Multimerization domains for the generation of homogenous multimers with immunoglobulin backbone

Abstract

The present invention relates to multimerization domains for the generation of homogenous multimers with immunoglobulin backbone, and polypeptides, conjugates and multimers comprising such multimerization domains. In particular, the present invention deals with multimers of IgM and IgG backbones having a tail domain which differs from the wild-type IgM tailpiece.

Description

[0001] Sanofi PAT23105

[0002] MULTIMERIZATION DOMAINS FOR THE GENERATION OF HOMOGENOUS MULTIMERS WITH IMMUNOGLOBULIN BACKBONE

[0003] The present invention relates to multimerization domains for the generation of homogenous multimers with immunoglobulin backbone, and polypeptides, conjugates and multimers comprising such multimerization domains.

[0004] BACKGROUND OF THE INVENTION

[0005] Naturally occurring IgM molecules consist of five IgM monomers and a Joining-chain (J-chain), forming a pentamer: IgM(5)J. The formation of the IgM(5)J-complex is dependent on C-terminal sequences of the IgM heavy chains called the tailpiece (TP). The TPs and the J- chain form an intermolecular beta-sheet network. Incorporation of the J-chain into this network is the bottleneck of recombinant expression of IgM(5)J. The function of the J-chain is binding to the polymeric immunoglobulin receptor (plgR). Upon binding to the plgR on the basolateral side of epithelial cells, the IgM(5)J complex is transported to the apical face of the epithelial cell and secreted, resulting a short half-life of IgM(5)J molecules. Recombinant IgM can also be expressed without J-chain, resulting in a mixture of IgM(5) pentamers and IgM(6) hexamers, which is highly undesired for biopharmaceutical products which often require high homogeneity. There remains a need in the art for an effective way to produce homogenous multimeric IgM molecules. A multimeric IgM backbone may also be useful as a platform for the development of novel biotherapeutics. Producing homogenous multimeric immunoglobulin-based molecules without the J-chain would significantly simplify protein production and lead to an enhanced plasma half-life due to lack of binding to plgR. Surprisingly, the inventors identified variant mammalian TPs and non-mammalian TPs that, when fused to IgM heavy chain sequences, lead to an increase in the formation of one type of multimers, usually hexamers, and a decrease in the formation of other multimers, thus promoting homogenous hexamerization. Besides production of homogenous multimeric proteins, the expression yields using these TP sequences were considerably high, which is a prerequisite for usage in the development of biotherapeutics. Thus, the polypeptides of the present invention provide inter alia for one or more of the following advantages: (i) homogenous formation of multimers; (ii) increased formation of hexamers; (iii) decreased formation of pentamers; (iv) simplified recombinant production; (v) high expression yields; (vi) increased half-life, (vii) no epithelial transcytosis, (viii) no secretion. SUMMARY OF THE INVENTION

[0006] In a first aspect, the invention relates to a polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C- terminus:

[0007] IGD1 - IGD2 - TD and wherein IGD1 is selected from IgM CH3 and an immunoglobulin constant domain comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide; IGD2 is an immunoglobulin constant domain; and TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers of the polypeptide compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and / or IGD2.

[0008] In a second aspect, the invention relates to a conjugate comprising a polypeptide according to the first aspect covalently linked to a diagnostic or therapeutic agent (F).

[0009] In a third aspect, the invention relates to a protein complex comprising or consisting of a first polypeptide according to the first aspect and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain.

[0010] In a fourth aspect, the invention relates to a dimer comprising or consisting of two identical or different polypeptides according to the first aspect, two identical or different conjugates according to the second aspect, a polypeptide according to the first aspect and a conjugate according to the second aspect, a polypeptide according to the first aspect and a protein complex according to the third aspect, a conjugate according to the second aspect and a protein complex according to the third aspect, or two identical or different protein complexes according to the third aspect.

[0011] In a fifth aspect, the invention relates to a multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect, in particular a dodecamer comprising or consisting of twelve identical or different polypeptides according to the first aspect, twelve identical or different conjugates according to the second aspect, twelve identical or different protein complexes according to the third aspect, or six identical or different dimers according to the fourth aspect.

[0012] In a sixth aspect, the invention relates to a composition comprising the multimer according to the fifth aspect.

[0013] In a seventh aspect, the invention relates to one or more polynucleotides encoding the polypeptide according to the first aspect, the polypeptides of the protein complex according to the third aspect, the polypeptides of the dimer according to the fourth aspect, or the polypeptides of the multimer according to the fifth aspect.

[0014] In an eighth aspect, the invention relates to one or more vectors comprising the one or more polynucleotides according to the seventh aspect.

[0015] In a ninth aspect, the invention relates to a cell comprising the one or more polynucleotides according to the seventh aspect or the one or more vectors according to the eighth aspect.

[0016] In a tenth aspect, the invention relates to a method for providing a composition according to the sixth aspect, wherein the method does not comprise a step of removing a multimer selected from a hexamer, octamer, decamer or dodecamer comprising polypeptides according to the first aspect.

[0017] In an eleventh aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound according to the first aspect, the conjugate according to the second aspect, the protein complex according to the third aspect, the dimer according to the fourth aspect, the multimer according to the fifth aspect, the composition according to the sixth aspect, the one or more polynucleotides according to the seventh aspect, the one or more vectors according to the eighth aspect, or the cell according to the ninth aspect.

[0018] DESCRIPTION OF THE INVENTION

[0019] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0020] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (TUPAC Recommendations)", H.G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0021] The practice of the present invention will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf, e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0022] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

[0023] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0024] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0025] Definitions

[0026] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of their use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0027] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e., the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps.

[0028] Whenever the specification defines a feature as “comprising or consisting of’, the embodiment “consisting of’ is preferred over the embodiment “comprising”.

[0029] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0030] The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

[0031] Terms such as “reducing” or “inhibiting” relate to the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level. The term “inhibit” or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero. Terms such as “increasing", “enhancing", “promoting” or “prolonging” preferably relate to an increase, enhancement, promotion or prolongation by about at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%, preferably at least 200% and in particular at least 300%. These terms may also relate to an increase, enhancement, promotion or prolongation from zero or a non-measurable or non-detectable level to a level of more than zero or a level which is measurable or detectable.

[0032] The term “extracellular portion” or “extracellular domain” in the context of the present invention preferably refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

[0033] The term “antigen binding protein” as used in the present invention refers to a polypeptide or a complex of two or more polypeptides that is able to specifically bind to an antigen. The term antigen binding protein includes antigen binding proteins of multiple different formats as described below, including soluble antigen binding proteins, membrane bound antigen binding proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins, single chain antigen binding proteins and antigen binding proteins comprising two or more chains, fusion proteins and chimeric proteins.

[0034] The term “antibody” can refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or refers to an antigen-binding portion thereof. The term “antibody” also includes single-chain antibodies, single-domain antibodies, all recombinant forms of antibodies, in particular of the antibodies described herein, e.g., antibodies expressed in prokaryotes or eukaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described below. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The terms “variable region” and “variable domain” as well as the terms “constant region” and “constant domain” are used interchangeably herein. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0035] The term “humanized antibody” refers to a molecule having an antigen-binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and / or sequence of a human immunoglobulin. The antigen-binding site may either comprise complete variable regions fused onto constant regions or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable regions. Antigen binding sites may be wild-type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

[0036] The terms “chimeric antigen binding protein” and “chimeric antibody” refer to those antigen binding proteins and antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antigen binding proteins or antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another. Typically the variable region of both light and heavy chains mimics the variable regions of antigen binding proteins or antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antigen binding proteins or antibodies derived from another. One clear advantage to such chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B-cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human subject when the antigen binding proteins or antibodies are injected than would the constant region from a non-human source. However, the definition is not limited to this particular example.

[0037] The term “antigen-binding portion” (or simply “binding portion") of an antigen binding protein, in particular an antibody, as used herein, refers to one or more fragments of an antigen binding protein, in particular an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antigen binding protein, in particular an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341 : 544-546), which consist of a VH domain; (vi) single domain antibodies, such as VHH domains or VNAR domains; (vii) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antigen binding protein, in particular an antibody, and thus by the term “antigen binding protein” as used in the claims. A further example are binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region.

[0038] The term “epitope” means a protein determinant capable of binding to an antigen binding protein, in particular an antibody, wherein the term “binding” herein preferably relates to a specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The term “epitope” preferably refers to an antigenic determinant in a molecule, i.e., to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term “epitope” includes B cell epitopes and T cell epitopes.

[0039] A “paratope” is the part of an antigen binding protein that binds to the epitope.

[0040] The term “bispecific molecule” is intended to include any agent, e.g., a monomeric or multimeric polypeptide or protein complex, which has two different binding specificities. For example, the molecule may bind to two different antigens, e.g. to two different cancer antigens, or to one cancer antigen and one effector cell. The term “multispecific molecule” or “heterospecific molecule” is intended to include any agent, which has more than two different binding specificities. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules comprising the polypeptide hexamerization domain of the first aspect.

[0041] The polypeptides described herein are preferably isolated. An “isolated polypeptide” as used herein, is intended to refer to a polypeptide which is substantially free of other polypeptides. Moreover, an isolated polypeptide may be substantially free of other cellular material and / or chemicals.

[0042] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0043] As used herein, “isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0044] As used herein, a first compound (e.g. an antibody or antigen-binding fragment thereof) is considered to “bind” to a second compound (e.g. a target protein), if it has a dissociation constant Ka to said second compound of 1 mM or less, preferably 100 pM or less, preferably 50 pM or less, preferably 30 pM or less, preferably 20 pM or less, preferably 10 pM or less, preferably 5 pM or less, more preferably 1 pM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, and even more preferably 10 nM or less.

[0045] Typically, antigen binding proteins and antigen-binding fragments thereof according to the invention bind with a sufficient binding affinity to their target, for example, with a Kd value of between 500 nM - 1 pM, i.e. 500 nM, 450 nM, 400nM, 350 nM, 300nM, 250 nM, 200 nM, 150 nM, lOOnM, 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM.

[0046] The term “binding” according to the invention preferably relates to a specific binding. “Specific binding” means that a compound (e.g., an antibody or antigen-binding fragment thereof) binds stronger to a target such as an epitope for which it is specific compared to the binding to another target. A compound binds stronger to a first target compared to a second target, if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant (Kd) for the target to which the compound binds specifically is more than 10-fold, preferably more than 20- fold, more preferably more than 50-fold, even more preferably more than 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (Kd) for the target to which the compound does not bind specifically.

[0047] As used herein, the term “Kd” (usually measured in “mol / L”, sometimes abbreviated as “M”) is intended to refer to the dissociation equilibrium constant of the particular interaction between a binding moiety (e.g. an antibody or antigen-binding fragment thereof) and a target molecule (e.g. an antigen or epitope thereof).

[0048] Methods for determining binding affinities of compounds, i.e. for determining the dissociation constant Kd, are known to a person of ordinary skill in the art and can be selected for instance from the following methods known in the art: Surface Plasmon Resonance (SPR) based technology, Bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Typically, the dissociation constant Kd is determined at 20°C, 25°C, 30°C, or 37°C. If not specifically indicated otherwise, the Kd values recited herein are determined by surface plasmon resonance spectroscopy (Biacore™) at room temperature (25°C).

[0049] As used herein the term “naturally occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. In describing a protein or peptide, structure and function herein, reference is made to amino acids. In the present specification, amino acid residues are expressed by using the following abbreviations. Also, unless explicitly otherwise indicated, the amino acid sequences of peptides and proteins are identified from N-terminal to C-terminal (left terminal to right terminal), the N-terminal being identified as a first residue. Amino acids are designated by their 3 -letter abbreviation, 1 -letter abbreviation, or full name, as follows. Ala : A : alanine; Asp : D : aspartic acid; Glu : E : glutamic acid ; Phe : F : phenylalanine; Gly : G : glycine; His : H : histidine; He : I : isoleucine; Lys : K : lysine; Leu : L : leucine; Met : M : methionine; Asn : N : asparagine; Pro : P: proline; Gin : Q : glutamine; Arg : R : arginine; Ser : S : serine; Thr : T : threonine; Vai : V : valine; Trp : W : tryptophan; Tyr : Y : tyrosine; Cys : C : cysteine.

[0050] The teaching given herein with respect to specific amino acid sequences, e.g. those shown in the sequence listing, is to be construed so as to also relate to variants of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino acid sequences exhibiting properties identical or similar to those of the specific amino acid sequences.

[0051] For the purposes of the present invention, “variants” of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants.

[0052] Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: rneedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0053] The terms “part", “fragment” and “portion” are used interchangeably herein and refer to a continuous or discontinuous fraction of a structure. With respect to a particular structure such as an amino acid sequence or protein or a nucleic acid sequence the terms “part", “fragment” and “portion” thereof may designate a continuous or a discontinuous fraction of said structure. Preferably, a “part", “fragment” and “portion” of a structure such as an amino acid sequence or a nucleic acid sequence preferably comprises, preferably consists of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% of the entire structure or amino acid sequence or nucleic acid sequence. A portion, a part or a fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, a part or a fragment of an epitope, peptide or protein is preferably immunologically equivalent to the epitope, peptide or protein it is derived from. If the portion, part or fragment is a discontinuous fraction said discontinuous fraction is preferably composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure, each part being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably not more than 4 parts of said amino acid sequence, wherein each part preferably comprises at least 5 continuous amino acids, at least 10 continuous amino acids, preferably at least 20 continuous amino acids, preferably at least 30 continuous amino acids of the amino acid sequence.

[0054] Embodiments

[0055] In the following different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0056] Polypeptide

[0057] In a first aspect, the invention relates to a polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C- terminus: IGD1 - IGD2 - TD. IGD1 is selected from IgM CH3 and an immunoglobulin constant domain comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide according to the first aspect. IGD2 is an immunoglobulin constant domain. TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers, in particular dodecamers of the polypeptide, compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and / or IGD2.

[0058] The expression “increases the formation of homogenous multimers” is meant to specify that the multimers that are formed are more homogenous, i.e. that the type of multimer which is the most frequent within all formed multimers, such as pentamers or hexamers, in particular hexamers, is more frequent compared to the entirety of different types of multimers which are formed, in comparison to the distribution of said multimers in a benchmark. For illustration, when a benchmark comprises 60% hexamers and 40% different multimers, a composition with increased formation of homogenous multimers can comprise, e.g., 70% hexamers and 30% different multimers or 80% hexamers and 20% different multimers.

[0059] The term “dodecamer” in the context of the present specification relates to a multimeric protein complex consisting of twelve subunits. A given multimeric protein complex can be described in more than one way, depending on what is considered as the subunit. Thus, IgM(6) can be described as a dodecamer, if a single IgM heavy chain or a complex of one IgM heavy chain and one IgM light chain (also referred to as “IgM halfmer”) is considered as subunit. Alternatively, IgM(6) can be described as a hexamer, if a complex of two IgM heavy chains and two IgM light chains (also referred to as “IgM monomer”) is considered as subunit. In the context of the present specification, multimers having a structure analogous to the structure of IgM(6) are referred to either as hexamers or dodecamers, depending on which subunit is considered in the context.

[0060] The term “decamer” in the context of the present specification relates to a multimeric protein complex consisting of ten subunits. IgM(5) can be described as a decamer, if a single IgM heavy chain or a complex of one IgM heavy chain and one IgM light chain (also referred to as “IgM halfmer”) is considered as subunit. Alternatively, IgM(5) can be described as a pentamer, if a complex of two IgM heavy chains and two IgM light chains (also referred to as “IgM monomer”) is considered as subunit. In the context of the present specification, multimers having a structure analogous to the structure of IgM(5) are referred to either as pentamers or decamers, depending on which subunit is considered in the context.

[0061] As used herein, the term “heterologous” is a relative term, which when used with reference to amino acid sequences or domains of a polypeptide indicates that the polypeptide comprises two or more portions / parts that are not found in the same relationship to each other in nature (e.g., a “fusion protein” where the two subsequences are encoded by a single nucleic acid sequence).

[0062] In the context of the present specification, the term “immunoglobulin domain” refers to a protein domain that exhibits a three-dimensional structure referred to as “immunoglobulin fold”: a 2-layer sandwich of 7-9 antiparallel P-strands arranged in two P-sheets with a Greek key topology. Immunoglobulin domains are a frequently used “building blocks” in naturally occurring proteins. Proteins containing immunoglobulin domains are subsumed into the immunoglobulin superfamily. Not only antibodies, but also cell adhesion molecules, T-cell receptors, Fcy-receptors and many more belong to this protein family. The immunoglobulin fold has been described thoroughly in a review article by Bork et al. ("The immunoglobulin fold. Structural classification, sequence patterns and common core ". September 1994; J. Mol. Biol. 242 (4): 309-20).

[0063] In the context of the present specification, the term “constant immunoglobulin domain” refers to a constant domain of an antibody, e.g. CHI, CH2 and CH3 of IgG, IgA or IgD, or CHI, CH2, CH3, and CH4 oflgM or IgE. Unlike a variable immunoglobulin domain, a constant immunoglobulin domain does not comprise hypervariable regions (complementarity determining regions).

[0064] Unless expressly stated otherwise, constant immunoglobulin domains of the present specification are in particular constant mammalian immunoglobulin domains, more particularly constant human immunoglobulin domains.

[0065] IGD1

[0066] IGD1 comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another polypeptide according to the first aspect. In particular, the two polypetides of the first aspect connected by this disulphide bond are each comprised in a separate dimer, wherein the two dimers are neighboring dimers in a multimeric, in particular a hexameric protein complex (see Fig. 1).

[0067] IGD1 can be IgM CH3. In such embodiments, the IgM CH3 sequence already comprises a Cys residue at position 291 of the IgM constant domain (according to SEQ ID NO: 23). In some embodiments, IGD1 comprises, essentially comprises or consists of an amino acid according to SEQ ID NO: 42 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 42, which comprises a Cys residue at position 291 of the IgM constant domain (according to SEQ ID NO: 23) and is capable of assembling into an immunoglobulin fold. In some embodiments, IGD1 comprises, essentially comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 42, which comprises a Cys residue at position 74 of SEQ ID NO: 42 and is capable of assembling into an immunoglobulin fold.

[0068] IGD1 can also be an immunoglobulin constant domain different from IgM. For example, IGD1 can be selected from IgG CH2, IgD CH2, IgE CH3, IgAl CH2 and IgA2 CH2. In such embodiments, the immunoglobulin constant domain comprises a substitution of an amino acid residue into a Cys residue at a suitable position. In some embodiments, IGD1 is IgG CH2 L328C, wherein the numbering of the amino acid position is according to EU numbering as set out by Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991), to which is referred throughout this document when EU numbering is mentioned). In particular, IGD1 can be IgGl CH2 L328C (part of SEQ ID NO: 16), IgG2 CH2 L328C, IgG3 CH2 L328C, or IgG4 CH2 L328C, wherein the numbering of the amino acid position is according to EU numbering. Position 328 according to EU numbering corresponds to position 98 according to the IMGT unique numbering for constant domains. In some embodiments, IGD1 comprises, essentially comprises or consists of an amino acid according to SEQ ID NO: 21 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 21, which comprises a Cys residue at position 328 of IgG according to EU numbering and is capable of assembling into an immunoglobulin fold.

[0069] IGD2

[0070] IGD2 is an immunoglobulin constant domain. In some embodiments, IGD2 is selected from IgM CH4, IgG CH3, IgAl CH3, IgA2 CH3, IgD CH3, and IgE CH4.

[0071] In some embodiments, IGD2 comprises, essentially comprises or consists of an amino acid according to SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 22, which is capable of assembling into an immunoglobulin fold.

[0072] In some embodiments, IGD2 comprises, essentially comprises or consists of an amino acid according to SEQ ID NO: 44 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 44, which is capable of assembling into an immunoglobulin fold.

[0073] TD

[0074] When recombinantly expressed in the absence of a J-chain, the polypeptide of the first aspect forms homogenous multimers. The homogenous formation of multimers in the absence of a J-chain is due to the TD of the polypeptides of the first aspect. In some embodiments, the formation of hexamers (dodecamers) is increased and the formation of pentamers (decamers) is decreased, compared to a human wild-type IgM tail piece. In other embodiments, the formation of pentamers is increased and the formation of multimers different from pentamers is decreased.

[0075] In the context of the present specification, the expression “IgM tail piece” or “IgA tail piece” refers to a polypeptide comprising, essentially comprising or consisting of the 19 most C -terminal amino acids of an IgM or IgA heavy chain. In some embodiments, the expression “IgM tail piece” or “IgA tail piece” refers to a polypeptide consisting of the 19 most C-terminal amino acids of an IgM or IgA heavy chain.

[0076] In some embodiments, TD is a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers compared to a human wildtype IgM tail piece. In particular, TD is a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of hexamers compared to a human wild-type IgM tail piece. The human wild-type IgM tail piece essentially comprises or consists of SEQ ID NO: 45.

[0077] In some embodiments, TD is a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and / or IGD2 comprised in the polypeptide of the first aspect. In other words, if TD is a tail piece sequence of a given non-mammalian species, e.g. trout, IGD1 and / or IGD2 are constant immunoglobulin domains of a different (mammalian or non-mammalian) species. Optionally, the non-mammalian tail piece sequence comprises a substitution that increases the formation of homogenous multimers, in particular decamers, of the polypeptide compared to the respective wild-type IgA or IgM tail piece sequence belonging to the same organism as the IGD1 and / or the IGD2 domain.

[0078] In some embodiments, TD comprises or consists of the sequence

[0079] KPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N; wherein if X4 is N, then Xi is A, X2 is V or H, X3 is L, V or I, X5 is V, X7 is Y and / or X9 is E.

[0080] In some embodiments, X4 is Q and Xi, X2, X3, X5, Xe, X7, Xs, X9, X10, Xu and X12 are defined as above.

[0081] In some embodiments, TD comprises or consists of the sequence

[0082] KPXiX2X3X4VSX5X6X7X8X9XioXiiXi2TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, but not N, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N.

[0083] In some embodiments, TD comprises or consists of the sequence KPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is Q, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N.

[0084] In some embodiments, TD comprises or consists of the sequence KPXiX2X3X4VSX5X6X7X8X9XioXiiXi2TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is G; wherein if X4 is N, then Xi is A, X2 is V or H, X3 is L, V or I, X5 is V, X7 is Y and / or X9 is E.

[0085] In some embodiments, TD comprises or consists of the sequence KPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is G; wherein if X4 is N, then Xi is A, X2 is V, X3 is L, V or I, X5 is V, X7 is Y and / or X9 is E.

[0086] In some embodiments, TD comprises or consists of the sequence KPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is G; wherein if X4 is N, then Xi is A, X2 is V or H, X3 is L, X5 is V, X7 is Y and / or X9 is E.

[0087] In some embodiments, TD comprises or consists of the sequence KPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4 is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is G; wherein if X4 is N, then Xi is V, X3 is L, X5 is V, X7 is Y and / or X9 is E.

[0088] In some embodiments, TD comprises or consists of the sequence KSTKPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 79) or KSTGKPX1X2X3X4VSX5X6X7X8X9X10X11X12TCY (SEQ ID NO: 80), wherein Xi to X12 are defined as for SEQ ID NO: 1. All embodiments recited for SEQ ID NO: 1 also apply to SEQ ID NO: 79 and SEQ ID NO: 80.

[0089] In some embodiments, the substitution in TD replaces an Asn residue with another amino acid, in particular a Gin. In some embodiments, the substitution in TD replaces an Asn residue at position 6 of SEQ ID NO: 1, at position 9 of SEQ ID NO: 79, or at position 10 of SEQ ID NO: 80 with another amino acid, in particular a Gin. In some embodiments, TD is an IgM tail piece sequence, and the substitution replaces an Asn residue located at position 440 of the IgM constant domain (according to SEQ ID NO: 23) with another amino acid, in particular a Gin. In some embodiments, TD is an IgAl tail piece sequence, and the substitution replaces an Asn residue located at position 340 of the IgAl constant domain (according to SEQ ID NO: 24) or at position 327 of the IgA2 constant domain (according to SEQ ID NO: 25), with another amino acid, in particular a Gin. The numbering of the amino acid positions of IgM, IgAl and IgA2 is according to the numbering of SEQ ID NOs: 23, 24, and 25. Without wishing to be bound by theory, the inventors assume that the removal of a glycosylation site from the TP via the substitution of a Cys residue, in particular the Cys residue at the described position, enhances the formation of hexamers compared to dimers.

[0090] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is S or L, X2 is Y or V, X3 is not N, X4 is V or I, X5 is M or L, Xe is A or G, X7 is S, A, G or N.

[0091] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is S or L, X2 is Y, X3 is Q, X4 is V, X5 is L, Xe is A, X7 is G.

[0092] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is L, X2 is Y or V, X3 is Q, X4 is V, X5 is L, Xe is A, X7 is G.

[0093] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is L, X2 is Y, X3 is not N, X4 is V, X5 is L, Xe is A, X7 is G.

[0094] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi L, X2 Y, X3 is Q, X4 is V or I, X5 is L, Xe is A, X7 is G.

[0095] In some embodiments, TD comprises or consists of the sequence KPTX1X2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is L, X2 is Y, X3 is Q, X4 is V, X5 is M or L, Xe is A, X7is G.

[0096] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is L, X2 is Y, X3 is Q, X4 is V, X5 is L, Xe is A or G, X7is G.

[0097] In some embodiments, TD comprises or consists of the sequence KPTXIX2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is L, X2 is Y, X3 is Q, X4 is V, X5 is L, Xe is A, X7is S, A, G or N.

[0098] In some embodiments, TD has a sequence selected from

[0099] (a) KPTLYQVSLVMSDTAGTCY (human IgM N440Q) (SEQ ID NO: 3),

[0100] (b) KPTHVQVSVVMAEVDGTCY (human IgAl N340Q) (SEQ ID NO: 4),

[0101] (c) KPTHIQVSVVMAEADGTCY (human IgA2 N327Q) (SEQ ID NO: 5),

[0102] (d) KPAVLQVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, N440Q, L443V, M445Y, D447E) (SEQ ID NO: 6),

[0103] (e) KPAVLNVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, L443V, M445Y, D447E) (SEQ ID NO: 7),

[0104] (f) NQPNLVNLSLNVPQRCMAQ (trout IgM tail piece) (SEQ ID NO: 8),

[0105] (g) PSFVNISLALMDTINSCQ (nurse shark IgM tail piece) (SEQ ID NO: 9),

[0106] (h) NRJDLNMNINQDSKCSA (Nile tilapia IgM tail piece) (SEQ ID NO: 10), and

[0107] (i) KPTNVNVSLVLSDTC (Xenopus laevis IgM tail piece) (SEQ ID NO: 11).

[0108] In some embodiments, TD comprises the amino acid residues KST or KSTG (SEQ ID NO: 81) N-terminal of SEQ ID NO: 1 to 7.

[0109] In some embodiments, TD is a human IgM, IgAl or IgA2 tail piece, in which an Asn residue at position 440 of the IgM constant domain (according to SEQ ID NO: 23), position 340 of the IgAl constant domain (according to SEQ ID NO: 24) or position 327 of the IgA2 constant domain (according to SEQ ID NO: 25), is substituted with another amino acid, in particular a Gin, wherein the numbering of the amino acid positions is according to the numbering of SEQ ID NOs: 23, 24, and 25.

[0110] In some embodiments, TD is a non-mammalian IgM tail piece, in particular selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. Optionally, the nonmammalian tail piece comprises a substitution that increases the formation of homogenous multimers of the polypeptide compared to the respective wild-type non-mammalian IgM tail piece, e.g. a substitution of an Asn residue with another amino acid, in particular a Gin.

[0111] Dimerization domain (D) In some embodiments, the polypeptide of the first aspect further comprises a dimerization domain (D), wherein the IGD1, IGD2, D and TD have the following structure from N-terminus to C-terminus: D - IGD1 - IGD2 - TD. In some embodiments, D is selected from an immunoglobulin constant domain, an immunoglobulin hinge region and a peptide linker.

[0112] In some embodiments, D comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of a D domain of another identical or different polypeptide according to the first aspect comprising a D domain (see Fig. 1 C).

[0113] In some embodiments, D is a third immunoglobulin domain (IGD3). In some embodiments, D is an IgM or IgE CH2 domain, in particular an IgM CH2 domain. In some embodiments, D is an IgM CH2 domain comprising, essentially comprising or consisting of the amino acid sequence according to SEQ ID NO: 41 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 41, which comprises a Cys residue at position 109 of SEQ ID NO: 41 and is capable of assembling into an immunoglobulin fold. In some embodiments, D is an IgG, IgA, or IgD hinge region, in particular an IgG hinge region. In some embodiments, D is a hinge region comprising, essentially comprising or consisting of the amino acid sequence according to any one of SEQ ID NOs: 82 to 85 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 82 to 85, which comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of another hinge region, in particular an identical hinge region.

[0114] IGD4

[0115] In some embodiments, the polypeptide of the first aspect further comprises a fourth immunoglobulin domain (IGD4), wherein the IGD1, IGD2, TD, D and IGD4 have the following structure from N-terminus to C-terminus: IGD4 - D - IGD1 - IGD2 - TD.

[0116] In some embodiments, IGD4 is a CHI or CL domain, in particular an IgM CHI domain or an IgG CHI domain.

[0117] In some embodiments, IGD1 is IgM CH3, IGD2 is IgM CH4, IGD3 (if present) is IgM CH2 and IGD4 (if present) is IgM CHI.

[0118] In some embodiments, IGD1 is IgG CH2 L328C, IGD2 is IgG CH3, IGD3 (if present) is a IgG hinge region and IGD4 (if present) is IgG CHI.

[0119] In some embodiments, the polypeptide comprises, essentially comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 86, which comprises a Cys residue at position 291 of the IgM constant domain (according to SEQ ID NO:23), and C-terminal of this sequence the amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 11, SEQ ID NO: 79 or SEQ ID NO: 80. In some embodiments, the polypeptide comprises, essentially comprises or consists of the amino acid sequence according to SEQ ID NO: 46 to SEQ ID NO: 52 or SEQ ID NO: 54 to SEQ ID NO: 56.

[0120] In some embodiments, the polypeptide comprises, essentially comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 89, which comprises a Cys residue at position 328 of IgG according to EU numbering, and C-terminal of this sequence the amino acid sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 11, SEQ ID NO: 79 or SEQ ID NO: 80. In some embodiments, the polypeptide comprises, essentially comprises or consists of the amino acid sequence according to SEQ ID NO: 58 to SEQ ID NO: 67.

[0121] In some embodiments, one or more of D, IGD1 and IGD2 comprise heterodimerization mutations, in particular knob mutations or hole mutations of knob-into-hole mutation pairs. The knob-into-hole technology refers to mutations in the interface of a CH3 dimer to create a “knob” on one CH3 domain and a “hole” on the other CH3 domain to promote heterodimerization. By way of non-limiting example, particular knob-into-hole mutations are T366S, T366Y, T366W, L368A, Y407V, and Y407T. Those knob-into-hole mutations can be further stabilized by the introduction of additional cysteine amino acid substitutions Y349C and S354C. Thus, in some embodiments, IGD2 is an IgG CH3 domain with knob mutations or hole mutations of knob- into-hole mutation pairs. The skilled person is aware of how to introduce heterodimerization mutations, in particular knob-into-hole mutations, into one or more of D, IGD1 and IGD2 to promote heterodimerization over homodimerization of two polypeptides of the first aspect.

[0122] In some embodiments, the polypeptide according to the first aspect further comprises an effector domain (E), which comprises a diagnostic or therapeutic polypeptide or part thereof.

[0123] In some embodiments, E is a diagnostic polypeptide selected from a fluorescent protein, a protein comprising a radioactive label, and a protein comprising a spin label.

[0124] In some embodiments, E is a therapeutic polypeptide selected from a cell cycle inhibiting protein, a proinflammatory protein, an anti-inflammatory protein and a protein which modifies the metabolism

[0125] In preferred embodiments, E is or comprises one or more antigen binding sites, preferably one or more immunoglobulin variable domains.

[0126] In some embodiments, E is or comprises a heavy chain variable domain according to SEQ ID NO: 19. In some embodiments, E is or comprises a single domain antibody, a VHH, a VNAR, or a single chain antibody.

[0127] Conjugate

[0128] In a second aspect, the invention relates to a conjugate comprising a polypeptide according to the first aspect covalently linked to a diagnostic or therapeutic agent (F).

[0129] In some embodiments, F is a nucleic acid. In some embodiments, F is a polypeptide. In some embodiments, F is non-polypeptide non-nucleic acid compound with a molecular weight of less than 1,5 kD.

[0130] In some embodiments, F is a diagnostic agent, in particular selected from a fluorescent dye, a fluorescence emitting isotope, a radioisotope, a spin-labeled agent and a contrast agent.

[0131] In some embodiments, F is a therapeutic agent. In some embodiments, F is a cytokine. In some embodiments, F is a toxin.

[0132] Protein complex

[0133] In a third aspect, the invention relates to a protein complex comprising or consisting of a first polypeptide according to the first aspect and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain.

[0134] In some embodiments, the first polypeptide has the structure: Va- IGD4 - D - IGD1 - IGD2 - TD, and the second polypeptide has the structure: Vb - C, wherein Vaand Vb, are immunoglobulin variable domains that together form an antigen binding site, IGD4 and C are immunoglobulin constant domains capable of heterodimerization, and D, IGD1, IGD2 and TD are as defined in the first aspect. In one embodiment, one of Vaor Vb is an immunoglobulin variable heavy domain (VH) and one of Vaor Vb is an immunoglobulin variable light domain (VL). In some embodiments, Vaand Vb are the variable domains of Adalimumab as comprised in the Adalimumab heavy and light chain sequences as comprised in SEQ ID NO: 17 and 18. Preferably, one of IGD4 and C is a CHI domain and one of IGD4 and C is a CL domain.

[0135] In some embodiments, the first polypeptide comprises or consists of a sequence according to SEQ ID NO: 30 to SEQ ID NO: 36 or SEQ ID NO: 38 to SEQ ID NO: 40, and the second polypeptide comprises or consists of a sequence according to SEQ ID NO: 17.

[0136] In some embodiments, first polypeptide comprises or consists of a sequence according to SEQ ID NO: 58 to SEQ ID NO: 67, and the second polypeptide comprises or consists of a sequence according to SEQ ID NO: 17. Dimer

[0137] In a fourth aspect, the invention relates to a dimer comprising or consisting of two identical or different polypeptides according to the first aspect, two identical or different conjugates according to the second aspect, a polypeptide according to the first aspect and a conjugate according to the second aspect, a polypeptide according to the first aspect and a protein complex according to the third aspect, a conjugate according to the second aspect and a protein complex according to the third aspect, or two identical or different protein complexes according to the third aspect.

[0138] In embodiments where the dimer of the fourth aspect comprises two different polypeptides the first aspect, two different conjugates of the second aspect or two different protein complexes of the third aspect, heterodimerization is preferably achieved by introducing heterodimerization mutations into one or more of D, IGD1 and IGD2 as described above (e.g. knob-into-hole mutations).

[0139] In addition, in embodiments where the dimer of the fourth aspect comprises two different protein complexes of the third aspect, in each protein complex, the first polypeptide (Va- IGD4 - D - IGD1 - IGD2 - TD) is preferably paired with a different second polypeptide (Vb - C). This differential pairing can e.g. be achieved by introducing modifications into the dimerizing Vaand Vb and / or IGD4 and C domains, as described in in Brinkmann and Kontermann (MAbs. (2017) 9(2): 182-212), such as CrossMab, orthogonal Fab, CR3, MUT4 and DuetMab. Further possibilities to overcome the light chain pairing problem are described in WO 2018 / 037092 Al and WO 2021 / 198204 Al.

[0140] Dodecamer

[0141] In a fifth aspect, the invention relates to a multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect. In one embodiment, the multimer is a dodecamer comprising or consisting of twelve identical or different polypeptides according to the first aspect, twelve identical or different conjugates according to the second aspect, twelve identical or different protein complexes according to the third aspect, or six identical or different dimers according to the fourth aspect. In another embodiment, the multimer is a decamer comprising or consisting of ten identical or different polypeptides according to the first aspect, ten identical or different conjugates according to the second aspect, ten identical or different protein complexes according to the third aspect, or five identical or different dimers according to the fourth aspect.

[0142] Exemplary formats of the multimer of the fifth aspect are shown in Figure 1. The multimer may consist of constants domain only (Fig IB, 1C). Exemplary dimerization domains (D) are an immunoglobulin domain or an immunoglobulin hinge region (Fig. 1C, left and right). The multimers can comprise Fab fragments at the N-terminus of each polypeptide (Fig. ID). Those Fab fragments can be the same throughout the multimer (Fig. ID, left) or different (Fig. ID, right), e,g. by using knob-into-hole mutations for heterodimerization. The multimers can comprise a single binding antibody domain, e.g. a VHH, at the N-terminus of each polypeptide (Fig. IE). Those single binding antibody domains (e.g. VHH) can be the same throughout the multimer (Fig. IE, left) or different (Fig. IE, right), e,g. by using knob-into-hole mutations for heterodimerization. . The multimers can comprise a scFv domain at the N-terminus of each polypeptide (Fig. IF). Those scFv can be the same throughout the multimer (Fig. IF, left) or different (Fig. IF, right), e,g. by using knob-into-hole mutations for heterodimerization. The multimers can comprise a dual targeting domain at the N-terminus of each polypeptide, e.g. a DVD domain (Fig. 1G) or CODV domain (Fig. 1 H). The multimer can comprise combinations of the above-mentioned elements (Fab fragment, single binding antibody domain, scFv, DVD domain, CODV domain), wherein half of the polypeptides of the multimer comprise at the N- terminus one element while the other half comprise the other element, e.g. by using knob-into- hole mutations for heterodimerization. Exemplary combinations are a multimer with a single binding domain and a Fab-domain (Fig 1 I) and a multimer with a CODV domain and a Fab- fragment (Fig 1 J). The multimer can comprise a protein or peptide sequence at their N-terminus of each polypeptide (Fig. 1 K.). The multimers described above can be conjugated to a chemical compound, such as a diagnostic or therapeutic agent (F) as described above. (Fig 1 L):

[0143] The multimer of the fifth aspect does not comprise a J-chain polypeptide.

[0144] Composition

[0145] In a sixth aspect, the invention relates to a composition comprising the multimer according to the fifth aspect.

[0146] In some embodiments, the multimer is a dodecamer. In some embodiments, the multimer is a dodecamer and the composition comprises less than 50%, less than 40%, less than 30%, less than 20%, 10%, less than 5%, less than 2% of a decamer comprising ten polypeptides according to the fifth aspect. In some embodiments, the multimer is a decamer. In some embodiments, the multimer is a decamer and the composition comprises less than 50%, less than 40%, less than 30%, less than 20%, 10%, less than 5%, less than 2% of a multimer selected from a hexamer, octamer, or dodecamer comprising polypeptides according to the fifth aspect.

[0147] Nucleic Acids and vectors

[0148] In a seventh aspect, the invention relates to one or more polynucleotides encoding the polypeptide according to the first aspect, the polypeptides of the protein complex according to the third aspect, the polypeptides of the dimer according to the fourth aspect, or the polypeptides of the multimer according to the fifth aspect.

[0149] The term “nucleic acid” or “nucleic acid molecule", as used herein, is intended to include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Nucleic acids include according to the invention genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the invention, a nucleic acid may be present as a single- stranded or double-stranded and linear or covalently circularly closed molecule.

[0150] The nucleic acids described according to the invention may be isolated. The term “isolated nucleic acid” means according to the invention that the nucleic acid was (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel-electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid which is available for manipulation by recombinant DNA techniques.

[0151] Nucleic acids may, according to the invention, be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In some embodiments, a nucleic acid is functionally linked to expression control sequences which may be homologous or heterologous with respect to said nucleic acid. The term “homologous” means that a nucleic acid is also functionally linked to the expression control sequence naturally and the term “heterologous” means that a nucleic acid is not functionally linked to the expression control sequence naturally.

[0152] A nucleic acid, such as a nucleic acid expressing RNA and / or protein or peptide, and an expression control sequence are “functionally” linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said nucleic acid is under the control or under the influence of said expression control sequence. If the nucleic acid is to be translated into a functional protein, then, with an expression control sequence functionally linked to a coding sequence, induction of said expression control sequence results in transcription of said nucleic acid, without causing a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide.

[0153] The term “expression control sequence” comprises according to the invention promoters, ribosome binding sites, enhancers and other control elements which regulate transcription of a gene or translation of a mRNA. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of expression control sequences may vary as a function of the species or cell type, but generally comprises 5'- untranscribed and 5'- and 3 '-untranslated sequences (5'-UTR; 3'-UTR) which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5'-untranscribed expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the functionally linked nucleic acid. Expression control sequences may also comprise enhancer sequences or upstream activator sequences.

[0154] According to the invention the term “promoter” or “promoter region” relates to a nucleic acid sequence which is located upstream (5') to the nucleic acid sequence being expressed and controls expression of the sequence by providing a recognition and binding site for RNA- polymerase. The “promoter region” may include further recognition and binding sites for further factors which are involved in the regulation of transcription of a gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. Furthermore, a promoter may be “inducible” and may initiate transcription in response to an inducing agent or may be “constitutive” if transcription is not controlled by an inducing agent. A gene which is under the control of an inducible promoter is not expressed or only expressed to a small extent if an inducing agent is absent. In the presence of the inducing agent the gene is switched on or the level of transcription is increased. This is mediated, in general, by binding of a specific transcription factor.

[0155] Suitable promoters include promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMV promoter, and artificial hybrid promoters thereof (e.g., CMV) where a part or parts are fused to a part or parts of promoters of genes of other cellular proteins such as e.g., human GAPDH (glyceraldehyde-3 -phosphate dehydrogenase), and including or not including (an) additional intron(s).

[0156] According to the invention, the term “expression” is used in its most general meaning and comprises the production of RNA or of RNA and protein / peptide. It also comprises partial expression of nucleic acids. Furthermore, expression may be carried out transiently or stably. In an eighth aspect, the invention relates to or more vectors comprising the one or more polynucleotides according to the seventh aspect.

[0157] The term “vector” is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and / or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and / or expressed in the cells. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes, but also liposomes. The term “plasmid” as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0158] Cells

[0159] In a ninth aspect, the invention relates to a cell comprising the one or more polynucleotides according to the seventh aspect or the one more vectors according to the eighth aspect.

[0160] Method for providing a composition

[0161] In a tenth aspect, the invention relates to a method for providing a composition according to the sixth aspect, wherein the method does not comprise a step of removing a multimer selected from a hexamer, octamer, decamer, or dodecamer comprising polypeptides according to the first aspect.

[0162] In some embodiments, the composition according to the sixth aspect essentially comprises dodecamers comprising twelve polypeptides according to the first aspect, and the method does not comprise a step of removing a decamer comprising ten polypeptides according to the first aspect.

[0163] Pharmaceutical composition

[0164] In an eleventh aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound according to the first aspect, the conjugate according to the second aspect, the protein complex according to the third aspect, the dimer according to the fourth aspect, the multimer according to the fifth aspect, the composition according to the sixth aspect, the one or more polynucleotides according to the seventh aspect, the one or more vectors according to the eighth aspect, or the cell according to the tenth aspect. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the pharmaceutical composition is formulated for cardiovascular, in particular intravenous or intraarterial administration.

[0165] The pharmaceutical compositions may be formulated with pharmaceutically acceptable diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment, the compositions include a combination of multiple (e.g., two or more) isolated antigen binding proteins. In another embodiment, the compositions include a combination of multiple (e.g., two or more) nucleic acids, vectors or host cells.

[0166] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable diluent” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for cardiovascular (e.g., intravenous or intraarterial), intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antigen binding protein, bispecific and multispecific molecule, nucleic acids, vectors, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0167] A “pharmaceutically acceptable substance” refers to a substance that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).

[0168] The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), saline and aqueous buffer solutions, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0169] The carrier or the composition of the present invention can also comprise pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts that may be comprised include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0170] The composition of the present invention may also comprise antioxidants. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0171] The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, for example, monostearate salts and gelatin.

[0172] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0173] Pharmaceutically compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0174] Sequences

[0175] Table 1.

[0176]

[0177] SHORT DESCRIPTION OF THE FIGURES

[0178] Figure 1: Structure of exemplary multimers comprising the polypeptide of the invention. A) Domains as represented in Figure 1. B) Hexamer comprising TD, IGD1 and IGD2. C) Hexamer further comprising a dimerization domain D (left: D = IGD3; right: D = hinge region). D) “Fab” hexamer (left: symmetric; right: asymmetric; top row: D = IGD3; bottom row: D = hinge region). E) “Single domain” hexamer (left: symmetric; right: asymmetric; top row: D = IGD3; bottom row: D = hinge region). F) “Single chain” hexamer (left: symmetric; right: asymmetric, D = IGD3). G) “DVD” hexamer, D = IGD3. H) “COVD” hexamer, D = IGD3. I) “single domain / Fab” mixed hexamer (top: D = IGD3; bottom: D = hinge region). J) “CODV / Fab” mixed hexamer, D = IGD3. K) “Fusion peptide” hexamer. L) “Conjugate” hexamer.

[0179] Figure 2: SDS-PAGE of culture supernatant at time of harvest of different monoclonal antibodies (mabs) as separated by a 4-12 % SDS-PAGE under reducing conditions. Ml : Bench mark protein ladder. The position of the bands corresponding to the light chains (LC) and heavy chains (HC) are indicated. The relative expression levels are indicated for each protein variant. The expression level of mabOOl (variant with wild-type IgM tailpiece) was set as 100%. For expression level evaluation the staining intensity of the light chains were measured using the software Lab Image ID L340.

[0180] Figure 3: SDS-PAGE of different mabs as separated by a 3-12 % native SDS- PAGE. Protein samples mabOOl - mab008 were purified and analyzed under non-reducing conditions in a 3-12% native PAGE and stained with Coomassie-blue. Ml : Bench mark protein ladder; M2: Native Mark. The protein bands of mabOOl corresponding to pentamer and hexamer are exemplary indicated.

[0181] Figure 4: Densitometric analysis of the main protein band area of the SDS-PAGE in Fig. 3. The first band represents the hexamer, the second signal represents the pentamer as indicated in A). Additional bands representing other multimers are not further specified. For estimation of the percentage of the main protein bands, the intensity of the stained protein bands was used. The considered area of the corresponding bands is colored in grey and set as 100%. The percentage of each protein band is given in the corresponding figures. Image analysis was done using the software Lab Image ID L340. The panel depict the following mabs: A) mabOOl (SEQ ID NO: 17, SEQ ID NO: 37), B) mab002 (SEQ ID NO: 17, SEQ ID NO: 36), C) mab003 (SEQ ID NO: 17, SEQ ID NO: 30), D) mab004 (SEQ ID NO: 17, SEQ ID NO: 31), E) mab005 (SEQ ID NO: 17, SEQ ID NO: 32), F) mab006 (SEQ ID NO: 17, SEQ ID NO: 33), G) mab007 (SEQ ID NO: 17, SEQ ID NO: 34), and H) mab 008 (SEQ ID NO: 17, SEQ ID NO: 35). EXAMPLES

[0182] Example 1: Protein expression and purification

[0183] Monoclonal antibodies (mabs) 001-008 were produced, each mab consisted of a heavy chain and a light chain. The sequences of the chain for each mab are indicated in table 2. The mabs were expressed in transiently transfected HEK293 cells. DNA coding for the different protein chains were cloned into an expression vector under a CMV promotor and a leader sequence directing the proteins into the culture supernatant. Sequence SEQ ID NO: 13 was used as leader sequence. In all transfections plasmids were used at an equimolar ratio. For expression, cells were grown in non-baffled shake flasks (Corning) at 110 rpm, 37°C, and 8%CO2 in FreeStyle F17 medium (Gibco supplemented with 6 mM glutamine and 0.02% Pluronic F-68 (Gibco). At the time of transfection, cell densities were approximately 1.2 x 10A6 cells / mL as determined with an automated cell counter (Nucleocounter NC-200). Before transfection, plasmids were mixed with linear polyethyleneimine (PEI) at a ratio of 1 :3 in OptiMem I medium (Thermo Fisher scientific) or Freestyle F17 medium (Gibco). The transfection mixtures were incubated 10 min at room temperature and then added to the corresponding cell cultures. Cultivation was continued for 6 days. After expression, cell cultures were centrifuged, cell pellets were discarded and the obtained culture supernatants were sterile filtered using a 0.22 pm filter. For relative expression analysis, an aliquot of each culture supernatant was analyzed using an SDS-PAGE (4-12 % NuPAGE BisTris gel with MES-running buffer, Invitrogen) under reducing conditions. Samples were mixed with sample buffer (LDS-sample buffer (4x), Invitrogen) containing 0.1 mM DTT and incubated at 95°C for 5 minutes before loading on the SDS-PAGE. Proteins were captured from the cleared supernatant using CaptureSelect IgM (Thermo Fisher Scientific) equilibrated in PBS (Gibco). Proteins were washed on the capture affinity matrix with 0.1 M citrate, pH 6.0 and eluted with 0.1 M citrate, pH 3.0. Proteins were desalted using a HiPrep desalting column (Cytivia) equilibrated in PBS (Gibco, #14190) with added 150 mM NaCl. Final purification was done using a size exclusion HiPrep Sephacryl S300 HR (Cytivia) column equilibrated in the same buffer. Protein fractions were checked by SDS-PAGE and fractions containing the desired proteins were pooled, concentrated (VivaSpin 20, 5 kDa cut off, Satorius) and stored at -80°C until further usage. For SDS-PAGE the following markes were used: Ml : Bench mark protein ladder (Invitrogen), M2: NativeMark (Thermo Fisher Scientific). For densitrometric analysis of the SDS.PAGE the software Lab Image ID L340 (Kapelan Bioimaging, Leipzig, Germany) was used. Table 2. mabOOl-mabOO8: The constructs comprised Adalimumab Fab, fused to IgM CH2-CH4 and differed in TD sequences.

[0184] Results

[0185] All antibody variants with different multimerization domains could be well expressed after transient transfection in in HEK293 cells. For expression level estimation, samples of culture supernatants were separated on a 4-12% SDS-PAGE under reducing conditions. Expression level of the mab-variant with unmodified IgM tailpiece (mabOOl) was set as 100 %. A densitrometrical analysis of each stained protein band of the light chain of each variant was used for that reason. Expression levels were at least comparable to the mabOOl . For most mab variants with different multimerization domain the expression level were highly improved as compared to mabOOl (Fig. 2.). Furthermore, all mab variants could be purified using the above two step purification procedure in a comparable manner.

[0186] Example 2: SDS-PAGE analysis

[0187] To assess the extent of formation of hexamers and pentamers, purified protein samples were analyzed by native PAGE analysis. Protein samples were mixed with 4 x LDS sample buffer (Thermo Fisher Scientific). Protein separation was done using a NativePAGE 3-12 % BisTris gel with MES running buffer (Invitrogen) for 80 min at constant voltage of 180 V. Used markers are Ml : BenchMark protein ladder and M2: NativeMark (Thermo Fisher Scientific). After running, gels were stained with Coomassie-blue (Instant Blue, Expedeon). Gels were scanned using a Mikrotek Bio-5000 plus scanner and gel analysis was done with the software Lab Image ID L340 (Kapelan Bio-Imaging).

[0188] Results

[0189] If IgM antibody heavy chains are expressed without the J-chain, an additional IgM monomer (consisting of two heavy chains and two light chains) can be incorporated into the pentameric assembly of an IgM molecule thus forming a hexamer. Usually this results in a mixture of pentamers and hexamers and possibly more multimers. Native PAGE was used to evaluate the amount of hexamers versus pentamers in a particular mab preparation. A corresponding gel is shown in Fig 3. For the mab with the wt IgM tailpiece (mabOOl) two bands corresponding to a pentamer and a hexamer are clearly visible (Fig. 3), as expected. For the other mab variants (mab002-mab008) with different dimerization domain, the ratio of pentamers vs. hexamer is strikingly shifted towards the pentameric or hexameric form, thus resulting in a much more homogenous protein preparation. For example, for mab007 and mab008 one main protein band is visible. To further asses the ratio between pentamer and hexamer formation the gel was analysed using a densitrometrical approach. The area of interest of the corresponding samples was analyzed accordingly (Fig. 4). The staining intensities of the main protein bands was set as 100% (shown as grey area under the intensity curve). The percentage of each main band was estimated using the software Labimage and the corresponding percentages are given in the figure. For mabOOl, a variant with unmodified dimerization domain, the hexameric form accounts for only 28% of the mab preparation. The pentameric form is the dominant form, comprising 58% of the mab preparation. In sharp contrast to this, the mab variants mab003- mab008 are much more homogenous, consisting of only one major isoform, which might represent a pentamer or a hexamer. For example, in mab 008 the main component of the protein preparation is the hexameric isoform, comprising 97% of the mab preparation.

[0190] ITEMS

[0191] 1. A polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C-terminus:

[0192] IGD1 - IGD2 - TD and wherein

[0193] (i) IGD1 is selected from IgM CH3 and an immunoglobulin constant domain, in particular IgG CH2, IgD CH2, IgE CH3, IgAl CH2 or IgA2 CH2, comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide according to item 1, in particular a polypeptide comprised in a multimer according to item 23 in an adjacent dimer;

[0194] (ii) IGD2 is an immunoglobulin constant domain, in particular selected from IgM CH4, IgG CH3, IgAl CH3, IgA2 CH3, IgD CH3, and IgE CH4; and

[0195] (iii) TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and / or IGD2.

[0196] 2. The polypeptide according to item 1, wherein the more homogenous multimers are dodecamers of the polypeptide.

[0197] 3. The polypeptide according to item 1 or 2, wherein IGD1 is selected from IgM CH3 and IgG CH2 L328C, wherein the numbering of the amino acid position is according to EU numbering.

[0198] 4. The polypeptide according to any one of items 1 to 3, wherein TD comprises or consists of the sequence

[0199] KPXIX2X3X4VSX5X6X7X8X9X10X11 X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N; wherein if X4is N, then Xi is A, X2 is V or H, X3 is L, V or I, X5 is V, X7 is Y and / or X9 is E.

[0200] 5. The polypeptide according to any one of items 1 to 4, wherein the substitution in TD replaces an Asn residue, in particular an Asn residue at position 6 of SEQ ID NO: 1, at position 440 of the IgM constant domain (according to SEQ ID NO:23), position 340 of the IgAl constant domain (according to SEQ ID NO:24) or position 327 of the IgA2 constant domain (according to SEQ ID NO:25), with another amino acid.

[0201] 6. The polypeptide according to any one of items 1 to 5, wherein TD comprises or consists of the sequence

[0202] KPTX1X2X3VSLX4X5SDTX6X7TCY (SEQ ID NO: 2), wherein Xi is S or L, preferably L, X2 is Y or V, preferably Y, X3 is not N, more preferably X3 is Q, X4 is V or I, preferably V, X5 is M or L, preferably L, Xe is A or G, preferably A, X7 is S, A, G or N, preferably G.

[0203] 7. The polypeptide according to any one of items 1 to 6, wherein TD has a sequence selected from

[0204] (a) KPTLYQVSLVMSDTAGTCY (human IgM N440Q) (SEQ ID NO: 3),

[0205] (b) KPTHVQVSVVMAEVDGTCY (human IgAl N340Q) (SEQ ID NO: 4),

[0206] (c) KPTHIQVSVVMAEADGTCY (human IgA2 N327Q) (SEQ ID NO: 5),

[0207] (d) KPAVLQVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, N440Q, L443V, M445Y, D447E) (SEQ ID NO: 6),

[0208] (e) KPAVLNVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, L443V, M445Y, D447E) (SEQ ID NO: 7),

[0209] (f) NQPNLVNLSLNVPQRCMAQ (trout IgM tail piece) (SEQ ID NO: 8),

[0210] (g) PSFVNISLALMDTINSCQ (nurse shark IgM tail piece) (SEQ ID NO: 9),

[0211] (h) NRJDLNMNINQDSKCSA (Nile tilapia IgM tail piece) (SEQ ID NO: 10), and

[0212] (i) KPTNVNVSLVLSDTC (Xenopus laevis IgM tail piece) (SEQ ID NO: 11).

[0213] 8. The polypeptide according to any one of items 1 to 7, further comprising a dimerization domain (D), wherein the IGD1, IGD2, D and TD have the following structure from N- terminus to C-terminus:

[0214] D - IGD1 - IGD2 - TD and D is selected from an immunoglobulin constant domain, an immunoglobulin hinge region and a peptide linker. 9. The polypeptide according to item 8, wherein D comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of a D domain of another identical or different polypeptide according to item 8.

[0215] 10. The polypeptide according to item 9, wherein D is a third immunoglobulin domain (IGD3).

[0216] 11. The polypeptide according to any one of items 8 to 10, further comprising a fourth immunoglobulin domain (IGD4), wherein the IGD1, IGD2, TD, D and IGD4 have the following structure from N-terminus to C-terminus:

[0217] IGD4 - D - IGD1 - IGD2 - TD.

[0218] 12. The polypeptide according to item 11, wherein IGD4 is a CHI or CL domain.

[0219] 13. The polypeptide according to any one of items 1 to 12, wherein one or more of D, IGD1 and IGD2 comprise heterodimerization mutations, in particular knob mutations or hole mutations of knob-into-hole mutation pairs.

[0220] 14. The polypeptide according to any one of items 1 to 13, further comprising an effector domain (E), which comprises a diagnostic or therapeutic polypeptide or part thereof.

[0221] 15. The polypeptide according to item 14, wherein E comprises one or more antigen binding sites.

[0222] 16. The polypeptide according to any one of items 14 to 15, wherein E comprises a single domain antibody or a single chain antibody.

[0223] 17. A conjugate comprising a polypeptide according to any one of items 1 to 16 covalently linked to a diagnostic or therapeutic agent (F).

[0224] 18. A protein complex comprising or consisting of a first polypeptide according to any one of items 11 to 17 and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain. The protein complex according to item 18, wherein the first polypeptide has the structure:

[0225] Va- IGD4 - D - IGD1 - IGD2 - TD, and the second polypeptide has the structure:

[0226] Vb - C, wherein

[0227] Vaand Vb, are immunoglobulin variable domains that together form an antigen binding site,

[0228] IGD4 and C are immunoglobulin constant domains capable of heterodimerization, and D, IGD1, IGD2 and TD are as defined in items 1 to 13. The protein complex according to item 18 or 19, wherein one of Vaor Vb is an immunoglobulin variable heavy domain (VH) and one of Vaor Vb is an immunoglobulin variable light domain (VL), and / or one of IGD4 and C is a CHI domain and one of IGD4 and C is a CL domain. A dimer comprising or consisting of two identical or different polypeptides according to any one of items 1 to 16, two identical or different conjugates according to item 17, a polypeptide according to any one of items 1 to 16 and a conjugate according to item 17, a polypeptide according to any one of items 1 to 17 and a protein complex according to any one of items 18 to 20, or two identical or different protein complexes according to any one of items 18 to 20. A multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect, in particular a dodecamer comprising or consisting of twelve identical or different polypeptides according to any one of items 1 to 16, twelve identical or different conjugates according to item 17, twelve identical or different protein complexes according to any one of items 18 to 20, or six identical or different dimers according to item 20. 22. The multimer according to item 21, which does not comprise a J-chain polypeptide.

[0229] 23. A composition comprising the multimer according to item 21 or 22.

[0230] 24. One or more polynucleotides encoding the polypeptide according to any one of items 1 to 16, the polypeptides of the protein complex according to any one of items 18 to 20, the polypeptides of the dimer according to item 21, or the polypeptides of the multimer according to item 22 or 23.

[0231] 25. One or more vectors comprising the one or more polynucleotides according to item 24.

[0232] 26. A cell comprising the one or more polynucleotides of item 24 or the one more vectors according to item 25.

[0233] 27. A method for providing a composition according to item 23, wherein the method does not comprise a step of removing a multimer selected from a hexamer, octamer, decamer or dodecamer comprising polypeptides according to any one of items 1 to 16.

[0234] 28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound according to any of items 1 to 16, the conjugate according to item 17, the protein complex according to any one of items 18 to 20, the dimer according to item 21, the multimer according to item 22, the composition according to item 23, the one or more polynucleotides according to item 24, the one or more vectors according to item 25, or the cell according to item 26.

Claims

CLAIMS1. A polypeptide comprising a first immunoglobulin domain (IGD1), a second immunoglobulin domain (IGD2) and a tail domain (TD), wherein the IGD1, IGD2 and TD have the following structure from N-terminus to C-terminus:IGD1 - IGD2 - TD and wherein(ii) IGD1 is selected fromIgM CH3 and an immunoglobulin constant domain, in particular IgG CH2, IgD CH2, IgE CH3, IgAl CH2 or IgA2 CH2, comprising a substitution of an amino acid residue into a Cys residue at a position allowing the formation of a disulphide bond with a Cys of an IGD1 of another identical or different polypeptide according to claim 1, in particular a polypeptide comprised in a multimer according to claim 11 in an adjacent dimer;(ii) IGD2 is an immunoglobulin constant domain, in particular selected from IgM CH4, IgG CH3, IgAl CH3, IgA2 CH3, IgD CH3, and IgE CH4; and(iii) TD is selected from a mammalian IgAl, IgA2 or IgM tail piece comprising a substitution that increases the formation of homogenous multimers, in particular dodecamers, compared to a human wild-type IgM tail piece, and a non-mammalian IgA or IgM tail piece that is heterologous to the IGD1 and / or IGD2.

2. The polypeptide according to claim 1, wherein IGD1 is selected from IgM CH3 and IgG CH2 L328C, wherein the numbering of the amino acid position is according to EU numbering.

3. The polypeptide according to claim 1 or 2, wherein TD comprises or consists of the sequenceKPXIX2X3X4VSX5X6X7X8X9X10X11 X12TCY (SEQ ID NO: 1), wherein Xi is A or T, X2 is V, L, S or H, X3 is Y, L, V or I, X4is any amino acid, X5 is L or V, Xe is V or I, X7 is Y, L or M, Xs is S or A, X9 is E or D, X10 is T, V or A, Xu is A, G or D, X12 is A, S, G or N;wherein if X4 is N, then Xi is A, X2 is V or H, X3 is L, V or I, X5 is V, X7 is Y and / or X9 is E.

4. The polypeptide according to any one of claims 1 to 3, wherein the substitution in TD replaces an Asn residue, in particular an Asn residue at position 6 of SEQ ID NO: 1, at position 440 of the IgM constant domain (according to SEQ ID NO: 23), position 340 of the IgAl constant domain (according to SEQ ID NO: 24) or position 327 of the IgA2 constant domain (according to SEQ ID NO: 25), with another amino acid.

5. The polypeptide according to any one of claims 1 to 4, wherein TD has a sequence selected from(a) KPTLYQVSLVMSDTAGTCY (human IgM N440Q) (SEQ ID NO: 3),(b) KPTHVQVSVVMAEVDGTCY (human IgAl N340Q) (SEQ ID NO: 4),(c) KPTHIQVSVVMAEADGTCY (human IgA2 N327Q) (SEQ ID NO: 5),(d) KPAVLQVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, N440Q, L443V, M445Y, D447E) (SEQ ID NO: 6),(e) KPAVLNVSVVYSETAGTCY (human IgM T437A, L438V, Y439L, L443V, M445Y, D447E) (SEQ ID NO: 7),(f) NQPNLVNLSLNVPQRCMAQ (trout IgM tail piece) (SEQ ID NO: 8),(g) PSFVNISLALMDTINSCQ (nurse shark IgM tail piece) (SEQ ID NO: 9),(h) NRJDLNMNINQDSKCSA (Nile tilapia IgM tail piece) (SEQ ID NO: 10), and(i) KPTNVNVSLVLSDTC (Xenopus laevis IgM tail piece) (SEQ ID NO: 11).

6. The polypeptide according to any one of claims 1 to 5, further comprising a dimerization domain (D), wherein the IGD1, IGD2, D and TD have the following structure from N- terminus to C-terminus:D - IGD1 - IGD2 - TD and D is selected from an immunoglobulin constant domain, an immunoglobulin hinge region and a peptide linker; in particular wherein D comprises a Cys residue at a position allowing the formation of a disulphide bond with a Cys of a D domain of another identical or different polypeptide according to claim 8: and / oris a third immunoglobulin domain (IGD3).

7. The polypeptide according to any one of claims 1 to 6, further comprising an effector domain (E), which comprises a diagnostic or therapeutic polypeptide or part thereof; in particular wherein E comprises one or more antigen binding sites; more particularly E comprises a single domain antibody or a single chain antibody.

8. A conjugate comprising a polypeptide according to any one of claims 1 to 7 covalently linked to a diagnostic or therapeutic agent (F).

9. A protein complex comprising or consisting of a first polypeptide according to any one of claims 1 to 7 and a second polypeptide comprising an immunoglobulin variable domain and an immunoglobulin constant domain.

10. A dimer comprising or consisting of two identical or different polypeptides according to any one of claims 1 to 7, two identical or different conjugates according to claim 8, a polypeptide according to any one of claims 1 to 7 and a conjugate according to claim 8, a polypeptide according to any one of claims 1 to 7 and a protein complex according to claim 9, or two identical or different protein complexes according to claim 9.

11. A multimer comprising or consisting of identical or different polypeptides according to the first aspect, identical or different conjugates according to the second aspect, identical or different protein complexes according to the third aspect, or identical or different dimers according to the fourth aspect, in particular a dodecamer comprising or consisting of twelve identical or different polypeptides according to any one of claims 1 to 7, twelve identical or different conjugates according to claim 8, twelve identical or different protein complexes according to claim 9, or six identical or different dimers according to claim10.

12. One or more polynucleotides encoding the polypeptide according to any one of claims 1 to 7, the polypeptides of the protein complex according to claim 9, the polypeptides of the dimer according to claim 10, or the polypeptides of the multimer according to claim11.

13. One or more vectors comprising the one or more polynucleotides according to claim 12.

14. A cell comprising the one or more polynucleotides according to claim 12 or the one more vectors according to claim 13.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound according to any of claims 1 to 7, the conjugate according to claim 8, the protein complex according to claim 9, the dimer according to claim 10, the multimer according to claim 11, the one or more polynucleotides according to claim 12, the one or more vectors according to claim 13, or the cell according to claim 14.

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