using a set of antigens or antibodies against the domain. The constructs were also tested for binding to IE (isolate 9215), and binding values (%) for a concentration of 1 μg/ml are shown.

Fig. 4. Example 3 Shown are SPR binding curves (surface plasmon resonance - SPR) of C4 and various controls to GM-CSF and collagen. C4 and C5, which use GCE536 as a scaffold, bind to GM-CSF, but only C4 then binds to collagen. Non-specific antibodies to FI174 and collagen-specific antibodies MGD ^UCA do not show any specific binding signal.

Fig. 5. Example 3 Shown are the SPR binding curves of C4 and various controls to GM-CSF and collagen. C4 and GCE536 bind to GM-CSF, but only C4 is subsequently recognized by collagen. The collagen-specific antibody MGD ^UCA binds only to collagen. The control antibody TT107 is a TT-specific monoclonal antibody that does not show any specific binding signal in the SPR experiment.

Fig. 6. Example 4. ELISA curves of binding of constructs C5, C5b, C6 and C6b with GM-CSF or anti-PD1 or anti-SLAM antibodies compared to scaffold antibody (GCE536). PD1-containing C5 and

- 43 046243

C5b and SLAM-containing C6 and C6b constructs are recognized by anti-PDl or anti-SLAM antibodies, respectively. All constructs bind to GM-CSF as GCE536. The presence of linkers in C5b and C6 does not affect binding.

Fig. 7. Example 6. ELISA curves of Strep-tactin antibody binding to the C9 construct compared to the C1b frame. A double Strep-tag inserted into the bend region of the C9 construct is specifically recognized by an antibody to Strep-tactin.

Fig. 8. Example 7. Schematic of four additional constructs (C9 to C12) and their comparison with the scaffold antibody (FI174).

Fig. 9. Example 8. SPR binding curves of C9 and C10 and various controls for H1 and TT. C9 and C10 show double binding to both H1 and TT. TT107 is a TT-specific monoclonal antibody.

Fig. 10. Example 8 SPR binding curves of C11 and C12 and various controls for the H1 and RSV F proteins. C11 and C12 show dual binding to the H1 and RSV F proteins. MPE8 is a protein-specific monoclonal antibody against RSV F.

Examples.

The following are specific examples illustrating various embodiments and objects of the present invention. However, the present invention is not limited in its scope to the examples provided. The following formulations and examples are provided to enable those skilled in the art to more clearly understand and apply the present invention. The following embodiments of the present invention are intended only to illustrate certain objects and methods of the present invention that are functionally equivalent and fall within the scope of the present invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description, figures and examples. All such modifications are consistent with the appended claims of the present invention.

Example 1. Development and construction of antibody variants by inserting various Ig-like functional domains into the bend region of the antibody framework.

To investigate the impact of different (additional) functional domains inserted into the antibody fold region on antibody specificity, seven different constructs (termed C2-C8) were constructed in which non-mutant LAIR1 (SEQ ID NO) was inserted into the antibody fold region used as a scaffold. : 12), mutant LAIR1 (SEQ ID NO: 13) or other Ig-like domains. Constructs C2-C3 have the same overall heavy chain constant region as Construct C1 (VH: SEQ ID NO: 5, VL: SEQ ID NO: 6, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 7). Constructs C4-C6 have the same complete heavy chain constant region as antibody GCE536 (VH: SEQ ID NO: 1, VL: SEQ ID NO: 2, heavy chain constant region: SEQ ID NO: 3, light chain constant kappa: SEQ ID NO: 4; Piccoli L. et al., Nature communications 6, 2015, 7375). Constructs C7-C8 have the same complete heavy chain constant region as construct C1b (VH: SEQ ID NO: 8, VL: SEQ ID NO: 9, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4). The light chain of the constructs was not modified from the scaffold antibodies. All constructs are finally expressed as monoclonal antibodies (heavy and light chains).

The resulting structures below are shown schematically in FIG. 2.

1. The designation C1 (VH: SEQ ID NO: 5, VL: SEQ ID NO: 6, heavy chain constant region: SEQ ID NO: 3, light chain lambda constant region: SEQ ID NO: 7) means a recombinant monospecific antibody intended for control. C1 is formed as follows (in the direction from N- to C-terminus): the expression product of the gene segment V (variable) heavy chain variable domain (VH3-30); expression product of the heavy chain variable domain (D) gene segment D (diversity); expression product of the heavy chain variable domain element J (junction) gene segment (JH6); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the light chain variable domain element V (variable) gene segment and the expression product of the light chain variable domain element J (junction) gene segment; expression product of the lambda light chain constant region gene segment C (constant).

2. Designation C1b (VH: SEQ ID NO: 8, VL: SEQ ID NO: 9, heavy chain constant region: SEQ ID NO: 3, light chain lambda constant region.

SEQ ID NO: 4) means a recombinant monospecific antibody for control purposes. C1 is formed as follows (in the direction from N- to C-terminus): the expression product of the gene segment V (variable) heavy chain variable domain (VH3-20); expression product of the heavy chain variable domain (D) gene segment D (diversity); expression product of the C1 heavy chain variable domain element (JH3) gene segment J (junction) element; expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the V (variable) lung variable domain gene segment

- 44 046243 chains and expression product of the light chain variable domain J (junction) gene segment element; expression product of the light chain constant region C (constant) gene segment.

3. In the C2 construct, the mutant LAIR-1 fragment (LAIR1 ^gen ; SEQ ID NO: 13) is inserted into the bend region of the recombinant monospecific antibody C1 (see above). In the LAIR1 mutant fragment, the binding of LAIR1 to collagen is abolished, but the LAIR1 mutant fragment binds strongly to red blood cells infected with P. falciparum (Tan J. et al. Nature 529, 2016, 105-109; WO 2016/207402 A1). Construct C2 (full heavy chain: SEQ ID NO: 53, complete light chain: SEQ ID NO: 54) is formed as follows (in the direction from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) C1 (VH3-30); expression product of the C1 heavy chain variable domain D (diversity) gene segment (D); expression product of the C1 heavy chain variable domain element J (junction) gene segment (JH6); expression product of the mutant fragment LAIR-1 MGD21 (LAIR1D ²¹ ); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the C1 light chain variable domain element V (variable) gene segment and the expression product of the C1 light chain variable domain element J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

4. In construct C3, a non-mutant fragment of LAIR-1 (LAIR1 ^gen ; SEQ ID NO: 12) is inserted into the bend region of the recombinant monospecific antibody C1 (see above). Construct C3 (full heavy chain: SEQ ID NO: 55, complete light chain: SEQ ID NO: 54) is formed as follows (in the direction from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) C1 (VH3-30); expression product of the C1 heavy chain variable domain D (diversity) gene segment (D); expression product of the C1 heavy chain variable domain element J (junction) gene segment (JH6); expression product of a non-mutant fragment of LAIR-1 (LAIR1 ^gen ); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the C1 light chain variable domain element V (variable) gene segment and the expression product of the C1 light chain variable domain element J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

5. In construct C4, a non-mutant LAIR-1 fragment (LAIR1 ^gen ; SEQ ID NO: 13) is inserted into the bend region of the GCE536 antibody.

Construct C4 (full heavy chain: SEQ ID NO: 56, complete light chain: SEQ ID NO: 54) is formed as follows (in the direction from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) GCE536 (VH1-46); expression product of the heavy chain variable domain D (diversity) gene segment GCE536 (D); the expression product of the GCE536 heavy chain variable domain element J (junction) gene segment (JH6); expression product of a non-mutant fragment of LAIR-1 (LAIR1 ^gen ); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the GCE536 light chain variable domain element V (variable) gene segment and the expression product of the GCE536 light chain variable domain element J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

6. In construct C5, the extracellular Ig-like domain of the PD1 molecule (PD1; SEQ ID NO: 14) is inserted into the fold region of the GCE536 antibody.

Construct C5 (full heavy chain: SEQ ID NO: 58, complete light chain: SEQ ID NO: 57) is formed (in the direction from N- to C-terminus): the expression product of the gene segment V (variable) heavy chain variable domain GCE536 ( VH1-46); expression product of the heavy chain variable domain D (diversity) gene segment GCE536 (D); expression product of the GCE536 heavy chain variable domain element J (junction) gene segment (JH6); expression product of the extracellular Ig-like domain of the PD1 molecule (PD1); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the GCE536 light chain variable domain element V (variable) gene segment and the expression product of the GCE536 light chain variable domain element J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

7. In the C5b construct, the extracellular Ig-like domain of the PD1 molecule (PD1; SEQ ID NO: 14) with flanking intronic sequences (15-mer JH-PD1; SEQ ID NO: 49 and 15-mer PD1CH1 linkers; SEQ ID NO: 50 ) is inserted into the bend region of the GCE536 antibody. Construct C5 (full heavy chain: SEQ ID NO: 59, complete light chain: SEQ ID NO: 57) is formed (in the direction from N- to C-terminus): the expression product of the gene segment V (variable) heavy chain variable domain GCE536 ( VH1-46); expression product of the heavy chain variable domain D (diversity) gene segment GCE536 (D); expression product of the GCE536 heavy chain variable domain J (junction) gene segment (JH6); linker expression product (15-mer JH-PD1); expression product of the extracellular Ig-like domain of the PD1 molecule (PD1); linker expression product (15mer PD1-CH); expression product of gene segment C (constant) constant region of severe

- 45 046243 chains (IgG1 isotype); and on a separate chain: the expression product of the GCE536 light chain variable domain element V (variable) gene segment and the expression product of the GCE536 light chain variable domain element J (junction) gene segment; expression product of the kappa light chain constant region gene segment C (constant).

8. In the C6 construct, the extracellular Ig-like domain of the SLAM molecule (SLAM; SEQ ID NO: 15) is inserted into the fold region of the GCE536 antibody.

Construct C6 (full heavy chain: SEQ ID NO: 60, complete light chain: SEQ ID NO: 57) is formed (in the direction from N- to C-terminus): the expression product of the gene segment V (variable) heavy chain variable domain GCE536 ( VH1-46); expression product of the heavy chain variable domain D (diversity) gene segment GCE536 (D); expression product of the GCE536 heavy chain variable domain J (junction) gene segment (JH6); expression product of the extracellular Ig-like domain of the SLAM molecule (SLAM); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the GCE536 light chain variable domain V (variable) gene segment and the expression product of the GCE536 light chain variable domain J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

9. In the C6 construct, the extracellular Ig-like domain of the SLAM molecule (SLAM; SEQ ID NO: 15) with flanking intronic sequences (15-mer JH-SLAM; SEQ ID NO: 51 and 15-mer SLAM-CH1 linkers; SEQ ID NO : 52) is inserted into the bend region of the GCE536 antibody. Construct C6 (full heavy chain: SEQ ID NO: 61, complete light chain: SEQ ID NO: 57) is formed (in order from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) GCE536 ( VH1-46); expression product of the heavy chain variable domain D (diversity) gene segment GCE536 (D); expression product of the GCE536 heavy chain variable domain J (junction) gene segment (JH6); linker expression product (15-mer JHSLAM); expression product of the extracellular Ig-like domain of the SLAM molecule (SLAM); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the GCE536 light chain variable domain V (variable) gene segment and the expression product of the GCE536 light chain variable domain J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

10. In the C7 construct, the extracellular Ig-like domain of the PD1 molecule (PD1; SEQ ID NO: 14) is inserted into the fold region of the recombinant monospecific antibody C1b (see above). Construct C7 (full heavy chain: SEQ ID NO: 62, complete light chain: SEQ ID NO: 63) is formed (from N- to C-terminus): the expression product of the heavy chain variable domain V (VH3) gene segment -20); the expression product of the heavy chain variable domain (D) gene segment D (diversity); expression product of the J (Joining) gene segment element of the heavy chain variable domain (JH3); expression product of the extracellular Ig-like domain of the PD1 molecule (PD1); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of a light chain variable domain V (variable) gene segment and the expression product of a light chain variable domain J (junction) gene segment element; expression product of the light chain constant region C (constant) gene segment.

11. In the C8 construct, the extracellular Ig-like domain of the SLAM molecule (SLAM; SEQ ID NO: 15) is inserted into the fold region of the recombinant monospecific antibody Clb (see above). Construct C8 (full heavy chain: SEQ ID NO: 64, complete light chain: SEQ ID NO: 63) is formed (from N- to C-terminus): the expression product of the heavy chain variable domain V (VH3) gene segment -20); the expression product of the heavy chain variable domain (D) gene segment D (diversity); expression product of the J (Joining) gene segment element of the heavy chain variable domain (JH3); expression product of the extracellular Ig-like domain of the SLAM molecule (SLAM); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of a light chain variable domain V (variable) gene segment and the expression product of a light chain variable domain J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

Example 2 Ig-like domains can be inserted into the fold region of antibodies, resulting in the formation of functional antibodies.

The eight antibody constructs described in Example 1 were produced recombinantly by transient transfection. To do this, the heavy and light chains of the antibodies are cloned into human IgG1, IgK and λλ expression vectors and expressed by transient transfection of Expi293F cells (ThermoFisher Scientific) using polyethylenimine (PEI). Cell lines are regularly tested for mycoplasma contamination.

Antibody constructs C1-C8 and control antibody GCE536 (see Example 1) were then tested using the 9215 IE staining method (infected red blood cells), and binding values (%) at concentration

- 46 046243 1 μg/ml antibodies are calculated by interpolating binding curves fitted to a sigmoidal curve model (Graphpad Prism 6). In addition, binding to recombinant human collagen, anti-human LAIR1 antibody, recombinant human GM-CSF, anti-PD1 and anti-SLAM antibodies was tested by ELISA. Briefly, total IgG was quantified using MaxiSorp 96-well plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, catalog no. 2040-01) using certified reference material 470 (product ERMs-DA470, Sigma -Aldrich) as standard. To test the specific binding of antibody constructs, ELISA plates are coated with 2 μg/ml recombinant human collagen type I (Millipore, catalog number CC050), 2 μg/ml anti-human LAIR1 antibody (clone DX26, BD Biosciences, catalog number 550810) 1 μg/ml recombinant human GM-CSF (Gentaur), 2 μg/ml anti-PD1 or anti-SLAM antibody (R&D Systems, catalog numbers AF1086 and AF164, respectively). The plates are blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies followed by goat anti-human IgG conjugated to an FcY fragment-specific AR (Jackson Immuno Research, 109-056098). The plates are then washed, substrate (p-NPP, Sigma) is added and the plates are read at 405 nm.

The results of various binding studies are shown in FIG. 3. Antibody constructs carrying LAIR1-stained IE are recognized by anti-LAIR1 antibody. Insertion of the Ig-like domains LAIR1, PD1, or SLAM into the bend region of the anti-GM-CSF antibody does not affect binding to GM-CSF (constructs C4-C6), therefore, this site allows the insertion of various domains without affecting the specificity of the parent antibody. In contrast, insertion of LAIR1 into CDR3 abolished binding to GM-CSF (data not shown).

Accordingly, different domains can be inserted into the bend region without affecting the specificity of the original antibody.

Example 3 Constructs containing an Ig-like domain in the bend region can simultaneously bind to two different antigens.

To test whether bispecific constructs bearing a binding site in the bend region are capable of simultaneously binding to both specificities, simultaneous binding was examined by surface plasmon resonance (SPR). To this end, the C4 bispecific construct, which has a V(D)J region specific for GM-CSF and carrying unmutated LAIR1 (binding site for collagen) in the fold region, is tested for simultaneous binding to GM-CSF and collagen by SPR.

In one experiment, GM-CSF is immobilized on the surface of a sensor chip and the constructs are injected followed by collagen injection.

Antibodies C5 and GCE536 (see above), MGD ^UCA antibody (contains non-mutant LAIR1 and is thus able to bind collagen; Tan J. et al., Nature 529, 2016, 105-109; VH: SEQ ID NO: 71, VL: SEQ ID NO: 72, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) and antibody FI174 (influenza H1 hemagglutinin specific; Pappas L. et al., Nature 516, 2014, 418-422; (VH: SEQ ID NO: 10, VL: SEQ ID NO: 11, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) were used as controls. Briefly, GMCSF (200 nM) was stabilized in 10 mM acetate buffer, pH 4.5 and immobilized on a pre-activated ProteOnsensor chip (Bio-Rad) by amine binding, unreacted groups are blocked by injection of 1 M ethanolamine HCl. HEPES buffer saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 surfactant) is used as a running buffer. All injections are performed at a flow rate of 100 μl/min. Monoclonal antibodies were diluted in HBS to 10 nM and applied to the chip for 240 s on a GM-CSF-coated chip, followed by injection of collagen (50 nM) for 120 s. HBS is injected into one channel of the chip and used as a control. Each monoclonal antibody binding to GM-CSF and collagen was assessed using a ProteONXPR36 instrument (Bio-Rad) and data processed using ProteOn Manager software.

The results are shown in Fig. 4. Both constructs C4 and C5, which use GCE536 as a scaffold (see Example 1), bind to GM-CSF. Of C4 and C5, only C4 (which carries LAIR1 in the bend region, whereas C5 carries the Ig-like domain of PD1 in the bend region) is then bound by collagen. The monospecific anti-GM-CSF antibody GCE536 binds only to GM-CSF and not to collagen. The anti-H1 antibody FI174 and the collagen-specific antibody MGD ^UCA do not exhibit any specific binding signal. Thus, our data indicate that the C4 bispecific construct binds to GM-CSF and then collagen binds to the LAIR1 domain of C4.

In the second experiment, Protein A is used to capture the construct followed by co-injection of the test materials (GM-CSF followed by the addition of collagen). In this experiment, antibodies GCE536 and MGD ^UCA (all described in examples 1 and 2) and antibody TT107 (specific

- 47 046243 for tetanus toxoid (TT); VH: SEQ ID NO: 73, VL: SEQ ID NO: 74, heavy chain constant region: SEQ ID NO: 3, light chain constant region: SEQ ID NO: 4) were used as controls. Briefly, protein A (25 μg/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized on a preactivated ProOontSnsensor chip (Bio-Rad) with ethyl(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) via amine coupling; unreacted groups are blocked by injection of 1 M ethanolamine HCl. HEPES buffer saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 surfactant) was used as a running buffer. All injections are performed at a flow rate of 100 μl/min. Monoclonal antibodies are diluted in HBS to 10 nM and injected for 240 sec onto the protein A-coated capture chip, followed by a simultaneous injection of GM-CSF collagen (50 nM), immediately followed by collagen (50 nM) for a total of 110 sec. HBS is injected into one channel of the chip as a control. Each binding of monoclonal antibodies to GMCSF and collagen is assessed using the ProteONXPR36 instrument (Bio-Rad) and the data is processed using the ProteOn Manager program.

The results are shown in Figure 5. Construct C4 and GCE536 bind to GM-CSF. However, only C4 is then recognized by collagen. The collagen-specific antibody MGD ^UCA binds only to collagen. The control antibody TT107 is a monoclonal antibody specific for TT (tetanus toxoid) that does not exhibit any specific binding signal in the SPR experiment. Thus, these results indicate that constructs containing an (additional) functional domain, such as an Ig-like domain, in the bend region can simultaneously bind to (i) the binding partner of the bend site and (ii) an antigen recognized by the variable domains framework antibody.

Example 4. Ig-like domains can be inserted together with flanking linkers into the fold region of antibodies, resulting in the formation of functional antibodies

Antibody constructs C5b and C6b contain the same domain (either PD1 or SLAM) inserted into the fold region of constructs C5 and C6, respectively, with two additional 15-mer amino acid linkers inserted between JH and the domain, and between the domain and CH1 . ELISA assays for binding to recombinant human GM-CSF, anti-PD1 and anti-SLAM antibodies. Briefly, total IgG was quantified using MaxiSorp 96-well plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, catalog no. 2040-01) using certified reference material 470 (product ERMs-DA470, Sigma -Aldrich) as standard. To test specific binding of antibody constructs, ELISA plates are coated with 1 μg/ml recombinant human GM-CSF (Gentaur), 2 μg/ml anti-PD1 or anti-SLAM antibodies (R&D Systems, AF1086 and AF164). The plates are blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies followed by goat anti-human IgG conjugated to FcY fragment-specific AR (Jackson Immuno Research, 109-056-098). The plates are then washed, substrate (p-NPP, Sigma) is added and the plates are read at 405 nm.

The results of various bindings are shown in FIG. 6. Insertion of Ig-like PD1 or SLAM domains with flanking linkers into the bend region of the GM-CSF antibody does not affect the binding of GMCSF; therefore, different domains with flanking linkers can also be inserted into this site without affecting the specificity of the original antibody. In addition, the presence of linkers did not change the Ig-like domain, since the anti-PD1 antibody recognized both C5 and C5b equally. Similar results were obtained for C6 and C6b, recognized by the anti-SLAM antibody. Summarizing the results obtained, it can be assumed that domains can be inserted into the bend region with additional linkers that remove such domains from the main antibody framework. Without being limited by any theory, it is believed that linkers may provide increased flexibility of the insert.

Example 5: Construction of an antibody containing a molecular tag inserted into the bend region.

To explore the possibility of using a tag inserted into the bend region of an antibody for antibody detection, a design (named C9) was developed in which a twin-Strep tag is inserted into the heavy chain bend region of the C1b framework antibody (compare with Example 1). The light chain of the construct is not modified compared to the framework antibody. As a result, the construct is expressed as a monoclonal antibody (heavy and light chains).

The structure of C9 is shown schematically in FIG. 2. In construct C9, a double Strep-tag (2XST; SEQ ID NO: 20) was inserted into the fold region of the recombinant monospecific antibody C1b (see above). Construct C9 (full heavy chain: SEQ ID NO: 65, complete light chain: SEQ ID NO: 63) is formed as follows (in N- to C-terminal direction): the expression product of the heavy chain variable domain (variable) gene segment V (VH3-20); expression product of the heavy chain variable domain (D) gene segment D (diversity); expression product of the heavy chain variable domain J (junction) gene segment element (JH3); product express

- 48 046243 these double Strep-tags (2XST); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the light chain variable domain V (variable) gene segment and the expression product of the light chain variable domain J (junction) gene segment; expression product of the kappa light chain constant region gene segment C (constant).

Example 6 Molecular tags can be inserted into the bend region of antibodies, resulting in the formation of functional antibodies.

The C9 antibody construct described in Example 5 is obtained by recombination by temporary transfection. To do this, the heavy and light chains of the antibodies are cloned into human IgG1 and IgK expression vectors and expressed by transient transfection in Expi293F cells (ThermoFisher Scientific) using polyethylenimine (PEI). Cell lines are tested daily for mycoplasma contamination.

The C9 antibody construct and the control antibody C1b (see Example 1) are then tested for recognition by the Strep-tag-specific Strep-tactin molecule by ELISA. Briefly, total IgG was counted using 96-well MaxiSorp plates (Nunc) coated with goat anti-human IgG (Southern Biotech, 2040-01), using Certified Reference Material 470 (ERMs) as a standard. DA470, Sigma-Aldrich). To specifically recognize the antibody construct, ELISA plates are coated with 10 μg/ml anti-human IgG (Southern Biotech), blocked with 1% bovine serum albumin (BSA) and incubated with titrated antibodies followed by AP-conjugated reagent. Strep-tactin (Iba Lifesciences). The plates are then washed, the substrate (p-NPP, Sigma) is added, and the plates are read at a wavelength of 405 nm.

The results are shown in Fig. 7. The C9 antibody construct, which carries the Strep-tag, is efficiently and specifically recognized by the Strep-tag-specific Strep-tactin, whereas the control antibody C1b, which does not carry the Strep-tag, is not recognized. These results indicate that molecular tags can be inserted into the bend region to allow antibody recognition and tracking.

Example 7: Construction of antibodies containing a single chain antibody variable domain (VHH) or a single chain variable fragment (ScFv) inserted into the bend region.

To investigate whether the insertion of (additional) functional domains other than Ig-like domains into the fold region results in functional multispecific antibodies, four new constructs (C9-C12) were developed. In four new C9-C12 constructs, a single-chain antibody variable domain (VHH) or a single-chain variable fragment (ScFv) specific for tetanus toxoid (TT) or respiratory syncytial virus (RSV) hybridization protein (F), respectively, was inserted into the fold region of the antibodies , specific for influenza H1 hemagglutinin (FI174; Pappas, L. et al., Nature 516, 2014, 418-422; VH: SEQ ID NO: 10, VL: SEQ ID NO: 11, heavy chain constant region: SEQ ID NO : 3, kappa light chain constant region: SEQ ID NO: 12/SEQ ID NO: 4). The light chain of the constructs is not modified compared to the FI174 framework antibody. Ultimately, all constructs are expressed as monoclonal antibodies (heavy and light chains). The following variants were obtained, schematically shown in Fig. 8:

1. In construct C10, anti-TT VHH (T3-VHH; Rossotti M.A. et al., mAbs 7, 2015, 820-828; SEQ ID NO: 16) is inserted into the FI174 bend region. Construct C10 (full heavy chain: SEQ ID NO: 66, complete light chain: SEQ ID NO: 67) is formed as follows (in the N- to C-terminal direction): the expression product of the heavy chain variable domain gene segment V (variable) FI174 ( VH); expression product of the heavy chain variable domain D (diversity) gene segment FI174 (D); expression product of the FI174 heavy chain variable domain J (junction) gene segment element (JH); anti-TT VHH expression product (T3-VHH; Rossotti M.A. et al., mAbs 7, 2015, 820-828); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and on a separate chain: the expression product of the FI174 light chain variable domain gene segment V (variable) and the expression product of the FI174 light chain variable domain gene segment J (Joining) element; expression product of the light chain constant region C (constant) gene segment.

2. In construct C11, anti-TT ScFv (TT39.7-ScFv; SEQ ID NO: 17) was inserted into the FI174 bend region. Construct C11 (full heavy chain: SEQ ID NO: 68, complete light chain: SEQ ID NO: 67) is formed as follows (in the direction from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) FI174 (VH); expression product of the heavy chain variable domain D (diversity) gene segment FI174 (D); expression product of the FI174 heavy chain variable domain J (junction) gene segment element (JH); anti-TT ScFv expression product (TT39.7-ScFv); expression product of the heavy chain constant region gene segment C (constant) (IgG1 isotype); and in a separate chain: the expression product of the FI174 light chain variable domain V (variable) gene segment and the expression product of the FI174 light chain variable domain J (junction) gene segment; expression product of the light chain constant region C (constant) gene segment.

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3. In the C12 construct, the anti-RSV protein F VHH (F4-VHH; Rossey I. et al., Nature communications 8, 2017, 14158; SEQ ID NO: 18) is inserted into the FI174 bend region. Construct C12 (full heavy chain: SEQ ID NO: 69, complete light chain: SEQ ID NO: 67) is formed as follows (from N- to C-terminus): expression product of the heavy chain variable domain V gene segment FI174 (VH); expression product of the heavy chain variable domain D (diversity) gene segment FI174 (D); expression product of the FI174 heavy chain variable domain gene segment J (junction) element (JH); expression product of the anti-RSV VHH F protein (F4VHH; Rossey I. et al., Nature communications 8, 2017, 14158; expression product of the C (constant) gene segment of the heavy chain constant region (IgG1 isotype); and in a separate chain: expression product the FI174 light chain variable domain gene segment V (variable) and the expression product of the FI174 light chain variable domain gene segment J (junction) element; the expression product of the light chain constant region gene segment C (constant).

4. In construct C13, the anti-RSV protein F ScFv (MPE8-ScFv; Corti D. et al., Nature 501, 2013, 439-443; SEQ ID NO: 19) is inserted into the FI174 bend region. Construct C13 (full heavy chain: SEQ ID NO: 70, complete light chain: SEQ ID NO: 67) is formed as follows (in the direction from N- to C-terminus): the expression product of the heavy chain variable domain gene segment V (variable) FI174 (VH); expression product of the heavy chain variable domain D (diversity) gene segment FI174 (D); expression product of the FI174 heavy chain variable domain gene segment J (junction) element (JH); expression product of the anti-RSV protein ScFv (MPE8-ScFv; Corti D. et al., Nature 501, 2013, 439-443; expression product of the C (constant) gene segment of the heavy chain constant region (IgG1 isotype); and in a separate chain: the expression product of the FI174 light chain variable domain gene segment V (variable) and the expression product of the FI174 light chain variable domain gene segment J (junction) element; the expression product of the light chain constant region gene segment C (constant).

Example 8 VHH and ScFv can be inserted into the hinge region, resulting in the formation of functional bispecific antibodies.

The four new antibody constructs described in Example 4 were produced by recombination via transient transfection. To do this, the heavy and light chains of the antibodies are cloned into human IgG1, IgK and Ig?t expression vectors and expressed by transient transfection of Expi293F cells (ThermoFisher Scientific) using polyethylenimine (PEI). The constructs are tested for dual binding to (i) H1 and (ii) TT or RSV F protein using surface plasmon resonance (SPR).

In the first experiment, constructs C10 and C11 (with TT-specific VHH or scFv in the FI174 bend region) were analyzed. For this purpose, Protein A is used to capture the constructs followed by simultaneous injection of the test material (H1 as the first test material, immediately followed by TT). The TT-specific antibody TT107 and the III-specific antibody FI174 are used as controls. Briefly, Protein A (25 μg/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized on a pre-activated ProteOnsensor chip (Bio- Rad) by amine coupling, unreacted groups are blocked by injection of 1 M ethanolamine HCl. HEPES buffer saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 surfactant) was used as a running buffer. All injections are performed at a flow rate of 100 μl/min. Monoclonal antibodies are diluted in HBS to 10 nM and applied to a Protein A-coated capture chip for 240 sec, followed by a simultaneous injection of California hemagglutinin H1 (50 nM), immediately followed by tetanus toxoid (50 nM) over 110 sec. HBS is injected into one channel of the chip as a control. Each monoclonal antibody binding to H1 and TT was assessed using a ProteONXPR36 instrument (Bio-Rad), and the data were processed using the ProteOn Manager program. The results are shown in Fig. 9. Constructs C10 and C11 (carrying a TT-specific domain (VHH or ScFv)) bind both H1 and TT, whereas control antibodies FI174 and TT107 recognize only H1 or only TT, respectively.

In the second experiment, constructs C12 and C13 (with RSV F protein-specific VHH or scFv in the FI174 bend region) are examined. To this end, protein A was used to capture constructs followed by co-injection of test materials (H1 as the first test material, immediately followed by RSV F protein). Protein-specific antibodies against RSV, the F-specific antibody MPE8 and the III-specific antibody FI174, are used as controls. Briefly, Protein A (25 μg/ml) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized on a pre-activated ProteOnsensor chip (Bio-Rad). by amine coupling, unreacted groups are blocked by injection of 1 M ethanolamine HCl. HEPES buffer saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 surfactant) was used as a running buffer. All injections are carried out at a flow rate

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100 µl/min. Monoclonal antibodies are diluted in HBS to 10 nM and applied to a capture protein A-coated chip for 240 sec, followed immediately by California hemagglutinin H1 (50 nM), followed immediately by RSV F protein (50 nM) for 110 sec. HBS is injected into one channel of the chip as a control. Each monoclonal antibody binding to H1 and RSV F was assessed using a ProteONXPR36 instrument (Bio-Rad) and the data were processed using the ProteOn Manager program. The results are shown in Fig. 10. Constructs C10 and C11 (carrying the RSV F protein-specific domain (VHH or ScFv)) bind both H1 and RSV F protein, whereas the control antibodies FI174 and MPE8 recognize only the H1 protein or only the RSV protein, respectively.

Thus, the results show that insertion of VHH or ScFv domains into the FI174 bend region does not affect binding to hemagglutin H1, indicating that different domains can be inserted into this site without affecting the specificity of the parent antibody, as shown above for Ig- similar domains. The dual simultaneous recognition of two different specific antigens by the VDJ region of the framework antibody and the VHH or ScFv domains inserted into the fold region indicates that it is possible to generate functional and bispecific antibodies bearing different types of fold region inserts.

Table of sequences and SEQ ID numbers (list of sequences)

SEQ ID NO Sequences Notes SEQ ID NO: 1 QLQL VQ S GTE VKKP G AS VK VS CKS S GY VFT S Y YL V WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRVTV FTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPPY LYF ALD VWGQGT AVT VS S GCE536 VH aa SEQ ID NO: 2 EIVLTQSPGTLLSLSPGETAILSCRASQSVSSSLLAWYQ QKPGQAPRLLIYGASNRATGIRGRFSGSGSGTDFTLT ISRLEPEDFVLYYCQHYGSRVTFGQGTKLEIK GCE536 VL aa SEQ ID NO: 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV FVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEK FISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Human HC IgGl constant region aa SEQ ID NO: 4 RT VAAP S VFIFPP SDEQLKSGT AS VVCLLNNFYPRE A KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Human LC kappa constant region aa SEQ ID NO: 5 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGIH WVRQAPGKGLEWLSLIENHGRKIYYAESVKGRITVS RDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRYT D AGGTHRT AYLD Y WGRGTL VT VS S Cl VH aa

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SEQ ID NO: 6 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQQ TSGQAPVLVIYEGTKRPSGIPERFSGSVSGAMATLMI SEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLTVL Cl VL aa SEQ ID NO: 7 GQPK A AP S VTLFPP S SEELQ ANK ATL VCLISDFYP GA VT VA WK AD S SP VK AG VETTTP SKQ SNNKY A AS S YL SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Human LC lambda constant region aa SEQ ID NO: 8 DVQLVESGGGVVRPGVSLRLSCVASGFSFKNYDMA WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRFLI SRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNTG RDHP YDL WGQGTMVT VS S Clb VH aa SEQ ID NO: 9 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQ QKPGKAPKLLIYKASSLGSGVPSRFSGSGSGTQFTLTI SSLQPDDFATYYCQQYNNYPYTFGQGTKLEIK Clb VL aa SEQ ID NO: 10 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALSW VRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRVTINA DESSTTVYLELSSLTSEDTAIYYCAGSATYYESRFDY WGQGTLVTVSS FI 174 VH aa SEQ ID NO: 11 EIVLTQSPGTLSLSPGARATLSCRASQSVSSSSLAWY QQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCHQYGDSRKTFGQGTKVEIK FI 174 VL aa SEQ ID NO: 12 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK unmutated LAIR1 fragment aa SEQ ID NO: 13 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNAGL FRCIYYKSRKWSEQSDYLELVVK mutated LAIR1 fragment aa SEQ ID NO: 14 DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSES FVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQ IKESLRAELRVT PD-1 fragment aa SEQ ID NO: 15 EQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAY SWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICTV SNPISNNSQTFSPWPGCRTDPS SLAM fragment aa SEQ ID NO: 16 MAQVQLVESGGGLVQAGGSLTLSCAASGSTSRSYA LGWFRQAPGKEREFVAHVGQTAEFAQGRFTISRDFA KNTVSLQMNDLKSDDTAIYYCVASNRGWSSPSRVSY WGQGTQVTVSS T3-VHH aa SEQ ID NO: 17 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSRVGVG WIRQPP GK ALE WL SLIY WDDEKH Y SP SLKNRVTISK DSSKNQVVLTLTDMDPVDTGTYYCAHRGVDTSGW GFDYWGQGALVTVSSGGGGSGGGGSGGGGSQSAL TQPASVSGSPGQSITISCSGAGSDVGGHNFVSWYQQ YPGKAPKLM IYDVKNRPSGVSYRFSGSKSGYTASLTI S GLQ AEDE AT YFC S S Y S S S STLIIFGGGTRLT VL TT39.7-scFv aa

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SEQ ID NO: 18 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGW FRQAPGKEREAVSCISGSGSTYYPDSVKGRFTISRD NAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGGCV HYGMD Y WGKGTQVT VS S F4-VHH aa SEQ ID NO: 19 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISASSSYSDYADSAKGRFTISRD NAKTSLFLQMNSLRAEDTAIYFCARARATGYSSITPY FDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVVT QPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQL PGTAPKLLIYDNNNRPSG VPDRFSASKSGTSASLAIT GLQAEDEADYYCQSYDRNLSGVFGTGTKVTVL MPE8-scFv aa SEQ ID NO: 20 SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK twin StrepTag aa SEQ ID NO: 21 GLNDIFEAQKIEWHE AviTag SEQ ID NO: 22 KRRWKKNFIAVSAANRFKKISSSGAL Calmodulin tag SEQ ID NO: 23 EEEEEE Polyglutamate tag SEQ ID NO: 24 GAPVPYPDPLEPR E-tag SEQ ID NO: 25 DYKDDDDK FLAG tag SEQ ID NO: 26 YPYDVPDYA ON-tag SEQ ID NO: 27 HHHHHH His-tag SEQ ID NO: 28 EQKLISEEDL Moose tag SEQ ID NO: 29 TKENPRSNQEESYDDNES NE-tag SEQ ID NO: 30 KET A A AKFERQHMD S S-mark SEQ ID NO: 31 MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQG QREP SBP tag SEQ ID NO: 32 SLAELLNAGLGGS Softag 1 SEQ ID NO: 33 TQDPSRVG Softag 3 SEQ ID NO: 34 WSHPQFEK Strep-tag SEQ ID NO: 35 CCPGCC TS label SEQ ID NO: 36 GKPIPNPLLGLDST V5 label SEQ ID NO: 37 YTDIEMNRLGK VSV tag SEQ ID NO: 38 DLYDDDDK Xpress label SEQ ID NO: 39 TDKDMTITFTNKKDAE Isopeptag SEQ ID NO: 40 AHIVMVDAYKPTK SpyTag SEQ ID NO: 41 KLGDIEFIKVNK SnoopTag SEQ ID NO: 42 EVHTNQDPLD Tyl label SEQ ID NO: 43 GGGGS linker SEQ ID NO: 44 GGGGSGGGGS linker SEQ ID NO: 45 GGGGSGGGGSGGGGS linker SEQ ID NO: 46 GGGGSGGGGSGGGGSGGGGS linker SEQ ID NO: 47 GGGGSGGGGSGGGGSGGGGSGGGGS linker SEQ ID NO: 48 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker SEQ ID NO: 49 FVGPPSPFLTSLHLS linker SEQ ID NO: 50 GAASEAPGQGLAEPV linker SEQ ID NO: 51 QGFTSVMAPFLPLLT linker SEQ ID NO: 52 GEYTGGSLCATLMSM linker

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SEQ ID NO: 53 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGIHW VRQAPGKGLEWLSLIENHGRKIYYAESVKGRITVSR DNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRYTD AGGTHRTAYLDYWGRGTLVTVSSEDLPRPSISAEPG FVIPLGSHVTFVCRGPVGVQTFRLERERNYLYSDTED VSQTSP SESE ARFRID S VNA GNAGLFRCIYYKSRK WS EQSDYLELVVKASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL Y SL S S V VT VP S S SLGTQT YICN VNHKP SNTK VDKR VE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK C2 heavy chain aa SEQ ID NO: 54 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQQ T S GQ AP VL VIYEGTKRP S GIPERF S GS VS G AM ATLMI SEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLTVLG QPK A AP S VTLFPP S SEELQ ANK ATL VCLISDF YP G AV TVAWKADSSPVKAGVETTTTPSKQSNNKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS C2/C3 light chain aa SEQ ID NO: 55 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGIH WVRQAPGKGLEWLSLIENHGRKIYYAESVKGRITVS RDNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRYT DAGGTHRTAYLDYWGRGTLVTVSSEDLPRPSISAEP GTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDT EDVSQASPSESEARFRIDSVSEG NAGPYRCIYYKPPK WSEQSDYLELLVKASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK C3 heavy chain aa

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SEQ ID NO: 56 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV W VRQ AP GQGLE WM ATI SP GD VNT S YEQRFQGR VT VTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPP YLYFALDVWGQGTAVTVSSEDLPRPSISAEPGTVIPL GSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQA SPSESEARFRIDSV SEGNAGPYRCIYYKPPKWSEQSD YLELLVKASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL S SWT VPS S SLGTQTYICNVNHKP SNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK C4 heavy chain aa SEQ ID NO: 57 EIVLTQSPGTLSLSPGETAILSCRASQSVSSSLLAWY QQKPGQAPRLLIYGASNRATGIRGRFSGSGSGTDFT LTISRLEPEDFVLYYCQHYGSRVTFGQGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC C4/C5/C6 light chain aa SEQ ID NO: 58 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV W VRQ AP GQGLE WM ATI SP GD VNT S YEQRFQGR VT VTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPP YLYF ALD VWGQGT AVT VS SD SPDRP WNPPTFSP AL LVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD KLAAFPED RSQPGQDCRFRVTQLPNGRDFHMSVVR ARRNDSGTYLCGAISLAPKAQIKESLRAELRVTAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV D VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GF YP SDI AVE WE SNGQPENNYKTTPP VLD SD GSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK C5 heavy chain aa

- 55 046243

SEQ ID NO: 59 QLQL VQ S GTE VKKP G AS VK VS CKS S GY VFT S Y YL V WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRVTV FTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPPY LYFALDVWGQGTAVTVSSFVGPPSPFLTSLHLSDSPD RPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLN WYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVTQLPN GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTGAASEAPGQGLAEPVASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQ S S GL Y SL S S V VT VP S S SLGTQT YICN VNHK PSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK C5b heavy chain aa SEQ ID NO: 60 QLQL VQ S GTE VKKP G AS VK VS CKS S GY VFT S Y YL V WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRVTV FTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPPY LYFALDVWGQGTAVTVSSFVGPPSPFLTSLHLSDSPD RPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLN WYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVTQLPN GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTGAASEAPGQGLAEPVASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH FFP AVLQ S S GL Y SL S S V VT VP S S SLGTQT YICN VNHK PSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK C6 heavy chain aa

- 56 046243

SEQ ID NO: 61 QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRVT VTTDASTNTVDMELRSLRSEDTAVYYCARGPRSKPP YLYFALDVWGQGTAVTVSSQGFTSVMAPFLPLLTE QVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAYS WSEKAGTHPLNP ANS SHLL SLTLGPQH ADNIYICT V SNPISNNSQTFSPWPGCRTDPSGEYTGGSLCATLMS MASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISK AKGQPREPQ VYTLPP SREEMTKNQ VSLT CL VKGF YP SDI AVE WE SNGQPENNYKTTPP VLD SD G SFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYT QKS LSLSPGK C6b heavy chain aa SEQ ID NO: 62 DVQLVESGGGVVRPGVSLRLSCVASGFSFKNYDMA WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRFL ISRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNT GRDHP YDL WGQGTMVT VS SD SPDRP WNPPTFSP AL LVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD KLAAFPEDRSQPGQDCRFRVTQLPNG RDFHMSVVR ARRNDSGTYLCGAISLAPKAQIKESLRAELRVTAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GF YP SDI AVE WE SNGQPENNYKTTPP VLD SD GSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK C7 heavy chain aa SEQ ID NO: 63 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWY QQKPGKAPKLLIYKASSLGSGVPSRFSGSGSGTQFTL TISSLQPDDFATYYCQQYNNYPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC C7/C8/C9 light chain aa

- 57 046243

SEQ ID NO: 64 DVQLVESGGGVVRPGVSLRLSCVASGFSFKNYDMA WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRFLI SRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNTG RDHPYDLWGQGTMVTVSSEQVSTPEIKVLNKTQEN GTCTLILGCTVEKGDHVAYSWSEKAGTHPLNPANSS HLLSLTLGPQHADNIYICTV SNPISNNSQTFSPWPGCR TDPSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP iektiskakgoprepovytlppsreemtknovsltcl VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK C8 heavy chain aa SEQ ID NO: 65 DVQLVESGGGVVRPGVSLRLSCVASGFSFKNYDMA WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRFL ISRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNT GRDHPYDLWGQGTMVTVSSSAWSHPQFEKGGGSG GGSGGSAWSHPQFEKASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAV LQ S S GL Y SL S S VVT VP S S SLGTQT YICN VNHKP SNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK C9 heavy chain aa SEQ ID NO: 66 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALSW VRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRVTINA DESSTTVYLELSSLTSEDTAIYYCAGSATYYESRFDY WGQGTLVTVSSMAQVQLVESGGGLVQAGGSLTLS CAASGSTSRSYALGWFRQAPGKEREFVAHVGQTAE FAQGRFTISRDFA KNTVSLQMNDLKSDDTAIYYCVA SNRGWSPSRVSYWGQGTQVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE vkf CIO heavy chain

- 58 046243

SEQ ID NO: 67 EIVLTQSPGTLSLSPGARATLSCRASQSVSSSSLAWY QQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCHQYGDSRKTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKAD YEKHK VY ACEVTHQGL S SP VTKSFNRGEC C10/C11/C12/C13 light chain aa SEQ ID NO: 68 PSLK NRVTISKDSSKNQVVLTLTDMDPVDTGTYYC AHRGVDTSGWGFDYWGQGALVTVSSGGGGSGGGG SGGGGSQSALTQPASVSGSPGQSITISCSGAGSDVGG HNFVSWYQQYPGKAPKLMIYDVKNRPSGVSYRFSG SKSGYTASLTISGLQAEDEATYFCSSYSSSSTLIIFGG GTRLTVLASTKGPSVFPLAPSSKS TSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL S SWT VPS S SLGTQTYICN VNHKP SNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK Cl 1 heavy chain aa SEQ ID NO: 69 SVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAT IRSSSWGGCVHYGMDYWGKGTQVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Cl2 heavy chain aa

- 59 046243

SEQ ID NO: 70 QVQLVQSGAEVRKPGSSVKVSCKTSGGIIRKYALSW VRQAPGQGLEWMGGIIAIFGTTNYAQKFQGRVTINA DESTSTVYLELSSLTSEDTAIYYCAGSATYYESRFDY WGQGTLVTVSSEVQLVESGGGLVKPGGSLRLSCAA SGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYSDY ADSAKGRFTISRDNA KTSLFLQMNSLRAEDTAIYFC ARARATGYSSITPYFDIWGQGTLVTVSSGGGGSGGG GSGGGGSQSVVTQPPSVSGAPGQRVTISCTGSSSNIG AGYDVHWYQQLPGTAPKLLIYDNNNRPSGVPDRFS ASKSGTSASLAITGLQAEDEADYYCQSYDRNLSGVF GTGTKVTVLASTKGPSVFPLAPSSKSTSGGTAAL K VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK C13 heavy chain aa SEQ ID NO: 71 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWS WVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISV DKSKNQFSLKLSSVTAADTAVYYCARASPLKSQRD TEDLPRPPSISAEPGTVIPLGSHVTFVCRGPVGVQTFR LERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNA GPYRCIYYKPPKWSEQ SDYLELLVKGEDVTWALPQ SQLDPRACPQGELPISTDIYYMDVWGKGTTVTVSS MGD ^uca VH aa SEQ ID NO: 72 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQ QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLT ISCLQSEDFATYYCQQYYSYPPDFGQGTRLEIK MGD ^UCA VL aa SEQ ID NO: 73 QVQLIQSGGGLVKPGGSLRLSCAASGFTFSDYYMS WIRQVPGKGLEWISVISATTGYTDYADSVKGRFTIS RDNAKNSVFLQMNSLRVDDMAVYYCAREVLGTA WFDYWGQGTLVTISS TT107 VH aa SEQ ID NO: 74 EIVLTQSPGTLLSLSPGERATLSCRASQSVTSNYLAWY QQKPGQAPRLLIYGVSRRATGIPDRFSGSGSGTDFAL TISRLEPEDFAVYYCQQYRSSPRTFGPGTKVEFK TT107 VL aa SEQ ID NO: 75 E VQL VES GGG V VRP GESLRL S C A AS GFIFNDFGMN W VRQPPGRGLEWVAGIKWRGGGVALVPSVTGRFTISG DNDKNSLYLQMTSLRDEDTAVYYCARDSGFRGGRG HAFDLWGQGTMVTISAEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVHTFRLERESRSTYNETEDVSQASPSE SEARFRIDSVSEGNAGPYRCI YYKPPKWSEQSDYLEL LVK MGJ1 VH

- 60 046243

SEQ ID NO: 76 EVQVVESGGRVARPGGSLRLSCAASGFHLDDYDMS WVRQPPGKGLEWVAGINWNGGRTGYADSVKGRLTI SRDNAKKFLYLEMKSLRAEDTALYYCARDPGYSSG RRNALDIWGQGTMVTVSLEDLPRPSISAEPGTVIPLG SHVTFVCRGPVGVQTFRLERESRFTYNDTEDVSQASP SESEARFRIDSVSEGNAGPYRCIYY KPPKWSEQSDYL ELLVK MGI2 VH SEQ ID NO: 77 E VQL VQ S GGG V VRP GGFLRL S C A AS GFTFENY A V A WVRQVAGKGLEWLCVINWDAGTTNYADSVKGRFTI SRDIVKNSLVLEMSSLRAEDTALYYCARDPVYGSDR GDVFDMWGQGTVVTVSSDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTFRLERESRSTYNETEDVSQASPSE SEARFRIDSVSEGNAGPYRC IYYKPPKWSEQSDYLEL LVK MGI3 VH SEQ ID NO: 78 DVQLVESGGGVVRPGVSLRLSCVASGFSFKNYDMA WVRQVPGKGLEWVCGINWNGSLRGYADSVKGRFLI SRDHAKDSLYLQMSRLRAEDTALYYCARDPGYNTG RDHPYDLWGQGTMVTVSSEDLPRPSISAEPGTVIPLG SHVTFVCRGPVGVQTFRLERESRSTYNETEDVSQVSP SESEARFRIDSVSEGNAGPYRCI YYKPPKWSEQSDYL ELLVK MGI5 VH SEQ ID NO: 79 EVQLVESGGRVVRPGESLRLSCEVSGVSINDYDMSW VRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLTGDDTAFYYCVRDPGESSGRG HIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSHV IFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEGNAGPY RCIYYKPPKWSEQSDYLELL VK MMI1 VH SEQ ID NO: 80 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGDTSGRG HIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSE SEARFRIDSVSEGNAGPYRCIY YKPPKWSEQSDYLEL LVK MMI2 VH SEQ ID NO: 81 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DNGKNSLYLQMNSLRGEDTALYYCVRDPGDRSGRG HIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSHV IFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEGNAGPYRC IYYKPPKWSEQSDYLELL VK MMI5 VH SEQ ID NO: 82 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMSW VRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGDSSGRG QIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSHV IFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEGNAGPYRC IYYKPPKWSEQSDYLELL VK MMI6 VH

- 61 046243

SEQ ID NO: 83 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMSW VRQRLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGESSGRG HIFNIWGQGTMVTISLEDLPRPSISAEPGTVIPLGSHV FFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEGNAGPYRCI YYKPPKWSEQSDYLELL VK MMI7 VH SEQ ID NO: 84 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGDTSGRG HIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSE SEARFRIDSVSEGNAGPYRCIY YKPPKWSEQSDYLEL LVK MMI8 VH SEQ ID NO: 85 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGDTSGRG HIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSE SEARFRIDSVSEGNAGPYRCIY YKPPKWSEQSDYLEL LVK MMJ10 VH SEQ ID NO: 86 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQFLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGEDTALYYCVRDPGDTSGRG HIFNVWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSH VTFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSE SEARFRIDSVSEGNAGPYRCIY YKPPKWSEQSDYLEL LGK MMJ16 VH SEQ ID NO: 87 EVQLVESGGGVVRPGESLRLSCEVSGVNINDYDMSW VRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DNGKNSLYLQMNSLRGEDTALYYCVRDPGDRSGRG HIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSHV FFVCRGPVGVQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEGNAGPYRC IYYKPPKWSEQSDYLELL VK MMI23 VH SEQ ID NO: 88 EVQLVESGGGVVRPGESLRLSCEVSGVSINDYDMSW VRQPLGKGLEWVSGIDRKGVGTGYADSVKGRFTISR DHAKNSLYLQMNSLRGADT ALYYC VRDPGD S SGRG HIFNIWGQGTMVTVSLEDLPRPSISAEPGTVIPLGSHV FFVCRGPVGQTFRLERESRSTYNETEDVSQVSPSES EARFRIDSVSEG NAGPYRCIYYKPPKWSEQSDYLELL VK MMI25 VH SEQ ID NO: 89 QVQLAQYGGGAVQPGGSLRLSCVVSGFRFSLYGIHW VRQAPGKGLEWLSLIENHGRKIYYAESVKGRITVSR DNFKNVAYLEMYRLSTEDTAIYYCARNDGLGRYTD AGGTHRTAYLDYWGRGTLVTVSSEDLPRPSISAEPG FVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTED VSQASPSESEARFRIDSVSEGNA GPYRCVYYKPPKWS EQSDYLDLLVK MGM1 VH

- 62 046243

SEQ ID NO: 90 Q VQL VE S GGD V VQP GGSLRL S C A VS GFKFNIYDIH W VRQAPGKGLEWVSFIRHDGNNQEYADSVKGRFTISR DNFKNIIDLQMHSLRTEDTALYYCATNQGSGGSDDT WETNRSAFFPHWGQGTLVTVSSDLPRPSISAEPGTVI PLGSHVTFVCRGPVGVQTFRLERESRSIYNDTEDVSQ ASPS ESEARFRIDSVSEGNAGPYRCVYYKPPKWSEES DYLELLVK MGM3 VH SEQ ID NO: 91 QVQLVESGGGVVQPGGSLRLSCEVSGFRFSTYGIHW ARQAPGKGLEWVAFIRYDGNNKSYADSVKGRFTISR DNSKNTLYLQMNSLRIEDTAVYYCAKNQASGGYDD FWGTYRSAYLDYWGQGTLVTVSSEDLPRPSISAEPG FVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTED VSQASPSESEARFRIDSVSEG NAGPYRCVYYKPPKWS EESDSLELLVK MGM4 VH SEQ ID NO: 92 Q VQL VE S GGGV VQP GGSLRL S CKMS GFKF S AFGIH W VRQAPGKGLEWVAFVRYDGGDKYYADSVKGRFTIS RDNSKNTVHLQLNSLKPADTAVYYCAKNQPSGQSD DTWGTSLSAYLDYWGQGTQVSVSPEDLPRPSISAEP GTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTE DVSQASPSESEARFRIDSVSEGNAGPYRCVYYKPPK WSEQSDYLELLVK MGM5 VH SEQ ID NO: 93 EAQVVDHGNRGRARDLEDIKKRRARDLEYEDLPRPPS ISAEPGTVIPLGSRVTFVCRGPVGVQTFRLERESRSKY NETEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYK PPKWSEHSDFLELLVK MGB2 VH SEQ ID NO: 94 VAEVEEHINKRRARDLEYEDLPRPSISAEPGTVIPLGS HVTFVCRGPVGVQTFRLERESRSRYNETEDVSQTSPS ESEARFRIDSVSEGNAGPYRCLYYKTPKWSEQSDFLE LLVK MGB43 VH SEQ ID NO: 95 EAEVVEHVNKRRARALEYEDLPRPSISAEPGTVIPLG SHVTFVCRGPVGVQTFRLERESRSRYTETEDVSQTSP SESEARFRIDSVSEGNAGPYRCLYYKPPKWSEQSDFL ELLVK MGB47 VH SEQ ID NO: 96 DFQMTQSPSTLSASVGDRVTITCRASQNVNTWLAW YQQAAGKAPKLLIYEASTLQSGVPSRFRGGGSGTEFT LTITSLQPEDFATYYCHQYKSHPPFTFGPGTKVDVR MGJ1 VL SEQ ID NO: 97 DIQMTQSPSTLSASVGDRVTITCRASQTISSWLAWYQ QKPGKAPKFLIYKASFLENGVPSRFSGSESGTEFTLTI NSLQPDDFATYYCQQYKSYPFTFGPGTKVEIK MGI2 VL SEQ ID NO: 98 DIQMTQSPSTLSASVGDRVTFTCGASQSITDCLAWYQ QKPGKDPKLLIYKASRLEAGVPARFSASGSGTEFTFTI RSMQPEDFATYYCQQCYSYSYPFTFGPGTKVDLK MGI3 VL SEQ ID NO: 99 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQ QKPGKAPKLLIYKASSLGSGVPSRFSGSGSGTQFTLTI SSLQPDDFATYYCQQYNNYPYTFGQGTKLEIK MGJ5 VL

- 63 046243

SEQ ID NO: 100 DIQMTQSPSTVSASIGDRVTITCRASQIIERSLAWYQQ KPGKSPKALIYKTSNLEDGVPSRFSGSGSGTDFTLTV SSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MM JI VL SEQ ID NO: 101 DIQMTQSPSTLSASIGDRVTITCRASQVIDRSLAWFQ QKPGKSPRPLIYKASTLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MMJ5 VL SEQ ID NO: 102 DIQMTQSPSTLSASIGDRVTITCRASQIIHRSLAWYQQ KPGKSPRALIYKASNLEGGVPSRFSGSGSGTDFTLTV SSLQPDDFAMYYCQQYDTYPFTFGPGTTVTLR MMJ6 VL SEQ ID NO: 103 DIQMTQSPSTLSASIGDRVTITCRASQSIDRSLAWYQ QKPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFADYYCQQYDTYPFTFGPGTTVTLR MMJ7 VL SEQ ID NO: 104 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQQ KPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLTV SSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MMJ8 VL SEQ ID NO: 105 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQQ KPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLTV SSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MMJ10 VL SEQ ID NO: 106 DIQMTQSPSTLSASIGDRVTITCRASQIIDRSLAWYQQ KPGKSPKALIYKASNLEGGVPSRFSGSGSGTDFTLTV SSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MMJ16 VL SEQ ID NO: 107 DIQMTQSPSTLSASIGDRVTITCRASQVIDRSLAWFQ QKPGKSPRPLIYKASTLEGGVPSRFSGSGSGTDFTLT VSSLQPDDFANYYCQQYDTYPFTFGPGTTVTLR MMJ23 VL SEQ ID NO: 108 DIQMTQSPSTLSASIGDRVTITCRASQNIDRSLAWYQ QKPGKSPKALIYKASNLEDGVPSRFSGSGSGTDFTLT VSSLQPDDFALYYCQQYDTYPFTFGPGTTVTLR MMJ25 VL SEQ ID NO: 109 SYEVTQPPSVSVSPGQAARITCSGDELPRTDISWYQQ TSGQAPVLVIYEGTKRPSGIPERFSGSVSGAMATLMI SEAQLEDEGDYYCFSIDTSGNHGGAFGTGTKLTVL MGM1 VL SEQ ID NO: 110 SYELIQPPSXSVSPGQTARITCSGEPLPRTSTSWYRQK SGQAPVLIIYEVSKRPSGIPERXSGSNTGTKATLFIVG AQVEDEGDYYCYSTNTSGGSRGAFGTGTSLTVL MGM3 VL SEQ ID NO: 111 SYELTQPPSVSVSPGQTARITCSGDAVPNTYTYWYQ QKS GQ AP VL VIYED SKRP S GIPERF S GS S S GTM ATFIIS GAQVEDEADYYCYSTDTSDDHRGAFGTGTKVTVL MGM4 VL SEQ ID NO: 112 SYELTQPPSVSVSPGQTARITCSGDALPRTFIYWYQQ KSRQ AR V V VI YED VKRP S GIPERF S GSIS GTQ ATLIITG AQVEDEADYYCYSTDTNNTHRGAFGTGTKVTVL MGM5 VL SEQ ID NO: 113 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVHTFRLE RESRSTYNETEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MGJ1 LAIR1 SEQ ID NO: 114 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRFTYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MGJ2 LAIR1

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Claims (57)

SEQ ID NO: 115 DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLER ESRSTYNETEDVSQASPSESEARFRIDSVSEGNAGPY RCIYYKPPKWSEQSDYLELLVK MGJ3 LAIR1 SEQ ID NO: 116 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MGJ5 LAIR1 SEQ ID NO: 117 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ1 LAIR1 SEQ ID NO: 118 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ2 LAIR1 SEQ ID NO: 119 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ5 LAIR1 SEQ ID NO: 120 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ6 LAIR1 SEQ ID NO: 121 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ7 LAIR1 SEQ ID NO: 122 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ8 LAIR1 SEQ ID NO: 123 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ10 LAIR1 SEQ ID NO: 124 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLGK MMJ16 LAIR1 SEQ ID NO: 125 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNETEDVSQVSPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEQSDYLELLVK MMJ23 LAIR1 SEQ ID NO: 126 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFR LERESRSTYNETEDVSQVSPSESEARFRIDSVSEGN AGPYRCIYYKPPKWSEQSDYLELLVK MMJ25 LAIR1 SEQ ID NO: 127 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCVYYKPPKWSEQSDYLDLLVK MGM1 LAIR1 SEQ ID NO: 128 DLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLER ESRSIYNDTEDVSQASPSESEARFRIDSVSEGNAGPYR CVYYKPPKWSEESDYLELLVK MGM3 LAIR1 SEQ ID NO: 129 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCVYYKPPKWSEESDSLELLVK MGM4 LAIR1 SEQ ID NO: 130 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGP YRCVYYKPPKWSEQSDYLELLVK MGM5 LAIR1 SEQ ID NO: 131 EDLPRPSISAEPGTVIPLGSRVTFVCRGPVGVQTFRLE RESRSKYNETEDVSQASPSESEARFRIDSVSEGNAGP YRCIYYKPPKWSEHSDFLELLVK MGB2 LAIR1 SEQ ID NO: 132 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSRYNETEDVSQTSPSESEARFRIDSVSEGNAGP YRCLYYKTPKWSEQSDFLELLVK MGB43 LAIR1 SEQ ID NO: 133 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLE RESRSRYTETEDVSQTSPSESEARFRIDSVSEGNAGPY RCLYYKPPKWSEQSDFLELLVK MGB47 LAIR1 CLAIM 1. An antibody or an antigen-binding fragment thereof with a functional domain in the bend region, which/which includes a first polypeptide chain and a second polypeptide chain, the first of which contains, in the N to C direction, (i) one or more variable domains, (ii) a functional domain , And - 65 046243 (iii) one or more constant domains, and said second polypeptide chain includes in the N to C direction (iv) one or more variable domains forming antigen binding sites with one or more variable domains of the first polypeptide chain, (v) a functional domain, and (vi) one or more constant domains, characterized in that said functional domain (ii) of the first polypeptide chain does not include a fragment of the second polypeptide chain, and said functional domain (v) of the second polypeptide chain does not include a fragment of the first polypeptide chain, with wherein (a) one or more constant domains of the first polypeptide chain are heavy chain constant domains containing at least a CH1 constant domain, one or more variable domains of the first polypeptide chain are heavy chain variable domains VH, the constant domain of the second polypeptide chain is a CL constant domain light chain, and one or more variable domains of the second polypeptide chain are VL light chain variable domains, or (b) the constant domains of the first polypeptide chain are a CL light chain constant domain, one or more variable domains of the first polypeptide chain are VL light chain variable domains, one or more constant domains of the second polypeptide chain - constant domains of the heavy chain, containing at least a constant domain CH1, and one or more variable domains of the second polypeptide chain - variable domains VH of the heavy chain. 2. An antibody or an antigen-binding fragment thereof with a functional domain in a fold region which/which includes a first polypeptide chain and a second polypeptide chain, the first of which includes in the N to C direction (i) one or more variable domains, (ii) a functional domain , and (iii) one or more constant domains, and said second polypeptide chain includes in the N to C direction (iv) one or more variable domains forming antigen binding sites with one or more variable domains of the first polypeptide chain, and (v) one or more constant domains, characterized in that the C-terminus of the outermost C-terminal variable domain of the second polypeptide chain is directly linked to the N-terminus of the outermost N-terminal constant domain of the second polypeptide chain, wherein (a) one or more constant domains of the first polypeptide chain chains are heavy chain constant domains containing at least a CH1 constant domain, one or more variable domains of a first polypeptide chain - heavy chain variable domains VH, a second polypeptide chain constant domain - a light chain constant domain CL, and one or more variable domains of a second polypeptide chain chain - variable domains of the VL light chain, or (b) the constant domains of the first polypeptide chain are the constant domain CL of the light chain, one or more variable domains of the first polypeptide chain are the variable domains of the VL light chain, one or more constant domains of the second polypeptide chain are constant domains heavy chain, containing at least a constant domain CH1, and one or more variable domains of the second polypeptide chain - variable domains VH of the heavy chain. 3. The antibody or antigen binding fragment thereof according to claim 1 or 2, wherein the CH1 domain and optionally any additional heavy chain constant domain is selected from the following classes: γ, α, μ, ε and δ. 4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the CL domain is selected from the following classes: k and λ. 5. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes or consists of Ig-like domain. 6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the Ig-like domain is selected from the Ig-like domain(s) PD1, SLAM, CD2, CD3, CD4, CD8, CD19, CD22, CD33, CD80 or CD86. 7. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a carrier domain, a reporter domain, tags, a domain localization, (independent) binding site, enzyme or enzymatic domain, receptor or functional fragment thereof, or ligand or functional fragment thereof. - 66 046243 8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a carrier domain. 9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the carrier domain is provided for conjugating the antibody or antigen-binding fragment thereof to a drug or imaging agent. 10. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a reporter domain. 11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the reporter domain contains the amino acid sequences of GFP, YFP, RFP, CFP, luciferase, beta-galactosidase or peroxidase. 12. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a localization domain. 13. The antibody or antigen binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes an enzyme or enzymatic domain, for example a catalytic domain. 14. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a label. 15. The antibody or antigen binding fragment thereof according to claim 14, wherein the label is an affinity tag, a solubilization tag, a chromatography tag, an epitope tag or a fluorescent tag. 16. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a receptor or a functional fragment thereof, for example an Ig-like receptor domain. 17. The antibody or antigen-binding fragment thereof according to claim 16, wherein the functional domain includes (i) a receptor selected from the group consisting of PD1, SLAM, LAIR1, CTLA4, BTLA, TIM-3, TIGIT, CD200R1, 2B4 (CD244) , TLT2, LILRB4, KIR2DL2, ICOS or CD28, or (ii) a functional fragment of a receptor, for example, an Ig-like domain of any one of the following receptors: PD1, SLAM, LAIR1, CTLA4, BTLA, TIM-3, TIGIT, CD200R1 , 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28. 18. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes a ligand or a functional fragment thereof. 19. The antibody or antigen-binding fragment thereof according to claim 18, wherein the ligand is a cytokine and/or a ligand of PD1, SLAM, LAIR1, CTLA4, BTLA, TIM-3, TIGIT, CD200R1, 2B4 (CD244), TLT2, LILRB4, KIR2DL2, ICOS or CD28. 20. An antibody, or an antigen-binding fragment thereof according to claim 18, in which the ligand is selected from the group consisting of PD-L1, PD-L2, B7-1, B7-2, B7-H4 (B7 homologue), galectin-9, receptor poliovirus (PVR), membrane glycoprotein OX-2, CD48, B7-NZ (homolog of B7), MHCI, ICOS-L. 21. The antibody or antigen-binding fragment thereof according to claim 18, wherein the ligand is selected from the group consisting of SIS family cytokines, SIG family cytokines, SCY family cytokines, members of the platelet factor 4 superfamily and intercrins, CC chemokine ligands CCL-1 through CCL-28, CXCL1-CXCL17, XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β), fractalkine (or CX ₃ CL1); interferons, for example type I IFN, type II IFN and type III IFN, in particular IFN-α, IFN-β, IFN-γ, IFN-ε, IFN-κ, IFN-ω, IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12); interleukins, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 , IL-13, IL-14, IL15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25 , IL-26, IL-27, IL-28, IL-29, IL-30, IL31, IL-32, IL-33, IL-34, IL-35 and IL-36; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF and interferon-gamma; tumor necrosis factors, for example CD40LG (TNFSF5); CD70 (TNFSF7); EDA; FASLG (TNFSF6); LTA (TNFSF1); LTB (TNFSF3); TNF, TNFa, TNFSF4 (OX40L); TNFSF8 (CD153); TNFSF9; TNFSF10 (TRAIL); TNFSF11 (RANKL); TNFSF12 (TWEAK); TNFSF13; TNFSF13B; TNFSF14; TNFSF15 and TNFSF18; and colony-stimulating factors, such as CSF1 (macrophage colony-stimulating factor), CSF2 (GM-CSF), CSF3 (G-CSF), as well as synthetic CSFs, such as promegapoietin. 22. The antibody or antigen-binding fragment thereof according to claim 7, wherein the functional domain (ii) of the first polypeptide chain and/or the functional domain (v) of the second polypeptide chain includes or consists of a (independent) binding site. 23. The antibody or antigen-binding fragment thereof according to claim 22, wherein the (independent) binding site - 67 046243 vaniya includes a receptor or a functional fragment thereof, including a binding site, a ligand or a functional fragment thereof, including a binding site, a CD molecule or a functional fragment thereof, a single chain antibody or an antigen binding fragment thereof, an antigen or a functional fragment thereof, or a label including the site binding. 24. The antibody or antigen binding fragment thereof according to claim 23, wherein the (independent) binding site comprises a single chain antibody (eg scFv or VHH) or an antigen binding fragment thereof, or an antigen or functional fragment thereof. 25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, wherein the functional domain includes an Ig-like domain, scFv, VHH or Strep-tag. 26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the functional domain comprises an amino acid sequence represented in any of SEQ ID NOs: 13-20, or a variant of the functional sequence thereof, which is at least 80% 85%, at least 90%, at least 95%, or at least 98% identical to the sequence. 27. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the first polypeptide chain comprises a single variable domain of (i) an N-terminal functional domain (ii) and the second polypeptide chain comprises a single variable domain of (iv) an N-terminal an optional functional domain (v) or an N-terminal one or more constant domains (vi), wherein a single variable domain (iv) of the second polypeptide chain forms an antigen binding site with a variable domain (i) of the first polypeptide chain. 28. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the first polypeptide chain includes two or more variable domains (i) an N-terminal functional domain (ii) and the second polypeptide chain includes two or more variable domains (iv ) N-terminal optional functional domain (v) or N-terminal one or more constant domains (vi), two or more variable domains (iv) of the second polypeptide chain, forming antigen binding sites with two or more variable domains (i) of the first polypeptide chain. 29. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, wherein the first polypeptide chain comprises a single chain antibody, e.g. scFv, or a single domain antibody, e.g. VHH, the N-terminus of the outermost N-terminal variable domain (i) and /or the C-terminus of the outermost C-terminal constant domain (iii). 30. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, wherein the second polypeptide chain comprises a single chain antibody, e.g. scFv, or a single domain antibody, e.g. VHH, the N-terminus of the outermost N-terminal variable domain (i) and /or the C-terminus of the outermost C-terminal constant domain (vi). 31. The antibody or antigen binding fragment thereof according to any one of claims 1 to 30, wherein the first polypeptide chain and the second polypeptide chain each comprise a single constant domain, in particular a CL domain and a CH1 domain, respectively. 32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the first polypeptide chain or the second polypeptide chain includes a single constant domain, in particular a CL domain; and the other first polypeptide chain or second polypeptide chain includes a CH1 domain and one or more additional constant domains, for example a CH2 and/or CH3 domain. 33. The antibody or antigen binding fragment thereof according to any one of claims 1 to 32, wherein the antibody or antigen binding fragment thereof comprises a portion of an Fc molecule or an Fc region. 34. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 33, wherein the first polypeptide chain and/or the second polypeptide chain contains/contain one or more linkers. 35. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the first polypeptide chain and/or the second polypeptide chain does not contain any linkers. 36. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, wherein the first polypeptide chain includes or consists of V-D-CH1, where V is the variable domain (i), D is functional domain (ii), and CH1 is the CH1 constant domain (iii). 37. The antibody or antigen-binding fragment thereof of claim 36, wherein the first polypeptide chain comprises or consists of V-D-CH1-CH2-CH3, wherein CH2 and CH3 are a CH2 constant domain and a CH3 constant domain, respectively. 38. The antibody or antigen binding fragment thereof according to any one of claims 35 to 37, wherein the first polypeptide chain includes or consists of (V)A-D-CH1, where A is an integer from 1 to 5. 39. An antibody or an antigen-binding fragment thereof to any one of claims 1 to 37, wherein the second polypeptide chain includes or consists of V-CL, where V is a variable domain (iv); and CL is a constant domain (vi). 40. The antibody or antigen-binding fragment thereof according to claim 39, wherein the second polypeptide chain - 68 046243 includes or consists of (V) _A -CL, where A means an integer from 1 to 5. 41. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, wherein the variable domain (i) of the first polypeptide chain includes or consists of an amino acid sequence presented in any of SEQ ID NO: 1, 5, 8 or 10, or a variant of its functional sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to that sequence. 42. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 41, wherein the variable domain (iv) of the second polypeptide chain includes or consists of an amino acid sequence represented in any of SEQ ID NO: 2, 6, 9 or 11, or a functional sequence variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to that sequence. 43. The antibody or antigen binding fragment thereof according to any one of claims 1 to 42, wherein the antibody or antigen binding fragment thereof comprises one or two first polypeptide chains and one or two second polypeptide chains. 44. The antibody or antigen binding fragment thereof according to any one of claims 1 to 43, wherein the first polypeptide bond and the second polypeptide bond are linked by a disulfide bond, thereby forming a pair. 45. An antibody or an antigen-binding fragment thereof according to claim 43 or claim 44, which/which comprises two first polypeptide chains and two second polypeptide chains, wherein the first two polypeptide chains and/or the two second polypeptide chains are linked by one or more, for example, two disulfide bonds. 46. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, which is bivalent for each specificity/antigen. 47. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 45, which is monovalent for each specificity (antigen). 48. The antibody or antigen-binding fragment thereof according to claim 46 or 47, wherein the antigen binding site(s) formed by one or more variable domains of the first pair of the first and second polypeptide chains, and the antigen binding site(s) formed by one or more variable domains of the second pair of the first and second polypeptide chains, are the same or different. 49. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 48, which/which includes at least two functional domains, which may be the same or different. 50. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 49, which/which comprises two first polypeptide chains and two second polypeptide chains forming a first and second pair of first and second polypeptide chains, wherein the first pair of first and second polypeptide chains comprise at least one functional domain(s) and/or a second pair of first and second polypeptide chains comprise at least one functional domain(s). 51. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, which is/is derived from an IgG-like antibody, Fab or F(ab) ₂ . 52. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, wherein the variable domains and/or constant domains are human or humanized. 53. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 52, wherein the (additional) functional domain(s) consists of or includes an amino acid sequence that is a human or humanized sequence. 54. The antibody or antigen binding fragment thereof according to any one of claims 1 to 53, wherein the first polypeptide chain includes or consists of an amino acid sequence that is any of the following SEQ ID NOs: 53, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 68, 69 or 70 or a functional variant of this sequence that is at least 80%, at least 85%, at least 90%, at least 95% or is at least 98% identical to this sequence, and/or the second polypeptide chain includes or consists of an amino acid sequence that is any of the following sequences of SEQ ID NO: 54, 57, 63 or 67 or a functional variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to this sequence. 55. A nucleic acid molecule comprising a polynucleotide encoding the first polypeptide chain of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 54. 56. A nucleic acid molecule comprising a polynucleotide encoding the second polypeptide chain of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 54. 57. A nucleic acid molecule, including a polynucleotide encoding the first polypeptide chain and the second polypeptide chain of an antibody or its antigen-binding fragment according to any -