Methods for generating multispecific antibodies from monospecific antibodies in vivo

Abstract

This paper reports a method for generating multispecific antibodies directly on the cell surface at the site of action via a half-antibody exchange reaction between two 2 / 3-IgG or two 2 / 3-BiFabs, wherein the 2 / 3-IgG or 2 / 3-BiFab halves are destabilized by asymmetric interfering mutations, thereby promoting the production of correctly assembled full-length bi- or multispecific antibodies. The method of the present invention can be performed even when the starting 2 / 3-IgG or 2 / 3-BiFab lacks hinge disulfide bonds.

Description

[0001] The present invention discloses a method for assembling multispecific (e.g., bispecific) antibodies in vivo on cells using a novel half-antibody exchange method. The method is even applicable to complete antibodies, i.e., antibodies comprising CH2-CH3 domains and thus having effector functions. Background of the Invention

[0003] Currently available technologies for biochemically converting monospecific antibody derivatives into assembled bispecific antibodies employ (i) half-antibody complementation reactions and (ii) IgG-IgG exchange reactions.

[0004] These techniques are disclosed in, for example, WO 2015 / 046467; Rispens et al., J. Biol. Chem. 289 (2014) 6098–6109; US 9,409,989; WO 2013 / 060867; WO 2011 / 131746; WO 2011 / 133886; WO 2011 / 143545; WO 2010 / 151792; Gunasekaran et al., J. Biol. Chem. 285 (2010) 19637–19646; WO 2009 / 041613; WO 2009 / 089004; WO 2008 / 119353; WO 2007 / 114325; US 8,765,412; US 8,642,745; WO 2006 / 047340; WO 2006 / 106905; WO 2005 / 042582; WO 2005 / 062916; WO 2005 / 000898; US 7,183,076; US 7,951,917; Segal, DM et al., Curr. Opin. Immunol. 11 (1999) 558–562; WO 98 / 50431; WO 98 / 04592; Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681; WO96 / 27011; Carter, P. et al., Immunotechnol. 2 (1996) 73; WO 93 / 11162 and Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553.

[0005] Existing methods for converting monospecific antibodies or antibody derivatives into bsAbs (bispecific antibodies) have drawbacks, such as limitations in the process and composition of the assembled bsAb products.

[0006] For example, half-antibody technology assembles monospecific and monovalent antibodies into bivalent IgG. The expression of the input molecule and the exchange reaction itself not only produce half antibodies, but also produce IgG-like bivalent (monospecific) antibody derivatives. Polymers will also be present in the input and in the output of the assembly reaction. Both (bivalent monospecific antibodies and polymers) need to be quantitatively removed from the assembled bsAb by complex purification methods, or (because quantitative removal is difficult to achieve in a high-throughput manner) they will "contaminate" the bsAb products to a certain extent.

[0007] For example, Fab-arm exchange technology assembles bispecific bivalent IgGs from monospecific bivalent IgG derivatives. Therefore, the input of the exchange reaction is bivalent (i.e., with avidity by default). In order to ensure that there is no remaining bivalent monospecific input in the exchange reaction, affinity or agonist antibody screening should be performed to ensure that any remaining bivalent input and any polymer that may be formed during the exchange reaction are completely removed. Due to the high similarity between the input and bsAb, complex procedures are required for quantitative removal (which is difficult to achieve high throughput), otherwise the remaining bivalent input and polymer will contaminate the final bsAb product to a certain extent.

[0008] Labrijn, AF et al. disclosed a method for efficiently generating stable bispecific IgG1 through controlled Fab-arm exchange (Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150).

[0009] WO 2014 / 081955 discloses heterodimeric antibodies and methods of use thereof.

[0010] WO 2009 / 089004 discloses a method for preparing antibody Fc heterodimeric molecules using the electrostatic steering effect. It discloses that of four unique charged residue pairs involved in domain-domain interactions (Asp356-Lys439', Glu357-Lys370', Lys392-Asp399', and Asp399-Lys409'), only Lys409-Asp399' is suitable for engineering because these two residues are structurally conserved and are buried residues. For the other three residue pairs, at least one partner residue is solvent exposed (%ASA > 10).

[0011] WO 2018 / 155611 discloses a combination of a first antigen-binding molecule and a second antigen-binding molecule that are not bound by covalent bonding, which, when mixed into a liquid, are more likely to form heterodimers rather than homodimers. One embodiment is disclosed in which the cysteine residues at one or both of positions 226 and 229 of the EU numbering system are replaced with other amino acids, and one or both of the first CH3 and the second CH3 are replaced with other amino acid residues at at least one of positions 357 or 397 of the EU numbering system.

[0012] Mayer, K. et al. (Int. J. Mol. Sci. 16 (2015) 27497-27506) disclosed TriFabs, trivalent IgG-type bispecific antibody derivatives, their design, production, characterization and application in targeted payload delivery. SUMMARY OF THE INVENTION

[0014] The present invention discloses a method for generating multispecific antibodies directly on the surface of target cells through a half-antibody exchange reaction. This allows, for example, the formation of functional binding sites directly at the desired site of action from inactive pro-binding sites. Such on-site formation eliminates the risk of systemic side effects.

[0015] It has been found that as starting materials, it is advantageous to use incomplete antibodies, such as 2 / 3-IgGs or 2 / 3-BiFabs comprising an antibody light chain, an antibody heavy chain and an antibody heavy chain fragment, wherein the heavy chain-heavy chain interaction is unstable due to asymmetric interference mutations, preferably in the heavy chain fragment, and wherein there is no heavy chain-heavy chain covalent / disulfide bond, such as a hinge region disulfide bond or a CH3 domain disulfide bond. It has been found that such asymmetric interference mutations promote the dissociation of the starting incomplete antibody on the one hand and the production of correctly assembled full-length bispecific antibodies on the other hand. The absence of heavy chain-heavy chain covalent bonds allows the reaction to proceed in the absence of a reducing agent, i.e., in vivo or under physiological conditions.

[0016] The method according to the present invention is performed in the absence of a reducing agent. Consequently, the starting antibodies lack heavy chain-heavy chain disulfide bonds, such as hinge region disulfide bonds. Therefore, the chain exchange reaction and method according to the present invention allow for the assembly of bispecific antibodies without initial reduction, making the method suitable for in vivo applications. Thus, naturally occurring intramolecular disulfide bonds between the heavy chains of the starting molecules can be removed, for example, by mutagenic PCR. Despite the lack of all intermolecular disulfide bonds between the heavy chains, stable, i.e., isolatable, antibodies can be correctly formed. Therefore, using these starting molecules, spontaneous chain exchange reactions can be utilized without reduction to achieve in vivo production of bispecific antibodies. In summary, the chain exchange method according to the present invention without reduction is capable of efficiently producing functional bispecific antibodies directly on the cell surface in vivo.

[0017] The present invention discloses a method for producing a (multispecific) binder / multimeric polypeptide, comprising the following steps:

[0018] - Incubate the following first conjugate and second conjugate,

[0019] a first binder (mono- or bispecific and heteromeric) / multimeric polypeptide comprising a first (monomeric) polypeptide and a second (monomeric) polypeptide, wherein both polypeptides comprise (in N-terminal to C-terminal direction) an antibody variable domain followed (immediately) by a human immunoglobulin (IgG1) CH3 domain,

[0020] wherein i) the variable domain of the first polypeptide is a heavy chain variable domain and the variable domain of the second polypeptide is a light chain variable domain, or ii) the variable domain of the first polypeptide is a light chain variable domain and the variable domain of the second polypeptide is a heavy chain variable domain,

[0021] wherein i) the CH3 domain of the first polypeptide comprises a knob mutation, and the CH3 domain of the second polypeptide comprises a hole mutation, or ii) the CH3 domain of the first polypeptide comprises a hole mutation, and the CH3 domain of the second polypeptide comprises a knob mutation,

[0022] wherein the first polypeptide comprises at least one functional binding site or at least part of a binding site,

[0023] wherein the second polypeptide comprises at least one / first interfering mutation in the CH3 domain selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V 397Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T), wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interact with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0024] wherein the first polypeptide and the second polypeptide non-covalently associate with each other / form a non-covalent dimer / are non-covalently associated with each other / are a non-covalent dimer, (whereby, when the second polypeptide and the first polypeptide form a heterodimer, the interfering mutation in the second polypeptide results in a destabilizing interaction)

[0025] as well as

[0026] a second binder (mono- or bispecific and heteromeric) / multimeric polypeptide comprising a third (monomeric) polypeptide and a fourth (monomeric) polypeptide, wherein both polypeptides comprise (in N-terminal to C-terminal direction) an antibody variable domain followed directly by a human immunoglobulin (IgG1) CH3 domain,

[0027] wherein i) the variable domain of the third polypeptide is a heavy chain variable domain, and the variable domain of the fourth polypeptide is a light chain variable domain, or ii) the variable domain of the third polypeptide is a light chain variable domain, and the variable domain of the fourth polypeptide is a heavy chain variable domain, wherein i) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the fourth polypeptide is a light chain variable domain, or ii) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the fourth polypeptide is a heavy chain variable domain,

[0028] wherein i) the CH3 domain of the third polypeptide comprises a knob mutation, and the CH3 domain of the fourth polypeptide comprises a hole mutation, or ii) the CH3 domain of the third polypeptide comprises a hole mutation, and the CH3 domain of the fourth polypeptide comprises a knob mutation, wherein i) where the first polypeptide comprises a hole mutation, the fourth polypeptide comprises a knob mutation, or ii) where the first polypeptide comprises a knob mutation, the fourth polypeptide comprises a hole mutation,

[0029] wherein the fourth polypeptide comprises at least one functional binding site or at least part of a binding site,

[0030] wherein the third polypeptide comprises at least one / a second interfering mutation in the CH3 domain selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399E 99K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T), wherein the amino acid sequence of the fourth polypeptide comprises a human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) in the wild-type immunoglobulin (IgG1) that interacts with the amino acid residue at the interfering mutation, wherein the mutation in the third polypeptide is at a different position than the mutation in the second polypeptide,

[0031] wherein the third polypeptide and the fourth polypeptide covalently or non-covalently associate with each other / form a covalent or non-covalent dimer / are non-covalently or covalently associated with each other / are a non-covalent or covalent dimer, (whereby, when the third polypeptide and the fourth polypeptide form a heterodimer, the interfering mutation in the third polypeptide results in a destabilizing interaction),

[0032] wherein the (first) interfering mutation in the second polypeptide and the (second) interfering mutation in the third polypeptide result in an attractive interaction when the second polypeptide and the third polypeptide form a heterodimer,

[0033] wherein the variable domain of the first polypeptide and the variable domain of the fourth polypeptide form a functional (antigen-binding) binding site (antibody variable domain pair (VH / VL pair)), and the variable domain of the second polypeptide and the variable domain of the third polypeptide form a non-functional (antigen-incapable) variable domain pair,

[0034] and

[0035] - Recovering the binder comprising the first polypeptide and the fourth polypeptide, thereby generating a (multispecific) binder / multimeric polypeptide.

[0036] In one embodiment, the first to fourth polypeptides each comprise, from N-terminal to C-terminal direction, i) the amino acid sequence DKTHTSPPS (SEQ ID NO: 66), ii) an antibody variable domain derived from a human IgG1 variable domain, and iii) a CH3 domain derived from a human IgG1 CH3 domain.

[0037] In one embodiment, i) the first and fourth polypeptides each further comprise a CH1 domain derived from a human IgG1 CH1 domain ((variant) human IgG1 CH1 structure), and (independently of each other) another (heavy chain or light chain) variable domain, or ii) the first or fourth polypeptide comprises a CH1 domain derived from a human IgG1 CH1 domain ((variant) human IgG1 CH1 domain), and the corresponding other polypeptide comprises a domain derived from a light chain constant domain ((variant) human κ or λ CL domain), and each polypeptide further comprises another variable domain. In one embodiment, the other variable domain of the first polypeptide and the other variable domain of the fourth polypeptide are (different) heavy chain variable domains. In one embodiment, the other variable domain of the first polypeptide is a heavy chain variable domain, and the other variable domain of the fourth polypeptide is a light chain variable domain, or vice versa.

[0038] In one embodiment, the first and fourth polypeptides may have the same or different N-terminal to C-terminal sequences, and if the first and fourth polypeptides are the same, they are selected from the group consisting of the following polypeptides; if the first and fourth polypeptides are different, they are independently selected from the group consisting of the following polypeptides, wherein the polypeptides comprise, from N-terminal to C-terminal direction:

[0039] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0040] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0041] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0042] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0043] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0044] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0045] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0046] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0047] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0048] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0049] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0050] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0051] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0052] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom),

[0053] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0054] In one embodiment, one of the first and fourth polypeptides comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a third heavy chain variable domain, a first light chain constant domain, a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain; and the other of the first and fourth polypeptides comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain. In one embodiment, the binder comprising a polypeptide comprising two heavy chain variable domains further comprises a first light chain and a (domain-exchanged) second light chain, wherein the first light chain comprises a first light chain variable domain and a second light chain constant domain (paired with the first heavy chain variable domain), the second light chain comprises a second light chain variable domain and a human IgG1 CH1 domain (a CH1 domain derived therefrom) (paired with the second heavy chain variable domain), and the other binder further comprises the first light chain.

[0055] In one embodiment, one of the first and fourth polypeptides comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a second light chain variable domain, a second human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CHI domain; and the other of the first and fourth polypeptides comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CHI domain. In one embodiment, the binder comprising a polypeptide comprising two variable domains further comprises a first light chain and a (domain-exchanged) second light chain, wherein the first light chain comprises a third light chain variable domain and a first light chain constant domain (paired with the first heavy chain variable domain), the second light chain comprises a third heavy chain variable domain and a second light chain constant domain (paired with the first light chain variable domain), and the other binder further comprises the first light chain.

[0056] In one embodiment, the first and second binders further comprise an antibody light chain.

[0057] In one embodiment,

[0058] The first conjugate comprises

[0059] - a first polypeptide selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0060] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0061] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0062] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0063] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0064] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0065] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0066] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0067] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0068] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0069] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0070] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0071] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0072] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0073] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0074] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0075] and contain a knob mutation or a hole mutation,

[0076] and

[0077] - A second polypeptide selected from the group consisting of polypeptides comprising, from the N- to the C-terminus:

[0078] The hinge region of SEQ ID NO: 66, the first heavy or light chain variable domain, the CH3 domain derived from the human IgG1 CH3 domain,

[0079] wherein i) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the second polypeptide is a heavy chain variable domain, or ii) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the second polypeptide is a light chain variable domain,

[0080] wherein if the first polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the first polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0081] and comprising a first interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399 A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0082] wherein the first polypeptide and the second polypeptide non-covalently associate with each other / form a non-covalent dimer, (whereby, when the second polypeptide and the first polypeptide form a heterodimer, an interfering mutation in the second polypeptide results in a destabilizing interaction),

[0083] and

[0084] - a third polypeptide comprising another light chain variable domain and a light chain constant domain,

[0085] wherein the third polypeptide is covalently bound to the first polypeptide via a disulfide bond,

[0086] and

[0087] The second combination comprises:

[0088] - A fourth polypeptide, which is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0089] a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain, wherein if the second polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the second polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0090] and comprises a second interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S 400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the fifth polypeptide comprises a wild-type amino acid residue of a human immunoglobulin (IgG1) at an amino acid position in the wild-type immunoglobulin (IgG1) that interacts with the amino acid residue at the interfering mutation, wherein the interfering mutation in the fourth polypeptide is at a different position than the interfering mutation in the second polypeptide,

[0091] and

[0092] - A fifth polypeptide, which is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0093] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0094] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0095] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0096] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0097] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0098] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0099] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0100] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0101] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0102] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0103] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0104] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0105] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0106] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0107] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0108] wherein if the fourth polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the fourth polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0109] wherein i) if the variable domain of the second polypeptide is a light chain variable domain, the variable domain of the fourth polypeptide is a heavy chain variable domain, or ii) if the variable domain of the second polypeptide is a heavy chain variable domain, the variable domain of the fourth polypeptide is a light chain variable domain,

[0110] wherein the fourth polypeptide and the fifth polypeptide non-covalently associate with each other / form a non-covalent dimer, (whereby, when the fourth polypeptide and the fifth polypeptide form a heterodimer, the interfering mutation in the fourth polypeptide results in a destabilizing interaction)

[0111] and

[0112] - a sixth polypeptide comprising a light chain variable domain and a light chain constant domain,

[0113] The sixth polypeptide is covalently bound to the fourth polypeptide via a disulfide bond.

[0114] In one embodiment, the incubating step is performed in the absence of a reducing agent.

[0115] In one embodiment, i) the second polypeptide and the third polypeptide, or ii) the second polypeptide and the fifth polypeptide further comprise a (C-terminal) tag. In one embodiment, the tag has the amino acid sequence HHHHHH (SEQ ID NO: 67) or HHHHHHHH (SEQ ID NO: 68), and recovery is performed by chromatography on a metal (nickel) chelate affinity chromatography column.

[0116] In one embodiment,

[0117] The first conjugate comprises

[0118] The first polypeptide is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0119] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0120] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0121] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0122] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0123] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0124] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0125] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0126] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0127] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0128] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0129] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0130] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0131] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0132] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0133] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0134] and contain a knob mutation or a hole mutation,

[0135] and

[0136] The second polypeptide is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0137] The hinge region of SEQ ID NO: 66, the first heavy or light chain variable domain, the CH3 domain derived from the human IgG1 CH3 domain,

[0138] wherein i) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the second polypeptide is a heavy chain variable domain, or ii) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the second polypeptide is a light chain variable domain,

[0139] wherein if the first polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the first polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0140] and comprising a first interfering mutation selected from the group consisting of D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K370E and K439E, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0141] wherein the first polypeptide and the second polypeptide non-covalently associate with each other / form a non-covalent dimer, (whereby, when the second polypeptide and the first polypeptide form a heterodimer, an interfering mutation in the second polypeptide results in a destabilizing interaction),

[0142] and

[0143] a third polypeptide comprising a light chain variable domain and a light chain constant domain,

[0144] wherein the third polypeptide is covalently bound to the first polypeptide via a disulfide bond,

[0145] and

[0146] The second conjugate comprises

[0147] The fourth polypeptide is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0148] The hinge region of SEQ ID NO: 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0149] wherein if the second polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the second polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0150] and comprises a second interfering mutation selected from the group consisting of D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K370E and K439E, wherein the amino acid sequence of the fifth polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1), wherein the interfering mutation in the fourth polypeptide is at a different position than the interfering mutation in the second polypeptide,

[0151] and

[0152] The fifth polypeptide is selected from the group consisting of polypeptides comprising, from the N-terminus to the C-terminus:

[0153] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0154] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0155] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0156] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0157] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0158] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0159] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0160] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0161] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0162] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0163] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0164] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0165] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0166] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0167] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0168] If the fourth polypeptide comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the fourth polypeptide comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0169] wherein the fourth polypeptide and the fifth polypeptide non-covalently associate with each other / form a non-covalent dimer, (wherein an interfering mutation in the fourth polypeptide results in a destabilizing interaction when the fourth polypeptide and the fifth polypeptide form a heterodimer),

[0170] wherein the variable domain of the first polypeptide and the variable domain of the fourth polypeptide form a functional (antigen-binding) binding site (antibody variable domain pair (VH / VL pair)), and the variable domain of the second polypeptide and the variable domain of the third polypeptide form a non-functional (antigen-incapable) variable domain pair,

[0171] and

[0172] a sixth polypeptide comprising a light chain variable domain and a light chain constant domain,

[0173] The sixth polypeptide is covalently bound to the fourth polypeptide via a disulfide bond.

[0174] One aspect of the present invention reports a method for identifying a combination of binders comprising the following steps:

[0175] - performing the method according to the invention on each combination of a first conjugate selected from the first batch of conjugates and a second conjugate selected from the second batch of conjugates, to produce a batch of conjugates,

[0176] - determining the simultaneous binding of each conjugate to at least two antigens by ELISA analysis, and

[0177] - Based on the results of ELISA, binders are selected from the batch of binders to identify a combination of binders.

[0178] One aspect of the present invention reports a multimeric polypeptide comprising a first polypeptide and a second polypeptide

[0179] wherein both polypeptides comprise (directly following each other from N-terminus to C-terminus, optionally with a peptide linker between the variable domain and the CH3 domain) the amino acid sequence DKTHTSPPS (SEQ ID NO: 66), an antibody variable domain and a human immunoglobulin (IgG1) CH3 domain,

[0180] wherein i) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the second polypeptide is a heavy chain variable domain, or ii) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the second polypeptide is a light chain variable domain,

[0181] wherein i) the CH3 domain of the first polypeptide comprises a knob mutation, and the CH3 domain of the second polypeptide comprises a hole mutation, or ii) the CH3 domain of the first polypeptide comprises a hole mutation, and the CH3 domain of the second polypeptide comprises a knob mutation,

[0182] wherein the first polypeptide comprises at least one functional binding site or at least part of a binding site,

[0183] wherein the second polypeptide comprises at least one interfering mutation in the CH3 domain selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V3 97Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T), wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0184] wherein the first polypeptide and the second polypeptide non-covalently associate with each other / form a non-covalent dimer, (wherein an interfering mutation in the second polypeptide results in a destabilizing interaction when the second polypeptide and the first polypeptide form a heterodimer),

[0185] The variable domain of the first polypeptide and the variable domain of the second polypeptide form a functional or non-functional (incapable of antigen binding) variable domain pair.

[0186] In one embodiment, the first polypeptide is selected from a polypeptide comprising, in N- to C-terminal direction:

[0187] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0188] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0189] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0190] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0191] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0192] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0193] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0194] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0195] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0196] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0197] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0198] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0199] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0200] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0201] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0202] (and includes knob mutation or hole mutation)

[0203] and

[0204] The second polypeptide is selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0205] the hinge region of SEQ ID NO: 66, the first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain comprising a knob mutation or a hole mutation,

[0206] and comprising an interfering mutation selected from the group consisting of: E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399 A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1).

[0207] In one embodiment, the multimeric polypeptide further comprises a third polypeptide comprising a (further) light chain variable domain and a light chain constant domain, wherein the third polypeptide is covalently bound to (the CH1 domain of) the first polypeptide via a disulfide bond.

[0208] One aspect of the present invention reports a composition comprising

[0209] A first heterotrimeric polypeptide comprising

[0210] - a first polypeptide selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0211] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0212] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0213] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0214] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0215] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0216] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0217] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0218] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0219] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0220] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0221] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0222] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0223] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0224] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0225] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0226] (including knob mutation or hole mutation)

[0227] and

[0228] - A second polypeptide selected from the group consisting of polypeptides comprising, from the N- to the C-terminus:

[0229] The hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0230] wherein i) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the second polypeptide is a heavy chain variable domain, or ii) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the second polypeptide is a light chain variable domain,

[0231] wherein if the first polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the first polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0232] and comprising an interfering mutation selected from E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A , D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0233] and

[0234] - a third polypeptide, which is a polypeptide comprising another light chain variable domain and a light chain constant domain, wherein the third polypeptide is covalently bound to the first polypeptide via a disulfide bond

[0235] as well as

[0236] A second heterotrimeric polypeptide comprises

[0237] - a first (fourth) polypeptide selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0238] a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0239] wherein if the second polypeptide of the first heterotrimeric polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the second polypeptide of the first heterotrimeric polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0240] and comprises a second interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S3400K, D4 01R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the second (fifth) polypeptide comprises a wild-type amino acid residue of a human immunoglobulin (IgG1) at an amino acid position in the wild-type immunoglobulin (IgG1) that interacts with the amino acid residue at the interfering mutation, wherein the interfering mutation in the first (fourth) polypeptide is at a different position than the interfering mutation in the second polypeptide of the first heterotrimer,

[0241] and

[0242] - a second (fifth) polypeptide selected from the group consisting of polypeptides comprising, from the N- to the C-terminus:

[0243] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0244] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0245] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0246] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0247] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0248] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0249] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0250] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0251] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0252] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0253] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0254] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0255] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0256] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0257] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0258] If the first (fourth) polypeptide comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the first (fourth) polypeptide comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0259] wherein i) if the variable domain of the second polypeptide is a light chain variable domain, the variable domain of the fifth polypeptide is a heavy chain variable domain, or ii) if the variable domain of the second polypeptide is a heavy chain variable domain, the variable domain of the fifth polypeptide is a light chain variable domain,

[0260] and

[0261] - a third (sixth) polypeptide, which is a polypeptide comprising a light chain variable domain and a light chain constant domain, wherein the sixth polypeptide is covalently bound to the first (fourth) polypeptide via a disulfide bond,

[0262] wherein i) the CH3 domain of the first polypeptide of the first heterotrimer comprises a knob mutation and the CH3 domain of the second polypeptide of the first heterotrimer comprises a hole mutation, or ii) the CH3 domain of the first polypeptide of the first heterotrimer comprises a hole mutation and the CH3 domain of the second polypeptide of the first heterotrimer comprises a knob mutation, wherein i) if the first polypeptide of the first heterotrimer comprises a hole mutation, the second (fifth) polypeptide of the second heterotrimer comprises a knob mutation, or ii) if the first polypeptide of the first heterotrimer comprises a knob mutation, the second (fifth) polypeptide of the second heterotrimer comprises a hole mutation,

[0263] wherein the second polypeptide of the first heterotrimer and the first polypeptide (fourth polypeptide) of the second heterotrimer do not comprise an interfering mutation at the same position / contain interfering mutations at different positions,

[0264] The variable domains of the first polypeptide and the fifth polypeptide form a functional (antigen-binding) binding site (antibody variable domain pair (VH / VL pair)), and the variable domains of the second polypeptide and the fourth polypeptide form a non-functional (antigen-incapable) variable domain pair.

[0265] One aspect of the present invention reports a multimeric polypeptide comprising a first polypeptide and a second polypeptide

[0266] Both polypeptides contain the human immunoglobulin (IgG1) CH3 domain.

[0267] wherein i) the CH3 domain of the first polypeptide comprises a knob mutation, and the CH3 domain of the second polypeptide comprises a hole mutation, or ii) the CH3 domain of the first polypeptide comprises a hole mutation, and the CH3 domain of the second polypeptide comprises a knob mutation,

[0268] wherein the first polypeptide comprises at least one functional binding site or at least part of a binding site,

[0269] wherein the second polypeptide comprises at least one interfering mutation in the CH3 domain selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V3 97Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T), wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0270] wherein the first polypeptide and the second polypeptide non-covalently or covalently associate with each other / form a non-covalent or covalent dimer, (whereby, when the second polypeptide and the first polypeptide form a heterodimer, the interfering mutation in the second polypeptide results in a destabilizing interaction).

[0271] In one embodiment, the first polypeptide is selected from a polypeptide comprising, in N- to C-terminal direction:

[0272] i) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0273] ii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0274] iii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a heavy chain variable domain,

[0275] iv) a first heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0276] v) a first heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second heavy chain variable domain,

[0277] vi) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0278] vii) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0279] viii) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second human IgG1 CH1 domain (a CH1 domain derived therefrom), and a light chain variable domain,

[0280] ix) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a light chain variable domain, and a second human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0281] x) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom),

[0282] xi) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a second heavy chain variable domain, or

[0283] xii) a first portion of the binding domain, optionally a first peptide linker, the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a second peptide linker, and a second portion of the binding domain, wherein the first portion of the binding domain and the second portion of the binding domain (of the same polypeptide) (bind and form a functional binding site that specifically binds to the target; in one embodiment, the first portion of the binding domain is an antibody heavy chain Fab fragment (VH-CH1 or CH1-VH) and the second portion of the binding domain is a light chain Fab fragment (VL-CL or CL-VL), or vice versa,

[0284] and contain a knob mutation or a hole mutation,

[0285] and

[0286] The second polypeptide is selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0287] The hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain comprising a knob mutation or a hole mutation,

[0288] and comprising an interfering mutation selected from the group consisting of: E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399 A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1).

[0289] In one embodiment, the multimeric polypeptide further comprises a third polypeptide comprising a light chain variable domain and a light chain constant domain, covalently bound to the first polypeptide via a disulfide bond.

[0290] One aspect of the present invention reports a composition comprising

[0291] A first heterotrimeric polypeptide comprising

[0292] - a first polypeptide selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0293] i) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0294] ii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0295] iii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a heavy chain variable domain,

[0296] iv) a first heavy chain variable domain, a first CH1 domain derived from a human IgG1 CH1 domain, a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a second CH1 domain derived from a human IgG1 CH1 domain,

[0297] v) a first heavy chain variable domain, a first CH1 domain derived from a human IgG1 CH1 domain, a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second CH1 domain derived from a human IgG1 CH1 domain, and a second heavy chain variable domain,

[0298] vi) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0299] vii) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0300] viii) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second human IgG1 CH1 domain (a CH1 domain derived therefrom), and a light chain variable domain,

[0301] ix) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a light chain variable domain, and a second human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0302] x) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom),

[0303] xi) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a second heavy chain variable domain, and

[0304] xii) a first portion of the binding domain, optionally a first peptide linker, the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a second peptide linker, and a second portion of the binding domain, wherein the first portion of the binding domain and the second portion of the binding domain (of the same polypeptide) (bind and form a functional binding site that specifically binds to the target; in one embodiment, the first portion of the binding domain is an antibody heavy chain Fab fragment (VH-CH1 or CH1-VH) and the second portion of the binding domain is a light chain Fab fragment (VL-CL or CL-VL), or vice versa,

[0305] and contain a knob mutation or a hole mutation,

[0306] and

[0307] - a second polypeptide selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0308] The hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0309] If the first polypeptide comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the first polypeptide comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0310] and comprising an interfering mutation selected from the group consisting of: E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399 A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0311] and

[0312] - a third polypeptide, which is a polypeptide comprising a light chain variable domain and a light chain constant domain, covalently bound to the first polypeptide via a disulfide bond,

[0313] as well as

[0314] A second heterotrimeric polypeptide comprises

[0315] - a first (fourth) polypeptide selected from the group consisting of polypeptides comprising, in N- to C-terminal direction:

[0316] the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0317] If the second polypeptide of the first heterotrimer comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the second polypeptide of the first heterotrimer comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0318] comprising a second interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S400K, D401R, F405 W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the second (fifth) polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interact with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1), wherein the interfering mutation in the first (fourth) polypeptide is at a different position than the interfering mutation in the second polypeptide of the first heterotrimer,

[0319] and

[0320] - a second (fifth) polypeptide selected from the group consisting of polypeptides comprising, from the N- to the C-terminus:

[0321] i) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0322] ii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0323] iii) the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a heavy chain variable domain,

[0324] iv) a first heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0325] v) a first heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second heavy chain variable domain,

[0326] vi) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0327] vii) a heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0328] viii) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second human IgG1 CH1 domain (a CH1 domain derived therefrom), and a light chain variable domain,

[0329] ix) a heavy chain variable domain, a first human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a light chain variable domain, and a second human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0330] x) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom),

[0331] xi) a first heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CHI domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a second heavy chain variable domain, or

[0332] xii) a first portion of the binding domain, optionally a first peptide linker, the hinge region of SEQ ID NO: 65 or 66, a CH2 domain derived from a human IgG1 CH2 domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a second peptide linker, and a second portion of the binding domain, wherein the first portion of the binding domain and the second portion of the binding domain (of the same polypeptide) (bind and form a functional binding site that specifically binds to the target; in one embodiment, the first portion of the binding domain is an antibody heavy chain Fab fragment (VH-CH1 or CH1-VH) and the second portion of the binding domain is a light chain Fab fragment (VL-CL or CL-VL), or vice versa,

[0333] If the first (fourth) polypeptide comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the first (fourth) polypeptide comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0334] and

[0335] - a third (sixth) polypeptide, which is a polypeptide comprising a light chain variable domain and a light chain constant domain, covalently bound to the first (fourth) polypeptide via a disulfide bond,

[0336] wherein i) the CH3 domain of the first polypeptide of the first heterotrimer comprises a knob mutation and the CH3 domain of the second polypeptide of the first heterotrimer comprises a hole mutation, or ii) the CH3 domain of the first polypeptide of the first heterotrimer comprises a hole mutation and the CH3 domain of the second polypeptide of the first heterotrimer comprises a knob mutation, wherein i) if the first polypeptide of the first heterotrimer comprises a hole mutation, the second polypeptide (fifth polypeptide) of the second heterotrimer comprises a knob mutation, or ii) if the first polypeptide of the first heterotrimer comprises a knob mutation, the second polypeptide (fifth polypeptide) of the second heterotrimer comprises a hole mutation,

[0337] The second polypeptide of the first heterotrimer and the first polypeptide (fourth polypeptide) of the second heterotrimer do not contain an interfering mutation at the same position / contain interfering mutations at different positions.

[0338] One aspect of the present invention reports a pharmaceutical formulation comprising a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds, or comprising a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, or comprising a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, as described herein, prepared by mixing such 2 / 3-IgG(s) or 2 / 3-BiFab(s).

[0339] One aspect of the present invention reports the use of a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds or a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds for use as a medicament.

[0340] One aspect of the present invention reports the use of a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds or a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds in the production or preparation of a drug.

[0341] One aspect of the present invention reports a method for treating a disease, which comprises administering to a diseased individual an effective amount of a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, or a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds.

[0342] In one embodiment, the composition comprises a first heterotrimeric polypeptide comprising a first, second, and third monomeric polypeptide, and a second heterotrimeric polypeptide comprising a fourth, fifth, and sixth monomeric polypeptide (i.e., each heterotrimeric polypeptide is composed of three (non-identical) monomeric polypeptides (comprising a total of six (non-identical) monomeric polypeptides (i.e., non-identical first, second, third, fourth, fifth, and sixth monomeric polypeptides))),

[0343] wherein the first, second, fourth and fifth (monomeric) polypeptides each comprise (from N-terminus to C-terminus) (i) the amino acid sequence DKTHTSPPS (SEQ ID NO: 66), (ii) a first antibody variable domain, and (iii) a human immunoglobulin (IgG1) CH3 domain, wherein (i), (ii) and (iii) are each independently linked to one another directly or via a peptide linker,

[0344] wherein the first antibody variable domains of i) the first and second (monomer) polypeptides, ii) the first and fifth (monomer) polypeptides, iii) the second and fourth (monomer) polypeptides and iv) the fifth and fourth (monomer) polypeptides are VH / VL pairs (i.e., the first variable domain of the first (monomer) polypeptide is a heavy chain variable domain or a light chain variable domain, whereby, if it is a heavy chain variable domain, the first variable domain of the second and fifth (monomer) polypeptides is a light chain variable domain, or if it is a light chain variable domain, the first variable domain of the second and fifth (monomer) polypeptides is a heavy chain variable domain, and if the first variable domain of the fifth (monomer) polypeptide is a heavy chain variable domain, the first variable domain of the fourth (monomer) polypeptide is a light chain variable domain, or if the first variable domain of the fifth (monomer) polypeptide is a light chain variable domain, the first variable domain of the fourth (monomer) polypeptide is a heavy chain variable domain),

[0345] wherein the CH3 domains of i) the first and fifth (monomeric) polypeptides, ii) the first and second (monomeric) polypeptides, iii) the second and fourth (monomeric) polypeptides and iv) the fifth and fourth (monomeric) polypeptides comprise a knob-hole mutation pair (i.e., the CH3 domain of the first (monomeric) polypeptide comprises a knob mutation or a hole mutation, wherein if it comprises a knob mutation, the CH3 domains of the second and fifth (monomeric) polypeptides comprise a hole mutation, or if it comprises a hole mutation, the CH3 domains of the second and fifth (monomeric) polypeptides comprise a knob mutation, and if the CH3 domain of the fifth (monomeric) polypeptide comprises a hole mutation, the CH3 domain of the fourth (monomeric) polypeptide comprises a knob mutation, or if the CH3 domain of the fifth (monomeric) polypeptide comprises a knob mutation, the CH3 domain of the fourth (monomeric) polypeptide comprises a hole mutation),

[0346] wherein the first (monomer) polypeptide and the fifth (monomer) polypeptide each independently of one another comprise a scFv or scFab or Fab at one or both of the N-terminus and the C-terminus,

[0347] wherein the second and fourth (monomer) polypeptides comprise at least one interfering mutation in the CH3 domain selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L369 368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C, and W366T), wherein the amino acid sequence of the first (monomer) polypeptide is in the wild-type immunoglobulin (IgG1) comprises a human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation of the second (monomeric) polypeptide, wherein the amino acid sequence of the fifth (monomeric) polypeptide comprises a human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation of the fourth (monomeric) polypeptide in the wild-type immunoglobulin (IgG1), wherein the interfering mutation of the second (monomeric) polypeptide and the interfering mutation of the fourth (monomeric) polypeptide are at different positions, wherein when the second polypeptide forms a heterodimer with the fourth polypeptide, the interfering mutation of the second (monomeric) polypeptide and the interfering mutation of the fourth (monomeric) polypeptide result in an attractive (charge interaction), wherein when the second (monomeric) polypeptide forms a heterodimer with the first (monomeric) polypeptide and the fourth (monomeric) polypeptide forms a heterodimer with the fifth (monomeric) polypeptide, the interfering mutations in the second polypeptide and the fourth (monomeric) polypeptide, respectively, result in a repulsive (charge interaction),

[0348] wherein the first and second (monomeric) polypeptides form a non-covalent dimer, the fourth and fifth (monomeric) polypeptides form a non-covalent dimer, the third and first (monomeric) polypeptides form a disulfide-linked dimer, and the sixth and fifth (monomeric) polypeptides form a disulfide-linked dimer,

[0349] wherein the third and sixth (monomer) polypeptides are antibody light chains.

[0350] In one embodiment, the first (monomeric) polypeptide is selected from polypeptides comprising, from N- to C-terminus:

[0351] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0352] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0353] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0354] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0355] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0356] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0357] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0358] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0359] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0360] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0361] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0362] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0363] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0364] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0365] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain.

[0366] In one embodiment, the second (monomeric) polypeptide is selected from polypeptides comprising, from N- to C-terminus:

[0367] The hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0368] wherein i) if the variable domain of the first polypeptide is a light chain variable domain, the variable domain of the second polypeptide is a heavy chain variable domain, or ii) if the variable domain of the first polypeptide is a heavy chain variable domain, the variable domain of the second polypeptide is a light chain variable domain,

[0369] wherein if the first polypeptide comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the first polypeptide comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0370] comprising an interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the first polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residue(s) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1),

[0371] and

[0372] A third polypeptide is a polypeptide comprising another light chain variable domain and a light chain constant domain, wherein the third polypeptide is covalently bound to the first polypeptide through a disulfide bond.

[0373] In one embodiment, the fourth (monomeric) polypeptide is selected from polypeptides comprising, from N- to C-terminus:

[0374] The hinge region of SEQ ID NO: 66, the first heavy or light chain variable domain, the CH3 domain derived from the human IgG1 CH3 domain,

[0375] wherein if the second polypeptide of the first heterotrimer comprises a hole mutation, the CH3 domain comprises a knob mutation, or if the second polypeptide of the first heterotrimer comprises a knob mutation, the CH3 domain comprises a hole mutation,

[0376] comprising a second interfering mutation selected from the group consisting of E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S400K, D401R, F405 W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T, wherein the amino acid sequence of the second (fifth) polypeptide comprises human immunoglobulin (IgG1) wild-type amino acid residues (one or more) at the amino acid position(s) that interacts with the amino acid residue at the interfering mutation in the wild-type immunoglobulin (IgG1), and wherein the interfering mutation in the first (fourth) polypeptide is at a different position from the interfering mutation in the second polypeptide of the first heterotrimer.

[0377] In one embodiment, the fifth (monomeric) polypeptide is selected from polypeptides comprising, from N- to C-terminus:

[0378] i) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, and a CH3 domain derived from a human IgG1 CH3 domain,

[0379] ii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a second heavy chain variable domain, and a human IgG1 CH1 domain (a CH1 domain derived therefrom),

[0380] iii) the hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, a human IgG1 CH1 domain (a CH1 domain derived therefrom), and a second heavy chain variable domain,

[0381] iv) scFv, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0382] v) scFab, optionally a peptide linker, a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain,

[0383] vi) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0384] vii) a hinge region of SEQ ID NO: 66, a heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0385] viii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0386] ix) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a third heavy chain variable domain,

[0387] x) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and an scFv,

[0388] xi) a second heavy chain variable domain, a human IgG1 CH1 domain (a CH1 domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CH3 domain, optionally a peptide linker, and a scFab,

[0389] xii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second human IgG1 CHI domain (a CHI domain derived therefrom), and a second light chain variable domain,

[0390] xiii) a second heavy chain variable domain, a first human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a second light chain variable domain, and a second human IgG1 CHI domain (a CHI domain derived therefrom),

[0391] xiv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a third heavy chain variable domain, and a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), or

[0392] xv) a second heavy chain variable domain, a human IgG1 CHI domain (a CHI domain derived therefrom), a hinge region of SEQ ID NO: 66, a first heavy or light chain variable domain, a CH3 domain derived from a human IgG1 CHI domain, optionally a peptide linker, a human IgG1 kappa or lambda light chain constant domain (a light chain constant domain derived therefrom), and a third heavy chain variable domain,

[0393] If the first (fourth) polypeptide comprises a hole mutation, then the polypeptide comprises a knob mutation, or if the first (fourth) polypeptide comprises a knob mutation, then the polypeptide comprises a hole mutation,

[0394] wherein i) if the variable domain of the second polypeptide is a light chain variable domain, the variable domain of the fifth polypeptide is a heavy chain variable domain, or ii) if the variable domain of the second polypeptide is a heavy chain variable domain, the variable domain of the fifth polypeptide is a light chain variable domain.

[0395] In one embodiment, the variable domains of the first polypeptide and the variable domains of the fifth polypeptide form a functional (antigen-binding) binding site (antibody variable domain pair (VH / VL pair)), and the variable domains of the second polypeptide and the variable domains of the fourth polypeptide form a non-functional (antigen-incapable) variable domain pair.

[0396] One aspect of the present invention is an (isolated) non-covalent complex / multimeric polypeptide comprising

[0397] - a first polypeptide comprising

[0398] i) from the N-terminus to the C-terminus, a) a first antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to a first target, and b) a first human immunoglobulin G CH3 domain,

[0399] and

[0400] ii) a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target, located at the N-terminus of the first antibody variable domain or the C-terminus of the first CH3 structure,

[0401] - a second polypeptide comprising

[0402] i) from the N-terminus to the C-terminus, a) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to a third target, and b) a second human immunoglobulin G CH3 domain,

[0403] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0404] as well as

[0405] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, wherein the first CH3 domain comprises

[0406] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0407] b) amino acid residue K at position 370, if the interfering mutation is E357K, or

[0408] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0409] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0410] and

[0411] ii) Optionally, a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target or the fourth target are located at the N-terminus of the second antibody variable domain or the C-terminus of the second CH3 domain, and this position is independent of the position of the variable domain pair of the first polypeptide,

[0412] All numbering is according to the Kabat EU index.

[0413] In one embodiment of all aspects the first CH3 domain and the second CH3 domain comprise amino acid sequence mutations disclosed herein to promote heterodimer formation, ie as outlined herein below in section D) Heterodimerization.

[0414] In one embodiment of all aspects the first CH3 domain and the second CH3 domain comprise additional mutations to promote heterodimer formation between said first CH3 domain and said second CH3 domain, said mutations being different from interfering mutations.

[0415] In one embodiment of all aspects,

[0416] The first CH3 domain comprises

[0417] a) T366W mutation, or

[0418] b) T366S / L368A / Y407V mutations,

[0419] as well as

[0420] The second CH3 domain comprises

[0421] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0422] b) T366W mutation, if the first CH3 domain comprises T366S / L368A / Y407V mutations.

[0423] One aspect of the present invention is an (isolated) non-covalent complex / multimeric polypeptide comprising

[0424] - a first polypeptide comprising

[0425] i) from the N-terminus to the C-terminus, a) a first antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to a first target, and b) a first human immunoglobulin G CH3 domain,

[0426] The first CH3 domain comprises

[0427] a) T366W mutation, or T366S / L368A / Y407V mutation, and

[0428] b) optionally a Y349C or S354C mutation,

[0429] and

[0430] ii) a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target, located at the N-terminus of the first antibody variable domain or the C-terminus of the first CH3 structure,

[0431] - a second polypeptide comprising

[0432] i) from the N-terminus to the C-terminus, a) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to a third target, and b) a second human immunoglobulin G CH3 domain,

[0433] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0434] and

[0435] The second CH3 domain comprises

[0436] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0437] b) T366W mutation, if the first CH3 domain contains T366S / L368A / Y407V mutations,

[0438] and

[0439] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, wherein the first CH3 domain comprises

[0440] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0441] b) amino acid residue K at position 470, if the interfering mutation is E357K, or

[0442] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0443] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0444] and

[0445] ii) Optionally, a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target or the fourth target are located at the N-terminus of the second antibody variable domain or the C-terminus of the second CH3 domain, and this position is independent of the position of the variable domain pair of the first polypeptide,

[0446] Therein, all numbering is based on the Kabat EU index.

[0447] One aspect of the present invention is an (isolated) non-covalent complex / multimeric polypeptide comprising

[0448] - a first polypeptide comprising

[0449] i) from N-terminus to C-terminus, a) a first human immunoglobulin G CH3 domain, and b) a first antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a first target,

[0450] and

[0451] ii) a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to a second target, located at the N-terminus of the first antibody CH3 domain or the C-terminus of the first antibody variable domain,

[0452] - a second polypeptide comprising

[0453] i) from N-terminal to C-terminal direction, a) a second human immunoglobulin G CH3 domain, and b) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a third target,

[0454] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0455] and

[0456] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, whereby the first CH3 domain comprises

[0457] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0458] b) amino acid residue K at position 470, if the interfering mutation is E357K, or

[0459] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0460] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0461] and

[0462] ii) Optionally, a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target or the fourth target are located at the N-terminus of the second CH3 domain or the C-terminus of the second variable domain, and this position is independent of the position of the variable domain pair of the first polypeptide,

[0463] Therein, all numbering is based on the Kabat EU index.

[0464] In one embodiment of all aspects the first CH3 domain and the second CH3 domain comprise amino acid sequence mutations disclosed herein to promote heterodimer formation, ie as outlined herein below in section D) Heterodimerization.

[0465] In one embodiment of all aspects the first CH3 domain and the second CH3 domain comprise additional mutations to promote heterodimer formation between said first CH3 domain and said second CH3 domain, said mutations being different from interfering mutations.

[0466] In one embodiment of all aspects,

[0467] The first CH3 domain comprises

[0468] a) T366W mutation, or

[0469] b) T366S / L368A / Y407V mutations,

[0470] as well as

[0471] The second CH3 domain comprises

[0472] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0473] b) T366W mutation, if the first CH3 domain comprises T366S / L368A / Y407V mutations.

[0474] One aspect of the present invention is an (isolated) non-covalent complex / multimeric polypeptide comprising

[0475] - a first polypeptide comprising

[0476] i) from N-terminus to C-terminus, a) a first human immunoglobulin G CH3 domain, and b) a first antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a first target,

[0477] The first CH3 domain comprises

[0478] a) T366W mutation, or T366S / L368A / Y407V mutation, and

[0479] b) optionally a Y349C or S354C mutation,

[0480] and

[0481] ii) a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to a second target, located at the N-terminus of the first antibody CH3 domain or the C-terminus of the first antibody variable domain,

[0482] - a second polypeptide comprising

[0483] i) from N-terminal to C-terminal direction, a) a second human immunoglobulin G CH3 domain, and b) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a third target,

[0484] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0485] as well as

[0486] The second CH3 domain comprises

[0487] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0488] b) T366W mutation, if the first CH3 domain contains T366S / L368A / Y407V mutations,

[0489] as well as

[0490] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, whereby the first CH3 domain comprises

[0491] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0492] b) amino acid residue K at position 470, if the interfering mutation is E357K, or

[0493] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0494] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0495] and

[0496] ii) Optionally, a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target or the fourth target are located at the N-terminus of the second CH3 domain or the C-terminus of the second variable domain, and this position is independent of the position of the variable domain pair of the first polypeptide,

[0497] Therein, all numbering is based on the Kabat EU index.

[0498] In one embodiment according to all aspects of the invention the first polypeptide and the second polypeptide are non-covalent dimers.

[0499] In one embodiment according to all aspects of the invention the first and second variable domains associate / bind and form a non-functional binding site.

[0500] In one embodiment according to all aspects of the invention, the first and second polypeptides comprise the amino acid sequence DKTHTSPPS (SEQ ID NO: 66) or DKTHT (SEQ ID NO: 94) or GGGS (SEQ ID NO: 69) or DKTHGGGGS (SEQ ID NO: 97) at the N-terminus of the first and second variable domains, respectively (if the first CH3 domain is located at the C-terminus of the first variable domain), or at the N-terminus of the first and second CH3 domains, respectively (if the first variable domain is located at the C-terminus of the first and second CH3 domains).

[0501] In one embodiment according to all aspects of the invention, the human immunoglobulin G is human IgG1 or human IgG2 or human IgG3 or human IgG4. In one embodiment according to all aspects of the invention, the human immunoglobulin G is human IgG1.

[0502] In one embodiment according to all aspects of the invention, the human immunoglobulin G CH3 domain is a human IgG1 CH3 domain or a human IgG2 CH3 domain or a human IgG3 CH3 domain or a human IgG4 CH3 domain.

[0503] In one embodiment according to all aspects of the present invention

[0504] i) the first CH3 domain comprises a T366W mutation and amino acid residue K at position 439,

[0505] and the second CH3 domain comprises a D356K interfering mutation and a T366S / L368A / Y407V mutation, or

[0506] ii) the first CH3 domain comprises a T366W mutation and an amino acid residue K at position 370,

[0507] and the second CH3 domain comprises an E357K interfering mutation and T366S / L368A / Y407V mutations, or

[0508] iii) the first CH3 domain comprises T366S / L368A / Y407V mutations and amino acid residue E at position 357,

[0509] and the second CH3 domain comprises a K370E interfering mutation and a T366W mutation, or

[0510] iv) the first CH3 domain comprises T366S / L368A / Y407V mutations and amino acid residue D at position 356,

[0511] and the second CH3 domain contains a K439E interfering mutation and a T366W mutation.

[0512] In one embodiment according to all aspects of the invention the first, second and third targets are different.

[0513] In one embodiment according to all aspects of the invention the first target or the third target is human CD3.

[0514] In one embodiment according to all aspects of the invention the pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to a second target is selected from the group consisting of: Fv, scFc, Fab, scFab, dsscFab, CrossFab, bispecific Fab, sdAb and VHH.

[0515] In one embodiment according to all aspects of the invention, the pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically binds to the fourth target, independent of the pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically binds to the second target, is selected from Fv, scFc, Fab, scFab, dsscFab, CrossFab, bispecific Fab, sdAb and VHH.

[0516] In one embodiment of all aspects of the invention, the first polypeptide comprises, from N-terminus to C-terminus:

[0517] an antibody heavy chain variable domain or an antibody light chain variable domain,

[0518] Human immunoglobulin G CH1 domain or human antibody light chain constant domain,

[0519] Alternatively, another antibody heavy chain variable domain or an antibody light chain variable domain, and another human immunoglobulin G CH1 domain or a human antibody light chain constant domain,

[0520] the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 94 or SEQ ID NO: 69 or SEQ ID NO: 77 or SEQ ID NO: 75 or SEQ ID NO: 76 or SEQ ID NO: 79 or SEQ ID NO: 97,

[0521] first antibody variable domain,

[0522] Optionally, human immunoglobulin G CH2 domain,

[0523] The first human immunoglobulin G CH3 domain,

[0524] Alternatively, the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 77 or SEQ ID NO: 75 or SEQ ID NO: 76 or SEQ ID NO: 79,

[0525] Alternatively, Fab or domain-swapped Fab or scFv or scFab.

[0526] In one embodiment of all aspects of the invention, the first polypeptide comprises, from N-terminus to C-terminus:

[0527] an antibody heavy chain variable domain or an antibody light chain variable domain,

[0528] Human immunoglobulin G CH1 domain or human antibody light chain constant domain,

[0529] Alternatively, another antibody heavy chain variable domain or an antibody light chain variable domain, and another human immunoglobulin G CH1 domain or a human antibody light chain constant domain,

[0530] the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 94 or SEQ ID NO: 69 or SEQ ID NO: 77 or SEQ ID NO: 75 or SEQ ID NO: 76 or SEQ ID NO: 79 or SEQ ID NO: 97,

[0531] Optionally, human immunoglobulin G CH2 domain,

[0532] The first human immunoglobulin G CH3 domain,

[0533] first antibody variable domain,

[0534] Alternatively, the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 77 or SEQ ID NO: 75 or SEQ ID NO: 76 or SEQ ID NO: 79,

[0535] Alternatively, Fab or domain-swapped Fab or scFv or scFab.

[0536] One aspect of the present invention is a composition comprising a first multimeric polypeptide according to the present invention and a second multimeric polypeptide according to the present invention, wherein

[0537] The second CH3 domain of the first multimeric polypeptide comprises a D356K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K439E mutation,

[0538] or

[0539] The second CH3 domain of the first multimeric polypeptide comprises an E357K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K370E mutation,

[0540] as well as

[0541] The first antibody variable domain of the first multimeric polypeptide and the first variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to the first target,

[0542] and

[0543] The second antibody variable domain of the first multimeric polypeptide and the second variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to a third target,

[0544] as well as

[0545] The second and fourth targets are independently of each other cell surface antigens.

[0546] In one embodiment of all composition aspects, the first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation, and the second CH3 domain of the first multimeric polypeptide and the first CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation.

[0547] In one embodiment of all composition aspects,

[0548] The first CH3 domain of the first polypeptide comprises

[0549] a) T366W mutation, or

[0550] b) T366S / L368A / Y407V mutations,

[0551] as well as

[0552] The second CH3 domain of the first polypeptide comprises

[0553] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0554] b) T366W mutation, if the first CH3 domain comprises T366S / L368A / Y407V mutations.

[0555] According to one aspect of the present invention is a (pharmaceutical) composition comprising a first multimeric polypeptide according to the present invention and a second multimeric polypeptide according to the present invention,

[0556] in

[0557] The first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide both comprise a T366W mutation or a T366S / L368A / Y407V mutation,

[0558] as well as

[0559] wherein the second CH3 domain of the first multimeric polypeptide comprises a D356K mutation and the second CH3 domain of the second multimeric polypeptide comprises a K439E mutation, or vice versa,

[0560] or

[0561] the second CH3 domain of the first multimeric polypeptide comprises an E357K mutation and the second CH3 domain of the second multimeric polypeptide comprises a K370E mutation, or vice versa,

[0562] as well as

[0563] Optionally, where

[0564] If the first antibody variable domain of the first multimeric polypeptide is an antibody heavy chain variable domain, the first antibody variable domain of the second multimeric polypeptide is an antibody light chain variable domain.

[0565] or

[0566] If the first antibody variable domain of the first multimeric polypeptide is an antibody light chain variable domain, and the first antibody variable domain of the second multimeric polypeptide is an antibody heavy chain variable domain,

[0567] as well as

[0568] in

[0569] The first antibody variable domain of the first multimeric polypeptide and the first variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to the first target,

[0570] and

[0571] The second antibody variable domain of the first multimeric polypeptide and the second variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to a third target,

[0572] as well as

[0573] wherein the second and fourth targets are independently cell surface antigens.

[0574] In one embodiment according to all aspects of the invention the first target or the third target is human CD3.

[0575] One aspect of the present invention is a multimeric polypeptide or a (pharmaceutical) composition according to the invention for use as a medicament.

[0576] One aspect of the present invention is a method of treatment comprising administering a multimeric polypeptide or a (pharmaceutical) composition according to the invention to an individual in need of such treatment. Detailed Description of the Invention

[0578] The present invention is based at least in part on the discovery that multispecific antibodies can be obtained by half-antibody exchange reactions, wherein as starting materials, non-complete antibodies, such as 2 / 3-IgGs or 2 / 3-BiFabs, are used, which comprise an antibody light chain, an antibody heavy chain, and an antibody heavy chain fragment, wherein the heavy chain-heavy chain interaction is destabilized by asymmetric interfering mutations, preferably in the heavy chain fragment, wherein the interfering mutations promote the dissociation of the starting non-complete antibody on the one hand and the production of correctly assembled full-length bi / multispecific antibodies on the other hand. It was further discovered that if the heavy chain-heavy chain disulfide bonds in the starting non-complete antibody are removed, then using such starting compounds, the method of the present invention can be performed even in the absence of a reducing agent (the generation of the starting material and the exchange reaction and the production of the multispecific antibody remain effective).

[0579] More specifically, the present invention is based, at least in part, on the discovery that multispecific antibodies and on-cell activation of binding sites can be obtained combinatorially using incomplete (i.e., non-bispecific) antibodies as starting materials through a half-antibody exchange reaction. Each starting molecule comprises a pair of antibody CH3 domains that bind to each other and form a dimer / multimer, a pair of antibody heavy chain variable domains and an antibody light chain variable domain that do not form a functional binding site, and at least one functional binding site for in vivo cell surface targeting. Thus, the CH3 domain pair can be part of a larger molecule (e.g., a pair of antibody heavy chains, a pair of fusion polypeptides, etc.). This CH3 domain pair with the modifications described herein defines the minimum structural elements required for the exchange reaction of the present invention. In the incomplete starting antibody, these CH3 domains still bind to each other (resulting in dimer or multimer formation), but the attraction between the CH3 domain pair can be reduced, i.e., destabilized, by asymmetric interfering (charge) mutations present only in one of the CH3 domains. The corresponding other CH3 domain still has wild-type residues at the positions that interact with the mutated positions in the bound wild-type CH3 domain pair. The interfering mutations only promote dissociation of the starting incomplete antibody and production of a properly assembled fully bispecific antibody in the presence of a second / other, more complementary, non-complete antibody. Studies have shown that the destabilized starting material can be isolated from cell culture supernatants despite the presence of (i) the destabilizing mutations between the CH3 domains and (ii) the absence of a disulfide bond between the two CH3 domains of the polypeptide comprising the starting non-complete bispecific antibody.

[0580] The present invention is based, at least in part, on the further discovery that, by using the aforementioned starting compounds, the exchange reaction can occur and form complete and functional bispecific antibodies in vivo in the absence of a reducing agent. Specifically, disulfide bonds between the CH3 domains contained in the starting polypeptides are not required. Thus, hinge disulfide bonds and other heavy chain-heavy chain disulfide bonds can be removed from the starting incomplete antibody.

[0581] I. Definition

[0582] As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbered according to Kabat." Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, while the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CH1, hinge, CH2 and CH3, in which case this is further clarified herein by reference to "numbered according to the Kabat EU index."

[0583] The CH3 domain in an antibody heavy chain can be altered using the "knob-into-holes" technique, which is described in detail with several examples in, for example, WO 96 / 027011; Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are altered to increase heterodimerization of the two CH3 domains and polypeptides containing them. One of the two CH3 domains (of the two heavy chains) can be a "knob" and the other a "hole." The introduction of a disulfide bridge can further stabilize the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increase production. However, this is not present in the molecules of the present invention.

[0584] The T366W mutation in the CH3 domain (of the antibody heavy chain) is denoted as a "knob mutation" or "mutated knob," and the T366S, L368A, and Y407V mutations in the CH3 domain (of the antibody heavy chain) are denoted as "hole mutations" or "mutated hole" (numbering according to the Kabat EU index). Additional interchain disulfide bridges between CH3 domains can also be utilized (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing an S354C mutation into a heavy chain CH3 domain with a "knob mutation" (denoted as a "knob-cys mutation" or "mutated knob-cys") and a Y349C mutation into a heavy chain CH3 domain with a "hole mutation" (denoted as a "hole-cys mutation" or "mutated hole-cys") (numbering according to the Kabat EU index). However, this is not present in the molecules of the present invention.

[0585] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0586] Useful methods and techniques that can be used to practice the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, eds., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press (1986); Watson, JD et al., Recombinant DNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Press (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc., NY (1987).

[0587] Derivatives of nucleic acids can be produced using recombinant DNA technology. Such derivatives can be modified at single or multiple nucleotide positions, for example, by substitution, alteration, exchange, deletion, or insertion. The modification or derivatization can be performed, for example, using site-directed mutagenesis. Those skilled in the art can readily implement these modifications (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD, and Higgins, SG, Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, UK).

[0588] It is important to note that, as used herein and in the appended claims, the singular forms "a," "a," and "the" include reference to the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes reference to a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0589] The term "MHCFcRP" refers to a mutant heavy chain Fc region polypeptide comprising at least an immunoglobulin constant domain 3 (CH3), wherein the CH3 comprises a knob mutation or a hole mutation and at least one interfering (i.e., destabilizing) mutation that introduces a single (i.e., single and / or additional) charge that is repulsive relative to the wild-type sequence. That is, when the MHCFcRP is paired with a polypeptide comprising a second CH3 domain, the second CH3 domain comprises a human immunoglobulin wild-type amino acid residue at one or more amino acid positions (in a wild-type immunoglobulin) that interact with the amino acid residue at the interfering mutation. In one embodiment, the interfering mutation is selected from E345R, Q347K, Y349W, Y349E, L351F, L351Y, S354E, S354V, D356S, D356A, D356K, E357S, E357A, E357L, E357F, E357K, K360S, K360E, Q362E, S364V, S364L, T366I, L368F, L368V, K370E, N390E, K392E, K392D, T394I, V397Y, D399A, D399K, S400K, D401R, F405W, Y407W, Y407L, Y407I, K409D, K409E, K409I, K439E, L441Y, Y349C, S366T, A368L, V407Y, S354C and W366T mutations (numbered according to the Kabat EU index). In a preferred embodiment, the interfering mutation is selected from the group consisting of D356K, E357K, K370E and K439E mutations.

[0590] The term "BiFab" refers to a molecule composed of two pairs of V1-C1 / V2-C2, where V represents an antibody variable domain and C represents an antibody constant domain, which are bound to each other. For example, the pairs can be VH1-CH1 / VL1-CL and VH2-CH31 / VL2-CH32. Similarly, "TriFab" refers to a molecule containing three pairs of V1-C1 / V2-C2, where V represents an antibody variable domain and C represents an antibody constant domain, which are bound to each other. For example, the pairs can be VH1-CH1 / VL1-CL, VH2-CH1 / VL2-CL, and VH3-CH31 / VL3-CH32.

[0591] The term "about" means a range of + / - 20% of the following numerical value. In one embodiment, the term "about" means a range of + / - 10% of the following numerical value. In one embodiment, the term "about" means a range of + / - 5% of the following numerical value.

[0592] The term "amino acid substitution" or "amino acid mutation" means that at least one amino acid residue in a predetermined parent amino acid sequence is replaced with a different "replacement" amino acid residue. The replacement residue(s) can be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and can be selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). In one embodiment, the replacement residue is not cysteine. Replacements with one or more non-naturally occurring amino acid residues are also encompassed within the definition of amino acid substitution herein. "Non-naturally occurring amino acid residues" refers to residues other than the naturally occurring amino acid residues listed above that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336. These non-naturally occurring amino acid residues can be produced using the procedures of Noren et al. (Science 244 (1989) 182) and / or Ellman et al. (supra). Briefly, these procedures involve chemical activation of suppressor tRNA with non-naturally occurring amino acid residues, followed by in vitro transcription and translation of RNA. Non-naturally occurring amino acids can also be incorporated into peptides by chemical peptide synthesis, which are then fused to recombinantly produced polypeptides (e.g., antibodies or antibody fragments).

[0593] The term "antibody-dependent cellular cytotoxicity (ADCC)" is a function mediated by Fc receptor binding and refers to target cell lysis mediated by the Fc region of an antibody in the presence of effector cells. In one embodiment, ADCC is measured as follows: in the presence of effector cells, such as freshly isolated PBMCs (peripheral blood mononuclear cells) or effector cells purified from buffy coats, such as monocytes or NK (natural killer) cells, a preparation of erythrocytes expressing a target (e.g., K562 cells expressing a recombinant target) is treated with a polypeptide comprising an Fc region as reported herein. The target cells are labeled with Cr-51 and subsequently incubated with the polypeptides reported herein. The labeled cells are incubated with effector cells, and the Cr-51 released in the supernatant is analyzed. Controls include target endothelial cells incubated with effector cells but without the polypeptides reported herein. The ability of the polypeptide to induce the initial step of mediating ADCC is investigated by measuring binding to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (substantially expressing FcγRIIIA). In a preferred embodiment, binding to FcγRs on NK cells is determined.

[0594] The term "CH1 domain" refers to the portion of an antibody heavy chain polypeptide that extends approximately from EU position 118 to EU position 215 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSC (SEQ ID NO: 27).

[0595] The term "CH2 domain" refers to the portion of an antibody heavy chain polypeptide that extends approximately from EU position 231 to EU position 340 (according to the EU numbering system of Kabat). In one embodiment, the CH2 domain comprises the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRWSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 28). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched sugar chains are intercalated between the two CH2 domains of an intact native Fc region. It is speculated that the sugars may provide an alternative to domain-domain pairing, contributing to the stabilization of the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206.

[0596] The term "CH3 domain" denotes the portion of an antibody heavy chain polypeptide that extends approximately from EU position 341 to EU position 446. In one embodiment, the CH3 domain comprises the amino acid sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 29).

[0597] The term "comprising" also includes "consisting of".

[0598] The term "complement dependent cytotoxicity (CDC)" refers to cell lysis caused by the Fc region of the antibodies reported herein in the presence of complement. In one embodiment, CDC is determined by treating human endothelial cells expressing the target with the polypeptides reported herein in the presence of complement. In one embodiment, the cells are labeled with calcein. CDC is present if the polypeptide induces 20% or more target cell lysis at a concentration of 30 μg / ml. Binding to complement factor C1q can be determined in an ELISA. In such an assay, in principle, a range of concentrations of the polypeptide are coated on an ELISA plate and purified human C1q or human serum is added. C1q binding is detected using an anti-C1q antibody followed by a peroxidase-labeled conjugate. Detection of binding (maximum binding Bmax) is measured using a peroxidase substrate The optical density at 405 nm (OD405) of (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) was determined.

[0599] As used herein, "treatment" (and its grammatical variations, "treat" or "treating" or "treatment") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be used for prevention or for the purpose of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies reported herein are used to delay the onset of the disease or slow the progression of the disease.

[0600] "Effector functions" refer to biological activities attributable to the Fc region of an antibody, which vary with the class of antibody from which the Fc region is derived. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as the B cell receptor); and B cell activation.

[0601] Fc receptor binding-dependent effector functions are mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate: the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) (presenting the Fc region) by antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for the IgG Fc region are called FcγRs. Fc receptor binding is described in, for example, Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; Gessner, JE et al., Ann. Hematol. 76 (1998) 231-248.

[0602] Cross-linking of receptors (FcγRs) on the Fc region of IgG antibodies can trigger a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and the release of inflammatory mediators, as well as the clearance of immune complexes and the regulation of antibody production. In humans, three classes of FcγRs have been characterized:

[0603] -FcγRI (CD64), which binds monomeric IgG with high affinity, is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one of amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbered according to Kabat's EU index) in the Fc region of IgG can reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduces binding to FcγRI by 10 3fold and abolished the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613–2624).

[0604] -FcγRII (CD32), binds complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is present on many cells involved in killing (such as macrophages, monocytes, neutrophils) and seems to be able to activate the killing process. FcγRIIB seems to play a role in the inhibition process and is found on B cells, macrophages, mast cells and eosinophils. On B cells, it seems to play a role in inhibiting further immunoglobulin production and isotype switching (for example to the IgE class). On macrophages, FcγRIIB inhibits phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B type can help inhibit the activation of these cells caused by the binding of IgE to their receptors. FcγRIIA was found to have reduced binding to, for example, an antibody comprising an IgG Fc region having a mutation in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index as in Kabat).

[0605] -FcγRIII (CD16), binds to IgG with medium to low affinity and exists in two types. FcγRIIIA is present on NK cells, macrophages, eosinophils and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. It was found that FcγRIIIA reduces the binding of antibodies such as those comprising the following IgG Fc region, wherein the IgG Fc region has a mutation in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbered according to the EU index of Kabat).

[0606] The location of the Fc receptor binding site on human IgG1, the mutation sites described above, and methods for determining binding to FcγRI and FcγRIIA are described in Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0607] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example, from about 221 to about 230 (EU numbering system according to Kabat), or from about 226 to about 230 (EU numbering system according to Kabat). Hinge regions of other IgG subclasses can be determined by aligning the hinge region cysteine residues with those of the IgG1 subclass sequence.

[0608] The hinge region is typically a dimer composed of two polypeptides with identical amino acid sequences. The hinge region typically has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 30), wherein X is S or P; or HTCPXCP (SEQ ID NO: 31), wherein X is S or P; or CPXCP (SEQ ID NO: 32), wherein X is S or P.

[0609] In one embodiment, the hinge region is free of internal disulfide bonds. This can be achieved by replacing the cysteine residues with serine residues in the sequence of SEQ ID NO: 32 (and similarly in SEQ ID NOs: 30 and 31), or by deleting the CPXC fragment (SEQ ID NO: 95) from the hinge region of SEQ ID NOs: 30, 31 or 32.

[0610] The term "peptide linker" refers to a linker of natural and / or synthetic origin. A peptide linker is composed of a linear chain of amino acids, wherein the 20 natural amino acids are monomeric building blocks, which are connected by peptide bonds. The chain length is 1 to 50 amino acid residues, preferably 1 to 28 amino acid residues, and particularly preferably 3 to 25 amino acid residues. A peptide linker may contain a repeating amino acid sequence or a sequence of a natural polypeptide. Peptide linkers have the function of ensuring that the domains of a fusion polypeptide can perform their biological activity by allowing the domains to fold and present correctly. Preferably, the peptide linker is a "synthetic peptide linker" and is designated as being rich in glycine, glutamine, and / or serine residues. These residues are arranged in small repeating units of, for example, up to 5 amino acids, such as GGGS (SEQ ID NO: 69), GGGGS (SEQ ID NO: 70), QQQG (SEQ ID NO: 71), QQQQG (SEQ ID NO: 72), SSSG (SEQ ID NO: 73), or SSSSG (SEQ ID NO: 74). This small repeating unit can be repeated two to five times to form a multimeric unit, for example (GGGS)2 (SEQ ID NO: 75), (GGGS)3 (SEQ ID NO: 76), (GGGS)4 (SEQ ID NO: 77), (GGGS)5 (SEQ ID NO: 78), (GGGGS)2 (SEQ ID NO: 79), (GGGGS)3 (SEQ ID NO: 80) or (GGGGS)4 (SEQ ID NO: 81). In one embodiment, the peptide linker is selected from the linkers of SEQ ID NOs: 69 to 82. In one embodiment, each peptide linker is independently selected from the linkers of SEQ ID NOs: 69 to 82. In a preferred embodiment, the peptide linker / each peptide linker (independently of each other) is selected from the linkers of SEQ ID NOs: 75 to 81. Up to six additional, arbitrary, natural amino acids may be added to the amino and / or carboxyl termini of the multimeric unit. Other synthetic peptide linkers can be composed of a single amino acid, repeated 10 to 20 times, and can contain up to six additional natural amino acids at the amino and / or carboxyl termini, such as the serine in the linker GSSSSSSSSSSSSSSSG (SEQ ID NO: 82). All peptide linkers can be encoded by nucleic acid molecules and can therefore be recombinantly expressed. Since the linker itself is a peptide, an antifusogenic peptide can be attached to the linker via a peptide bond formed between the two amino acids.

[0611] "Antibody fragments" are molecules that are not complete antibodies, which comprise a portion of an intact antibody and bind to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2; double-chain antibodies (diabodies); linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 93 / 16185; US 5,571,894 and US 5,587,458.

[0612] Diabodies are antibody fragments with two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 0 404 097; WO 1993 / 01161; Hudson, PJ et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat. Med. 9 (2003) 129-134.

[0613] Single domain antibodies are antibody fragments that contain all or part of an antibody heavy chain variable domain, or all or part of a light chain variable domain. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516).

[0614] Antibody fragments can be prepared by a variety of techniques including, but not limited to, protease digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage).

[0615] The term "antibody fragment" also includes "dual-acting Fab" or "DAF," which contain one antigen-binding site that binds to two different antigens (see, eg, US 2008 / 0069820).

[0616] "Monospecific antibody" refers to an antibody with a single binding specificity for one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2), or combinations thereof (eg, full-length antibodies plus additional scFv or Fab fragments).

[0617] "Multispecific antibody" means an antibody having binding specificity for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies) or combinations thereof (e.g., full-length antibodies plus additional scFv or Fab fragments). Engineered antibodies with two, three or more (e.g., four) functional antigen-binding sites have also been reported (see, e.g., US 2002 / 0004587 A1). A multispecific antibody is a bispecific antibody. Multispecific antibodies can also be prepared by constructing an electrostatic steering effect for generating antibody Fc-heterodimer molecules (WO 2009 / 089004).

[0618] The term "binding" refers to the binding of a binding site to its target, for example, the binding of an antibody binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain to a corresponding antigen. This binding can be achieved using, for example, Assay (GE Healthcare, Uppsala, Sweden). That is, the term "binding (antigen)" can refer to the binding of an antibody in an in vitro assay. In one embodiment, binding is determined in a binding assay in which the antibody is bound to a surface and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). Binding refers to, for example, binding affinity (K D ) is 10 -8 M or less, in certain embodiments 10 -13 to 10 -8 M, in certain embodiments, 10 -13 to 10 -9 M. The term "binding" also includes the term "specific binding".

[0619] Binding can be investigated by BIAcore assay (GE Healthcare Biosensor AB, Uppsala, Sweden). The affinity of binding is measured by the term k a (for antibody / antigen complexes, the association rate constant of the antibody), k d (dissociation constant) and K D (k d / k a ) to define.

[0620] For example, in In one possible embodiment of the assay, the antigen is bound to a surface and binding at the antibody binding site is measured by surface plasmon resonance (SPR). The affinity of the binding is defined by the terms ka (association constant: rate constant for complex formation), kd (dissociation constant: rate constant for complex dissociation), and KD (kd / ka). Alternatively, the binding signal of the SPR sensorgram can be directly compared to the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0621] The term "binding site" refers to any protein entity that exhibits binding specificity to a target. This can be, for example, a receptor, a receptor ligand, anticalin, affibody, antibody, etc. Therefore, the term "binding site" as used herein refers to a polypeptide that can specifically bind to a second polypeptide or can be specifically bound by a second polypeptide. In one embodiment, the binding site is a polypeptide selected from the group consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, a pair of antibody heavy chain and antibody light chain variable domains, a receptor or a functional fragment thereof, a receptor ligand or a functional fragment thereof, an enzyme or a substrate thereof.

[0622] In the case of antibodies, the binding site comprises at least three HVRs (e.g., in the case of VHH) or six HVRs (e.g., in naturally occurring antibodies, i.e., natural antibodies). In general, the antibody amino acid residues responsible for antigen binding will form the binding site. These residues are generally contained in the antibody heavy chain variable domain and the associated (cognate) antibody light chain variable domain pair. The antigen binding site of an antibody comprises amino acid residues from "hypervariable regions" or "HVRs." The "framework" or "FR" region is the variable domain region outside the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise FR1, HVR1 / CDR1, FR2, HVR2 / CDR2, FR3, HVR3 / CDR3, and FR4 regions (immunoglobulin frameworks) from N-terminus to C-terminus. In particular, the HVR3 / CDR3 region of the heavy chain variable domain is the region that contributes most to antigen binding, which defines the binding specificity of the antibody. A "functional binding site" is capable of specifically binding to its target. The term "specific binding" refers to the binding of a binding site to its target in an in vitro assay, in one embodiment a binding assay. Such a binding assay can be any assay that detects a binding event. For example, an assay in which an antibody is bound to a surface and surface plasmon resonance (SPR) is used to measure the binding of the antigen to the antibody. Alternatively, a bridge ELISA can be used. Binding refers to the binding of an antibody (conjugate) to its target with a specific affinity of 10. -8 M or smaller binding affinity (K D ), in certain embodiments 10 -13 to 10 -8 M, in certain embodiments, 10 -13 to 10- 9 M.

[0623] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Several of these classes are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to these different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0624] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The Fc region consists of two heavy chain Fc region polypeptides, which can be covalently linked to each other via interchain disulfide bonds formed by cysteine residues in the hinge region.

[0625] The multimeric polypeptides / binders reported herein may comprise a complete Fc region, in one embodiment, of human origin, but without the hinge region cysteine residues. In one embodiment, the Fc region comprises all portions of a human constant region, but without the hinge region cysteine residues. The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. While the effect of an antibody on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site within the Fc region. This binding site is known in the art and is described in Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324; and EP 0 307 434. This binding site includes, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the EU index of Kabat). Antibodies of IgG1, IgG2 and IgG3 subclasses typically exhibit complement activation, C1q binding and C3 activation, while IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. "Fc region of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of the antibody. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is an Fc region of the human IgG4 subclass, comprising S228P and / or L235E mutations (numbered according to the EU index of Kabat). In one embodiment, the Fc region is an Fc region of the human IgG1 subclass, comprising L234A and L235A and optionally P329G mutations (numbered according to the EU index of Kabat).

[0626] The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains (each chain comprising a light chain variable domain and a light chain constant domain) and two full-length antibody heavy chains (each chain comprising a heavy chain variable domain, a first constant domain, a hinge domain, a second constant domain, and a third constant domain). A full-length antibody may comprise other domains, such as additional scFv or scFab conjugated to one or more chains of the full-length antibody. These conjugates are also encompassed by the term full-length antibody.

[0627] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and progeny derived therefrom (regardless of the number of subcultures). The nucleic acid of the progeny may not be completely identical to that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the original transformed cell are also included herein.

[0628] The term "derived from" means that a variant amino acid sequence is derived from a parent amino acid sequence by introducing an alteration / mutation at at least one position. Thus, the derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position. In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from 1 to 15 amino acid residues at the corresponding positions. In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from 1 to 10 amino acid residues at the corresponding positions. In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from 1 to 6 amino acid residues at the corresponding positions. Likewise, the derived amino acid sequence has a high degree of amino acid sequence identity with its parent amino acid sequence. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 80% or more amino acid sequence identity. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 90% or more amino acid sequence identity. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 95% or more amino acid sequence identity.

[0629] In one embodiment, one or both heavy chain Fc-region polypeptides are derived from the Fc-region polypeptide of SEQ ID NO: 01 and have at least one amino acid mutation or deletion compared to the Fc-region polypeptide of SEQ ID NO: 01. In one embodiment, the Fc-region polypeptide comprises / has about 1 to 10 amino acid mutations or deletions, in one embodiment, about 1 to about 5 amino acid mutations or deletions. In one embodiment, the Fc-region polypeptide has at least 80% homology to the human Fc-region polypeptide of SEQ ID NO: 01. In one embodiment, the Fc-region polypeptide has at least 90% homology to the human Fc-region polypeptide of SEQ ID NO: 01. In one embodiment, the Fc-region polypeptide has at least 95% homology to the human Fc-region polypeptide of SEQ ID NO: 01.

[0630] An Fc-region polypeptide derived from the human Fc-region polypeptide of SEQ ID NO: 01 or 02 or 03 or 04 is further defined by the amino acid changes it contains. Thus, for example, the term P329G refers to an Fc-region polypeptide derived from a human Fc-region polypeptide having a proline to glycine mutation at amino acid position 329 relative to the human Fc-region polypeptide of SEQ ID NO: 01 or 02 or 03 or 04.

[0631] The human IgG1 Fc region polypeptide comprises the following amino acid sequence:

[0632] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:01).

[0633] The following Fc regions are variants derived from the wild-type human IgG1 Fc region.

[0634] An Fc region polypeptide derived from a human IgG1 Fc region having L234A and L235A mutations comprises the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 05).

[0635] An Fc region polypeptide derived from a human IgG1 Fc region having Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0636] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:06).

[0637] An Fc region polypeptide derived from a human IgG1 Fc region having S354C and T366W mutations, comprising the following amino acid sequence:

[0638] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:07).

[0639] An Fc region polypeptide derived from a human IgG1 Fc region having L234A, L235A mutations and Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0640] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:08).

[0641] An Fc region polypeptide derived from a human IgG1 Fc region having L234A, L235A, S354C, and T366W mutations, comprising the following amino acid sequence:

[0642] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:09).

[0643] An Fc region polypeptide derived from a human IgG1 Fc region having a P329G mutation, comprising the following amino acid sequence:

[0644] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:10).

[0645] An Fc region polypeptide derived from a human IgG1 Fc region having L234A, L235A mutations, and P329G mutations, comprising the following amino acid sequence:

[0646] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:11).

[0647] An Fc region polypeptide derived from a human IgG1 Fc region having a P329G mutation and Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0648] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:12).

[0649] An Fc region polypeptide derived from a human IgG1 Fc region having a P329G mutation and S354C and T366W mutations, comprising the following amino acid sequence:

[0650] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:13).

[0651] An Fc region polypeptide derived from a human IgG1 Fc region having L234A, L235A, P329G and Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0652] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:14).

[0653] An Fc region polypeptide derived from a human IgG1 Fc region having L234A, L235A, P329G mutations and S354C, T366W mutations, comprising the following amino acid sequence:

[0654] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:15).

[0655] The human IgG4 Fc region polypeptide comprises the following amino acid sequence:

[0656] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:04).

[0657] The following Fc regions are variants derived from the wild-type human IgG4 Fc region.

[0658] An Fc region polypeptide derived from a human IgG4 Fc region having S228P and L235E mutations, comprising the following amino acid sequence:

[0659] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:16).

[0660] An Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, and P329G mutations, comprising the following amino acid sequence:

[0661] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:17).

[0662] An Fc region polypeptide derived from a human IgG4 Fc region having S354C and T366W mutations, comprising the following amino acid sequence:

[0663] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:18).

[0664] An Fc region polypeptide derived from a human IgG4 Fc region having Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0665] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:19).

[0666] An Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, S354C, and T366W mutations, comprising the following amino acid sequence:

[0667] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:20).

[0668] An Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E and Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0669] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:21).

[0670] An Fc region polypeptide derived from a human IgG4 Fc region having a P329G mutation, comprising the following amino acid sequence:

[0671] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:22).

[0672] An Fc region polypeptide derived from a human IgG4 Fc region having P329G, Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0673] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:23).

[0674] An Fc region polypeptide derived from a human IgG4 Fc region having P329G, S354C, and T366W mutations, comprising the following amino acid sequence:

[0675] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:24).

[0676] An Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, P329G and Y349C, T366S, L368A, and Y407V mutations, comprising the following amino acid sequence:

[0677] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:25).

[0678] An Fc region polypeptide derived from a human IgG4 Fc region having S228P, L235E, P329G and S354C, T366W mutations, comprising the following amino acid sequence:

[0679] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIE KTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL(SEQ ID NO:26).

[0680] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. Alternatively, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to a humanized antibody.

[0681] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that comprises stretches of amino acid residues that are highly variable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or comprise antigen contact residues ("antigen contact points"). Generally, an antibody comprises six HVRs; three in the heavy chain variable domain VH (H1, H2, H3) and three in the light chain variable domain VL (L1, L2, L3).

[0682] HVR includes

[0683] (a) Hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917);

[0684] (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Press 91-3242.);

[0685] (c) antigenic contact points located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0686] (d) a combination of (a), (b) and / or (c), comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0687] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0688] The term "light chain" refers to the shorter polypeptide chain of a native IgG antibody. Antibody light chains are classified into two types, designated kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The human kappa light chain constant domain is shown in SEQ ID NO: 33, and the human lambda light chain constant domain is shown in SEQ ID NO: 34.

[0689] The term "paratope" refers to the portion of a given antibody molecule that is required for specific binding between a target and a binding site. The paratope can be contiguous, i.e., formed by adjacent amino acid residues present in the binding site, or discontinuous, i.e., formed by amino acid residues that are located at different sequential positions in the primary sequence, e.g., the amino acid sequence of HVRs / CDRs, but are close together in the three-dimensional structure adopted by the binding site.

[0690] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0691] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0692] The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, except for the following possible variant antibodies that are usually present in trace amounts, each individual antibody that makes up the group is identical and / or binds to the same epitope, wherein the variant antibody, for example, contains a naturally occurring mutation or appears during the production of a monoclonal antibody product. Unlike polyclonal antibody products (which typically include different antibodies for different determinants (epitopes)), each monoclonal antibody of a monoclonal antibody product is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" represents the characteristic of an antibody obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0693] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable domain (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be one of two types, referred to as κ and λ.

[0694] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no other ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

[0695] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0696] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric, humanized and human) that are prepared, expressed, created or isolated by recombinant means. This includes antibodies isolated from host cells such as NSO, HEK, BHK or CHO cells, or antibodies expressed using recombinant expression plasmids transfected into host cells.

[0697] The term "valent" as used in this application denotes the presence of a specific number of binding sites in an (antibody) molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" denote the presence of two, four, and six binding sites, respectively, in an (antibody) molecule. The bispecific antibodies reported herein are, in a preferred embodiment, "trivalent." The trispecific antibodies reported herein are, in a preferred embodiment, "trivalent."

[0698] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to its antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domains of antibodies that bind to that antigen. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0699] The term "variant" refers to a molecule with an amino acid sequence that is different from the amino acid sequence of the parent molecule. Typically, such a molecule has one or more changes, insertions, or deletions. In one embodiment, a modified antibody or modified fusion polypeptide comprises an amino acid sequence that comprises at least a portion of a non-naturally occurring Fc region. Such molecules have a sequence identity of less than 100% with the parent domain or Fc region. In one embodiment, the variant has an amino acid sequence with about 75% to less than 100% amino acid sequence identity, particularly about 80% to less than 100%, particularly about 85% to less than 100%, particularly about 90% to 100%, and particularly about 95% to less than 100% sequence identity with the amino acid sequence of the parent domain or Fc region. In one embodiment, the parent domain or Fc region and the variant domain or Fc region differ by one (single), two, or three amino acid residues.

[0700] The term "domain swapping" as used herein means that in a pair of antibody heavy chain VH-CH1 fragments and their corresponding cognate antibody light chains, i.e. in the antibody binding arm (i.e. in the Fab fragment), the domain order deviates from the natural order, wherein at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three general types of domain swapping, (i) swapping of CH1 and CL domains, which results in domain swap light chains having the order of VL-CH1 domains, and domain swap heavy chain fragments having the order of VH-CL domains (or full-length antibody heavy chains having the order of VH-CL-hinge-CH2-CH3 domains), (ii) swapping of VH and VL domains, which results in domain swap light chains having the order of VH-CL domains and domain swap heavy chain fragments having the order of VL-CH1 domains, and (iii) domain swapping of complete light chains (VL-CL) and complete VH-CH1 heavy chain fragments ("Fab swapping"), which results in domain swap light chains having the order of VH-CH1 domains and domain swap heavy chain fragments having the order of VL-CL domains (all domain orders described above are indicated in N-terminal to C-terminal direction).

[0701] As used herein, the term "interchangeable" refers to the aforementioned domain crossover for the respective heavy and light chain domains. Thus, when the CH1 and CL domains are "interchangeable," this refers to the domain crossover mentioned in item (i) and the resulting order of the heavy and light chain domains. Thus, when the VH and VL domains are "interchangeable," this refers to the domain crossover mentioned in item (ii); and when the CH1 and CL domains are "interchangeable" and the VH1 and VL domains are "interchangeable," this refers to the domain crossover mentioned in item (iii). Bispecific antibodies comprising domain swapping are described in, for example, WO 2009 / 080251; WO 2009 / 080252; WO 2009 / 080253; WO 2009 / 080254 and Schaefer, W. et al., Proc. Natl. Acad. Sci USA 108 (2011) 11187-11192.

[0702] The multispecific antibodies produced by the methods reported herein may also comprise Fab fragments comprising a domain exchange of the CH1 and CL domains mentioned in item (i) above, or a domain exchange of the VH and VL domains mentioned in item (ii) above. Fab fragments that specifically bind to the same antigen can be constructed to have the same domain order. Thus, if a multispecific antibody comprises multiple Fab fragments with domain exchange, the Fab fragments can specifically bind to the same antigen. II. Production of bi / multispecific antibodies according to the exchange reaction of the present invention

[0703] A) Method for converting monospecific monovalent IgG derivatives into bispecific IgG

[0704] The exchange methods described below can be used to convert monospecific (monovalent) antibodies or antibody fragments into bivalent bispecific antibodies (bsAbs) or to convert existing multispecific antibodies into higher-order multispecific antibodies, such as bispecific antibodies into tri- or tetraspecific antibodies.

[0705] Two non-functional half antibodies (i.e., monospecific for cellular targets only) are used as starting materials. For example, a 2 / 3 IgG can be used. The 2 / 3-IgG consists of a heavy chain with a first set of knob-in-hole (KiH) mutations, a complementary light chain, and an Fc region that is complementary to the Fc region of the heavy chain via a corresponding, complementary second set of knob-in-hole mutations. The complementary Fc region can be, for example, an Fc region heavy chain fragment or a second heavy chain (optionally, without binding specificity). To further facilitate the correct assembly of the desired bispecific (multispecific) antibody, the complementary Fc region contains, in addition to the complementary second set of KiH mutations, additional interfering (destabilizing) repulsive charge mutations. In addition, the complementary Fc region can contain an affinity tag (e.g., a His6 or C tag) for efficient removal of unwanted emissions and by-products after production. The second non-functional monospecific antibody contains complementary interfering mutations. Once the half antibodies are exchanged with each other, for example, when bound to interact on the cell surface, the two interfering mutations will be converted into attractive mutations.

[0706] The on-cell exchange reaction / method that can be performed using the multimeric molecules according to the present invention comprises the following steps:

[0707] - Bringing a first (starting) multimeric polypeptide (comprising the first and second polypeptides) and a second (starting) polypeptide (comprising the third and fourth polypeptides) into close proximity on the cell surface such that the second and third polypeptides cross-exchange to form a third multimeric polypeptide (comprising the first and fourth polypeptides) and a fourth multimeric polypeptide (comprising the second and third polypeptides).

[0708] in

[0709] i) the second polypeptide comprises a (first interfering) mutation resulting in the first multimeric polypeptide being destabilized compared to a (multimeric) polypeptide that is identical to the first multimeric polypeptide except for said mutation in the second polypeptide,

[0710] ii) the third polypeptide comprises a (second interfering) mutation resulting in the second multimeric polypeptide being destabilized compared to a (multimeric) polypeptide that is identical to the second multimeric polypeptide except for said mutation in the third polypeptide,

[0711] iii) the (first interfering) mutation in the second polypeptide and the (second interfering) mutation in the third polypeptide result in a stabilization of the third (exchanged) multimeric polypeptide comprising the second polypeptide and the third polypeptide compared to the first (starting) multimeric polypeptide and / or the second (starting) multimeric polypeptide,

[0712] iv) the fourth (exchanged) multimeric polypeptide is more stable than the first (starting) multimeric polypeptide and / or the second (starting) multimeric polypeptide,

[0713] v) the first multimeric polypeptide and the second multimeric polypeptide each comprise only a portion of one or both new binding sites that is non-functional (i.e., unable to bind to its target), and

[0714] vi) the third and / or fourth multimeric polypeptide comprises the one or two new binding sites in a functional form, i.e. in a form allowing specific binding to their respective targets, wherein the one or two new functional binding sites are generated / activated by exchanging the second and third polypeptide between the first multimeric polypeptide and the second multimeric polypeptide, i.e. by bringing together non-functional parts of the one or two new binding sites to form the one or two new functional binding sites.

[0715] Thus, the present invention is based, at least in part, on the discovery that the addition of a single (unilateral, non-pairing) destabilizing (interfering) mutation to a (hetero)multimeric polypeptide is sufficient to promote polypeptide chain exchange with a second (hetero)multimeric polypeptide also comprising a single (unilateral, non-pairing) destabilizing (interfering) mutation, because: upon exchange and recombination, the destabilizing mutation is converted into an attractive mutation, and the two newly formed exchanged (hetero)multimeric polypeptides have improved stability (i.e., lower CH3-CH3 binding free energy) compared to the starting (hetero)multimeric polypeptides. The only requirement being that the destabilizing (interfering) mutation is introduced at a position where the interaction can occur once the corresponding polypeptides are bound to each other.

[0716] This method can be applied to any (hetero)multimeric polypeptide that meets the above criteria.

[0717] However, the method according to the invention is particularly useful in the pharmaceutical field.

[0718] Different methods for producing (hetero)multimeric polypeptides are known in the art. Any of these methods can be used, as long as the mutations required for forming the starting (hetero)multimeric polypeptide do not interfere with or overlap with the (interfering) destabilizing mutations required for the exchange reaction according to the present invention.

[0719] Back to the pharmaceutical field, antibodies are the most widely used binders. Antibodies dimerize through the interaction of their constant regions, especially the CH3 domains of their heavy chains.

[0720] Therefore, the present invention is based at least in part on the discovery that introducing a single destabilizing mutation in one CH3 domain of a pair of CH3 domains is sufficient to implement the methods according to the present invention. More specifically, it has been found that introducing a first destabilizing mutation at position 357 in only one CH3 domain of a first starting (hetero)multimeric polypeptide and a second destabilizing mutation at position 370 in only one CH3 domain of a second starting polypeptide can promote spontaneous exchange of polypeptide chains between the two starting (hetero)multimeric polypeptides when these polypeptides are spatially close. One of the resulting exchanged polypeptides comprises a CH3 domain pair having mutations at positions 357 and 370, respectively, resulting in stabilization of the exchanged (hetero)multimer. A similar situation can be achieved by mutations at positions 356 and 439. All positions are numbered according to the EU index as in Kabat. A preferred pair of mutations is E357K and K370E. Another preferred pair of mutations is D356K and K439E. The method according to the invention can be applied to any IgG subclass, such as IgG1, IgG2, IgG3 and IgG4, since the residues mentioned are highly conserved. In a preferred embodiment, the CH3 domain belongs to the IgG1 subclass.

[0721] The present invention is based at least in part on the following discovery: the polypeptide chains of the starting (hetero) multimeric polypeptide do not need to be covalently linked to each other, for example, by disulfide bonds to allow the formation and separation of the starting (hetero) multimeric polypeptide. In more detail, since the starting polypeptide is already a heterodimer, they will include additional mutations for heterodimerization. It has been found that these mutations are sufficient to stabilize the starting heterodimer, even in the presence of specific destabilizing (interfering) single unilateral mutations. Therefore, the covalent linkage of the chains in the starting (hetero) multimeric polypeptide is no longer required. Therefore, in one embodiment, in the case of a starting (hetero) multimeric polypeptide comprising a hinge region, these hinge regions comprise C226S and C229S mutations or lack the entire CPXC (SEQ ID NO: 95) sequence (numbered according to the Kabat EU index).

[0722] By eliminating the disulfide bonds between the Fc-region-containing chains of the (hetero)multimeric starting polypeptide, the exchange reaction can be initiated without a reducing agent. This allows the exchange reaction to occur under mild in vivo conditions. Furthermore, other disulfide bonds may be present in the starting (hetero)multimeric polypeptide, such as in a Fab fragment, as long as they do not interfere with the exchange reaction (covalently binding the CH3 domain-containing polypeptides together).

[0723] The present invention is based at least in part on the discovery that exchanged (hetero)multimeric polypeptides, such as those containing only functional and target binding sites, can be further stabilized by disulfide bond formation only after the exchange reaction. For example, well-established mutations for forming (hetero)multimeric antibodies are knobs-into-hole mutations. These mutations exist in two variants: without additional disulfide bonds and with additional disulfide bonds. Thus, an alternative starting (hetero)multimeric polypeptide comprises a knobs-into-hole mutation for forming the starting (hetero)multimeric polypeptide, and the knobs-into-hole cysteine residue is only provided in the polypeptide chain containing the target binding site. Thus, only in the exchanged (hetero)multimeric polypeptide containing two target binding sites are the two cysteine residues located in corresponding matching positions required for disulfide bond formation present. Thus, disulfide bonds are only formed in the exchanged product. This results in further stabilization of the exchanged (hetero)multimeric polypeptide of interest, thereby preventing dissociation and / or reverse reactions.

[0724] As used herein, the term "(hetero)multimer" refers to a polypeptide comprising at least two polypeptide chains that are partially or completely different in amino acid sequence and that meet the requirements of the present invention. The term also encompasses polypeptides comprising three or more polypeptide chains, as long as at least two of them are (hetero)multimers according to the present invention. Thus, the term "multimer" refers to a polypeptide comprising at least three polypeptide chains, at least two of which are (hetero)multimers and that meet the requirements of the present invention.

[0725] The term "interference mutation" refers to a mutation that causes the (hetero)dimeric polypeptide to destabilize. This destabilization is usually achieved by changing the charge of the amino acid residues, for example, by exchanging a positively charged amino acid residue with a negatively charged amino acid residue, or vice versa. This exchange can result in similar charges at the interaction positions of the CH3-CH3 domains, and thus lead to charge repulsion. A pair of preferred mutations are E357K and K370E. Another pair of preferred mutations are D356K and K439E. In addition, the method according to the present invention can be applied to any IgG subclass, such as IgG1, IgG2, IgG3 and IgG4, because these residues are highly conserved. In a preferred embodiment, the CH3 domain belongs to the IgG1 subclass.

[0726] WO 2009 / 089004 (incorporated herein by reference) discloses a method for evaluating the effect of CH3 domain mutations on dimer stability. It outlines how to use EGAD software to estimate CH3-CH3 domain binding free energy (see also Pokala, N. and Handel, TM, J. Mol. Biol. 347 (2005) 203-227, which is incorporated herein by reference in its entirety):

[0727] EGAD can be used to roughly compare the binding free energies of various mutations made at the CH3 domain interface. The binding free energy of a mutant is defined as ΔΔGmut = μ(ΔGmut - ΔGwt) (mut = mutant, wt = wild type). Here, μ (typically = 0.1) is a scaling factor used to normalize the expected change in binding affinity to a slope of 1 when compared to the experimental energy. The dissociation free energy (ΔG) is defined as the energy difference between the complex (ΔGbound) and the free state (ΔGfree).

[0728] The present invention is exemplified below using a specific, exemplary starting material, 2 / 3-IgG. This is presented as an example of the general underlying concept and should not be construed as limiting the present invention. The true scope of the invention is set forth in the claims.

[0729] Figure 1 The design and modular composition of 2 / 3-IgG used as an exemplary starting compound in the method according to the present invention are shown. 2 / 3-IgG is composed of three separate chains: a light chain (usually a full-length light chain comprising a light chain variable domain and a light chain constant domain), a heavy chain (usually a full-length heavy chain comprising a heavy chain variable domain and all heavy chain constant domains, including a hinge region with or without cysteine residues), and a complementary heavy chain Fc region polypeptide (usually a heavy chain Fc region fragment, comprising at least a portion of a hinge and CH2-CH3, with no cysteine residues in the hinge region). The variable domains of the light and heavy chains constitute a functional binding site, i.e., a VH / VL pair.

[0730] The design and modular composition of 2 / 3-BiFabs used as exemplary starting compounds in the methods according to the present invention are also similar. 2 / 3-BiFabs are composed of three separate chains: a light chain (typically a full-length light chain comprising a light chain variable domain and a light chain constant domain), a heavy chain (typically a full-length heavy chain comprising a first heavy chain variable domain, a CH1 domain, a second variable domain, and a CH3 domain, including a hinge region with or without cysteine residues), and a complementary heavy chain polypeptide (typically a heavy chain fragment comprising a hinge-variable domain-CH3, with or without cysteine residues in the hinge region). The variable domain of the light chain and the first variable domain of the heavy chain form a functional binding site, i.e., a VH / VL pair, while the second variable domain of the heavy chain and the variable domain of the complementary heavy chain Fc region polypeptide also form a VH / VL pair, which is typically non-functional, i.e., has no binding ability.

[0731] The heavy chain (typically of the human IgG1 subclass) comprises i) a knob mutation or a hole mutation (the T366W mutation in the CH3 domain of the antibody heavy chain is denoted as a "knob mutation" and the T366S, L368A and Y407V mutations in the CH3 domain of the antibody heavy chain are denoted as a "hole mutation" (numbering according to the Kabat EU index), or ii) a knob-cys- mutation or a hole-cys- mutation (the T366W and S354C mutations in the CH3 domain of the antibody heavy chain are denoted as a "knob-cys- mutation" and the T366S, L368A, Y407V, Y349C mutations in the CH3 domain of the antibody heavy chain are denoted as a "hole-cys- mutation"; the reversal is also possible: T366W / Y349C and T366S / L368A / Y407V / S354C in the CH3 domain (numbering according to the Kabat EU index) can achieve the formation of knob-into-hole Fc region heterodimers.

[0732] The complementary heavy chain Fc region polypeptide or complementary heavy chain polypeptide may also be denoted as "dummy-Fc" or "dummy-HC", i.e., an IgG1 derivative lacking VH and CH1, starting at the N-terminus with a hinge region sequence (or a fragment thereof), followed by a CH2 domain or a variable domain, followed by a CH3 domain, and optionally comprising a purification tag at its C-terminus, such as a His6 or His8 or C tag. In addition, such a complementary polypeptide comprises a knob mutation or a hole mutation in its CH3 domain, depending on the mutation in the heavy chain. In addition to the knob mutation or the hole mutation, the complementary polypeptide comprises at least one interfering (i.e., destabilizing) mutation that introduces one (i.e., a single additional) or multiple charges that are repulsive relative to the wild-type sequence. For example, a D356K or E357K mutation, respectively, is combined with a K370E or K439E mutation (see Figure 2 ). This "mutant heavy chain Fc region polypeptide" is denoted as MHC FcRP in the following.

[0733] The heavy chain and MHC FcRP can form two types of heterodimers, depending on the distribution of knob-in-hole mutations:

[0734] i) heavy chain-knob::MHCFcRP-hole, and

[0735] ii) Heavy chain-hole::MHCFcRP-knob.

[0736] Thus, 2 / 3-IgG and 2 / 3-BiFabs are heteromers with bound light chains, i.e., heterotrimers. However, this is "defective" because the charge mutations in MHC FcRP have no matching counterparts in the heavy chain, and if present in the heavy chain, the charge-interfering mutations in MHC FcRP have no matching heavy chain counterparts.

[0737] 2 / 3-IgG is a monovalent, non-dimeric / aggregated, one-arm antibody derivative that can be expressed and purified with a yield similar to that of normal IgG (see Figure 3). This ensures the monovalence of the starting material. If bivalent 2 / 3-IgG is used, it can be monospecific and bispecific.

[0738] However, the polypeptides constituting these defective 2 / 3-IgGs can be rearranged into bispecific antibodies, e.g. Figure 4 shown.

[0739] The exchange reaction between the two starting molecules is driven by the better complementarity of the KiH (knob-in-hole) heavy chains (H chains) (no charge repulsion and, optionally, disulfide bond formation if free cysteine residues are present) and the better complementarity of the two MHC FcRPs. In the reaction, the Fc region complex of the two starting molecules dissociates and exchanges polypeptides to form two more favorable complexes. This drives the following reaction:

[0740] 2 / 3-IgG(A)-tag+2 / 3-IgG(B)-tag

[0741] (Starting heterodimeric polypeptide)

[0742] →

[0743] bsAb(AB)+MHCFcRP(A)-MHCFcRP(B)-tag

[0744] (Exchanged heterodimeric polypeptides).

[0745] In such Figure 4 In the example shown, the starting 2 / 3 heavy chains of IgG A (knob-cys) and the starting 2 / 3 heavy chains of IgG B (hole-cys) form matched bispecific antibody heterodimers (2xHC+2xLC).

[0746] When using 2 / 3-IgG without hinge-disulfide bonds in an on-cell / in vivo setting, no reduction step is required. Chain rearrangements occur spontaneously.

[0747] The same applies to 2 / 3-BiFabs.

[0748] See Examples 1 to 5.

[0749] B) Methods for converting monovalent and / or bivalent mono- or bispecific IgG derivatives into bi-, tri- or tetravalent bi-, tri- or tetraspecific antibodies

[0750] With the method according to the invention, not only can different binding specificities be combined, but these combinations can also be produced simultaneously in different formats and with different valencies. This can be achieved by expanding the starting materials used in the method described in the previous section.

[0751] For example, starting from the 2 / 3 IgG described in the previous section, the MHC FcRP remains unchanged, but the heavy chains are used in different formats. Such formats can be, for example, chains with one binding site at the C-terminus or N-terminus or with two binding sites (one at the N-terminus and one at the C-terminus) (see e.g. Figure 9 and 10 ).

[0752] In this example, in the method according to the invention, different starting formats (e.g., with an N-terminal binding site, a C-terminal binding site, or both an N- and a C-terminal binding site) are combined with each other, allowing the generation of different antibody formats (nine in the present example, see Figure 11 ).

[0753] For the generation of multispecific antibodies, the crossover-driven principle (converting defective input molecules into matching output molecules) remains unchanged. The MHC FcRP is also retained. Therefore, only the heavy chain is altered.

[0754] For example, Figure 1 and 9 -10 shows three different starting molecules (2 / 3-IgG with N-terminal, C-terminal, and N- and C-terminal binding sites) that have been combined with one another in an exchange reaction, i.e., in the method according to the invention, to generate nine different bispecific formats. These differ in valency, geometry, and position of the individual binding sites.

[0755] Without being bound by theory, it is believed that the exchange reaction based on the transient separation of two different 2 / 3 IgG or 2 / 3-BiFab defective heteromultimers should result in a product containing a preferentially matched Fc region heterodimer. Thus, this exchange converts the 2 / 3-IgG into a four-chain IgG (different format) and the corresponding Fc region heterodimer.

[0756] If hinge disulfide bonds are present in vitro, the exchange reaction can be initiated by a reduction step to break the interchain hinge disulfide bonds, whereas this can be omitted in cells / in vivo where hinge disulfide bonds are absent. Chain rearrangements occur spontaneously.

[0757] See Examples 6 and 7.

[0758] C) Reaction without Fc-Fc interchain disulfide bonds and without reduction step

[0759] The inter-heavy chain disulfide bonds stabilize the antibody and define the flexibility of the Fab arms connected to the hinge. Exchange methods for starting antibodies containing the hinge region require reducing these disulfide bonds before or during the exchange reaction and removing the reducing agent after the exchange reaction is complete (see Examples 3 and 7).

[0760] Therefore, we report here the exchange reactions of starting molecules that have hinge regions but no interchain disulfide bonds.

[0761] As an example of this, 2 / 3 IgG and 2 / 3-BiFabs have been produced in which all disulfide bonds in the Fc region have been eliminated. It has been found that even without these interchain disulfide bonds, such disulfide-deficient 2 / 3 molecules can be produced and purified in an efficient manner (see, e.g., Figure 15 20). When such disulfide-deficient 2 / 3 molecules are used as starting molecules, the method according to the present invention can be performed in vivo because a reduction step is no longer necessary, making these molecules particularly suitable for on-cell exchange reactions and in vivo applications. The multispecific antibodies generated from these disulfide-deficient 2 / 3-IgG and 2 / 3-BiFabs are functional and stable, held together by non-covalent Fc-Fc interactions without interchain disulfide bonds (see Figure 15 and Figure 16 ).

[0762] Thus, eliminating Fc-Fc interchain disulfide bonds allows for corresponding Fc region mismatch-driven exchange reactions without the need for reduction and reoxidation, i.e., under physiological conditions. This facilitates both manufacturing and (high-throughput) screening procedures. This also allows domain exchange reactions to occur under physiological conditions (including on the surface of living cells).

[0763] See Example 8.

[0764] In one embodiment, the hinge region is disulfide-free. In the sequence of SEQ ID NO: 32, the disulfide-free hinge region comprises a substitution of a serine residue for a cysteine residue.

[0765] In addition to removing disulfide bonds, the hinge region can also be shortened. By using such modified hinge regions, bispecific antibodies can be obtained that provide different distances between the binding sites (see Figure 51 ). Thus, in one embodiment, the hinge region has the amino acid sequence of SEQ ID NO: 31 (HTCPXCP, X=S or P) or SEQ ID NO: 95 (HTSPXSP, X=S or P) or SEQ ID NO: 94 (HTPAPE; CPXC of SEQ ID NO: 31 is deleted) or DKTHGGGGS (SEQ ID NO: 97).

[0766] III. Method according to the present invention

[0767] Here we report a method for on-cell assembly / half-antibody exchange of two differentially targeted antibody-prodrug derivatives, thereby generating new functions, preferably with therapeutic effects, directly on the cell surface at the desired site of action.

[0768] The methods according to the present invention are exemplified below using specific, exemplary starting materials, namely 2 / 3-IgG and 2 / 3-BiFab. These are presented merely as examples of the general underlying concepts and should not be construed as limiting the present invention. The true scope of the invention is set forth in the claims.

[0769] A) Monovalent monospecific antibodies are converted to bivalent IgG on cells

[0770] Compared with bivalent antibodies, monovalent antibodies can show that the binding ability to cell surface is reduced. Not subject to theoretical constraints, it is believed that its reason is that the apparent affinity to cell surface caused by bivalence enhances, i.e., avidity effect (avidity effect). By combining identical antigen / epitope with two binding sites / two arms of antibody, it is possible to achieve the combination and / or retention of avidity enhancement on the cell surface. In this monospecific arrangement, compared with the cell with low antigen density (reducing the possibility of two-arm combination), bivalent combination can increase the binding specificity of the cell carrying a large amount of associated antigens (increasing the possibility of two-arm combination).

[0771] Avidity-enhanced binding and / or retention on the cell surface can also be achieved by binding different antigens with different binding sites / different arms of a bispecific antibody. Without being bound by theory, it is believed that in this bispecific setting, bivalent binding results in increased binding specificity for cells that present and / or express two antigens (two binding sites / two arms binding) compared to cells that present and / or express only one antigen (only one binding site / one arm can bind).

[0772] The art for addressing avidity-mediated binding improvements employs preformed bsAbs that recognize two targets. To achieve specificity, each monovalent arm must have a sufficiently low affinity to avoid sufficient binding strength resulting from monovalent binding to overwhelm the avidity effect. If this prerequisite is met, bsAbs can bind to cells that present and / or express both antigens with increased specificity.

[0773] One drawback of currently available affinity-driven binding improvement concepts is that the density of the two antigens on the cell surface may not be an essential prerequisite for the binding of preformed bsAbs themselves (as long as both are present and accessible). Antigen density may only determine the amount of bsAb bound to cells expressing the antigen.

[0774] Therefore, entities with very high potency (and / or potential toxicity issues) carry the risk of binding to—and affecting—cells presenting / expressing both high and low levels of both target antigens. For example, bsAbs with inherent or additive cytotoxic functions may affect not only tumor cells displaying high antigen levels, but also non-target normal cells displaying low antigen levels.

[0775] The present invention relates to a method for converting monovalent monospecific antibodies on cells by bispecific antibodies, which can solve the above problems because the exchange reaction requires the physical interaction of two monospecific exchange partners. The probability of this interaction depends on concentration, i.e., the probability of interaction and exchange is low during low concentration, and the probability of interaction and exchange is higher during increased concentration. Therefore, for example, separate (continuous) application of monospecific antibodies with different specificities will result in the increase accumulation (with low affinity) of two monospecific components on cells showing a larger amount of two antigens. Thus, this cell will not only accumulate each monospecific antibody that increases concentration, but will more effectively convert them into bsAbs. Then, these bsAbs will be retained on the target cell due to the affinity binding of its two binding sites to cell surface antigens, and the unexchanged precursor antibody will dissociate.

[0776] B) Generation of BiFabs with prodrug function and conversion to functional TriFabs in cells

[0777] The driving force behind the exchange reaction reported here is the conversion of two input molecules with "defective" CH3 interfaces into two products with matching CH3 interfaces. The design of these CH3 interfaces dictates the composition of the MHC FcRPs. MHC FcRPs comprise a CH2 domain and a CH3 domain. However, all mutations that contribute to the specific composition of MHC FcRPs are located in the CH3 domain. Therefore, the CH2 domain of an MHC FcRP, as well as the CH2 domain of the relevant heavy chain, can be replaced with other, distinct domains. These must still allow (or even support) heavy chain-MHC FcRP heterodimerization to generate "defective" molecules.

[0778] The possibility of replacing the CH2 domain of IgG class molecules with other domains capable of heterodimerization has been demonstrated in the form of TriFab (see WO 2016 / 087416; Figure 18By swapping the CH2 domains for VH and VL, respectively, TriFabs exhibit bispecific functionality. The Fc-like "stem" of these molecules is held together by an intact KiH CH3 domain. Because the KiH CH3 domain is compatible with the modified CH3 domain of MHC FcRP, this also enables the generation of MHC FcRP-containing 2 / 3-BiFab analogs with swap-permitting properties.

[0779] TriFabs can have a functional Fv replacing the CH2 domain of the Fc region, wherein one CH2 domain is replaced by a VH domain and the other by a complementary VL domain. 2 / 3-BiFab derivatives can comprise MHC FcRPs with irrelevant, i.e., non-cognate VH or VL domains (in the former CH2 position), i.e., these domains do not bind to the target. 2 / 3-BiFabs can retain one functional monovalent binding arm and contain half of the third binding site in the stem region (see Figure 18 The exchange reaction of two complementary 2 / 3-BiFab molecules not only reconstitutes the TriFab form, but also results in the reconstitution of an additional third binding function (CH2 replacement) at the "stem position." Thus, the exchange reaction converts two 2 / 3-BiFab prodrugs into fully functional TriFabs.

[0780] Eliminating the Fc-Fc interchain disulfide bonds (hinge region and CH3 domain) of the aforementioned 2 / 3-IgG allows for MHC FcRP-driven exchange reactions without the need for controlled reduction and reoxidation. Thus, exchange reactions of this molecule can occur under physiological conditions, particularly those in which a single (monospecific) entity is bound to the target cell surface. Applying the same principle, 2 / 3-BiFabs can accumulate on target cells after binding of their functional Fab arms to their functional binding sites. If two complementary 2 / 3-BiFabs (both carrying binding-inactive but complementary stem Fvs) bind to the surface of the same cell, chain exchange reactions occur directly on the cell surface. This exchange occurs on the cell / in situ / in vivo, i.e., directly on the cell surface at the intended site of action, yielding a fully functional TriFab with at least dual or even triple specificity directly on the cell surface, i.e., at the site of action. The activation principle of the 2 / 3-BiFab-derived prodrug will result in binding function only on the surface of cells expressing one or more target antigens at sufficient density. This results in functionality only on the desired cells (including, very high efficacy and / or potential PK or toxicity issues).

[0781] Using the methods of the present invention, low affinity monospecific binders can be used to generate high affinity avidity binders directly on cells and simultaneously activate therapeutic binding sites.

[0782] IV. Multispecific Molecules Used in the Methods of the Invention

[0783] A) Multimeric polypeptides

[0784] The multimeric polypeptides used in the on-cell exchange reaction / method according to the present invention are defined as follows:

[0785] - comprising a first polypeptide and a second polypeptide, wherein each polypeptide comprises an immunoglobulin CH3 domain and a non-functional partial binding site, wherein the non-functional partial binding site is located at the N-terminus or C-terminus of the CH3 domain of both polypeptides; and wherein at least one of the polypeptides comprises a functional binding site that specifically binds to a cell surface target, preferably a non-internalized cell surface target,

[0786] - comprises a (interfering) mutation in only one CH3 domain, resulting in a destabilized multimeric polypeptide compared to a polypeptide which is identical to the destabilized multimeric polypeptide except for said mutation in the second polypeptide,

[0787] - contain mutations required for heterodimer formation, and

[0788] - There is no disulfide bond between the first and second polypeptides.

[0789] Thus, the present invention is based, at least in part, on the discovery that the addition of a single (unilateral, non-pairing) destabilizing (interfering) mutation to a (hetero)multimeric polypeptide is sufficient to promote polypeptide chain exchange with a second (hetero)multimeric polypeptide also comprising a single (unilateral, non-pairing) destabilizing (interfering) mutation, since the two newly formed exchanged (hetero)multimeric polypeptides have improved stability (i.e., lower CH3-CH3 binding free energy) compared to the starting (hetero)multimeric polypeptides due to the conversion of the destabilizing mutation into an attractive mutation after exchange and recombination. The only requirement being that the destabilizing (interfering) mutation be introduced at a position where the corresponding polypeptides can interact with each other when bound to each other.

[0790] This method can be applied to any (hetero)multimeric polypeptide that meets the above criteria.

[0791] Thus, the multimeric polypeptide used in the method according to the invention may comprise further domains or polypeptides.

[0792] In one embodiment, the multimeric polypeptide according to the present invention further comprises an immunoglobulin CH2 domain in the first and second polypeptides, at the (direct) N-terminus or (direct) C-terminus of the CH3 domain. In a preferred embodiment, the additional immunoglobulin CH2 domain is at the N-terminus of the CH3 domain.

[0793] In one embodiment, the multimeric polypeptide comprises a hinge region and C226S and C229S mutations, or a hinge region without the CPXC (SEQ ID NO: 95) sequence (numbering according to the Kabat EU index), or no hinge region.

[0794] Different methods for producing (hetero)multimeric polypeptides are known in the art. Any of these methods can be used, as long as the mutations required to form the starting (hetero)multimeric polypeptide do not interfere with or overlap with the (interfering) destabilizing mutations required for the exchange reaction according to the present invention.

[0795] Therefore, the present invention is based at least in part on the discovery that, in order to perform the method according to the present invention, it is sufficient to introduce a single destabilizing mutation in one CH3 domain of a pair of CH3 domains. More specifically, it has been found that introducing a first destabilizing mutation at position 357 in only one CH3 domain of a first starting (hetero)multimeric polypeptide and a second destabilizing mutation at position 370 in only one CH3 domain of a second starting polypeptide can promote spontaneous exchange of polypeptide chains between the two starting (hetero)multimeric polypeptides when the two starting polypeptides are in close spatial proximity. One of the resulting exchanged polypeptides comprises a CH3 domain pair having mutations at positions 357 and 370, respectively, resulting in stabilization of the exchanged (hetero)multimer. A similar situation can be achieved by mutations at positions 356 and 439. All positions are numbered according to the EU index as in Kabat. A preferred pair of mutations is E357K and K370E. Another preferred pair of mutations is D356K and K439E. The method according to the invention can be applied to any IgG subclass, such as IgG1, IgG2, IgG3 and IgG4, since these said residues are highly conserved.In a preferred embodiment, the CH3 domain is of the IgG1 subclass.

[0796] In one embodiment, the multimeric polypeptide comprises

[0797] - a first polypeptide comprising

[0798] i) from the N-terminus to the C-terminus, a) a first antibody variable domain selected from a pair of antibody light chain variable domain and heavy chain variable domain that specifically bind to a first target, and b) a first human immunoglobulin G CH3 domain,

[0799] The first CH3 domain comprises

[0800] a) T366W mutation, or

[0801] b) T366S / L368A / Y407V mutations,

[0802] and

[0803] ii) a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to the second target, located at the N-terminus of the first antibody variable domain or the C-terminus of the first CH3 domain

[0804] - a second polypeptide comprising

[0805] i) from the N-terminus to the C-terminus, a) a second antibody variable domain selected from a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to a third target, and b) a second human immunoglobulin G CH3 domain,

[0806] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0807] as well as

[0808] The second CH3 domain comprises

[0809] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0810] b) T366W mutation, if the first CH3 domain contains T366S / L368A / Y407V mutations,

[0811] as well as

[0812] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, whereby the first CH3 domain comprises

[0813] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0814] b) amino acid residue K at position 370, if the interfering mutation is E357K, or

[0815] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0816] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0817] and

[0818] ii) optionally, a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to the second or fourth target, located at the N-terminus of the second antibody variable domain or the C-terminus of the second CH3 domain,

[0819] All numbering herein is according to the Kabat EU index.

[0820] In one embodiment, the multimeric polypeptide comprises

[0821] - a first polypeptide comprising

[0822] i) from N-terminal to C-terminal direction, a) a first human immunoglobulin G CH3 domain, and b) a first antibody variable domain selected from a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to a first target,

[0823] The first CH3 domain comprises

[0824] a) T366W mutation, or

[0825] b) T366S / L368A / Y407V mutations,

[0826] and

[0827] ii) a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to a second target, located at the N-terminus of the first CH3 domain or the C-terminus of the first variable domain,

[0828] - a second polypeptide comprising

[0829] i) from N-terminal to C-terminal direction, a) a second human immunoglobulin G CH3 domain, and b) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a third target,

[0830] wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain, or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain,

[0831] as well as

[0832] The second CH3 domain comprises

[0833] a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or

[0834] b) T366W mutation, if the first CH3 domain contains T366S / L368A / Y407V mutations,

[0835] as well as

[0836] wherein the second CH3 domain comprises an interfering mutation selected from D356K, E357K, K370E and K439E, whereby the first CH3 domain comprises

[0837] a) amino acid residue K at position 439, if the interfering mutation is D356K, or

[0838] b) amino acid residue K at position 370, if the interfering mutation is E357K, or

[0839] c) amino acid residue E at position 357, if the interfering mutation is K370E, or

[0840] d) amino acid residue D at position 356, if the interfering mutation is K439E,

[0841] and

[0842] ii) Optionally, a pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second or fourth target are located at the N-terminus of the second CH3 domain or the C-terminus of the second variable domain,

[0843] All numbering herein is according to the Kabat EU index.

[0844] In one embodiment of all aspects of the invention the first polypeptide and the second polypeptide are a non-covalent dimer.

[0845] In one embodiment of all aspects of the invention the first variable domain and the second variable domain form a non-functional binding site.

[0846] In one embodiment of all aspects of the invention the first and second polypeptides each comprise the amino acid sequence DKTHTSPPS (SEQ ID NO: 66) or DKTHT (SEQ ID NO: 94) or GGGS (SEQ ID NO: 69) or DKTHGGGGS (SEQ ID NO: 97) located at the N-terminus of the first and second variable domains.

[0847] In one embodiment of all aspects of the invention

[0848] i) the first CH3 domain comprises a T366W mutation and amino acid residue K at position 439,

[0849] and the second CH3 domain comprises a D356K interfering mutation and a T366S / L368A / Y407V mutation, or

[0850] ii) the first CH3 domain comprises a T366W mutation and an amino acid residue K at position 370,

[0851] and the second CH3 domain comprises an E357K interfering mutation and T366S / L368A / Y407V mutations, or

[0852] iii) the first CH3 domain comprises T366S / L368A / Y407V mutations and amino acid residue E at position 357,

[0853] and the second CH3 domain comprises a K370E interfering mutation and a T366W mutation, or

[0854] iv) the first CH3 domain comprises T366S / L368A / Y407V mutations and amino acid residue D at position 356,

[0855] and the second CH3 domain contains a K439E interfering mutation and a T366W mutation.

[0856] In one embodiment of all aspects of the invention the first, second and third targets are different.

[0857] In one embodiment of all aspects of the invention the first target or the third target is human CD3.

[0858] In one embodiment of all aspects the second and fourth target (if present) are the same or different and are both located on the surface of the same (target) cell.

[0859] In one embodiment of all aspects of the invention the pair of antibody light and heavy chain variable domains that specifically bind to a second target is selected from the group consisting of Fv, scFc, Fab, scFab, dsscFab, CrossFab, bispecific Fab, sdAb and VHH.

[0860] In one embodiment of all aspects of the invention, the pair of antibody light chain and heavy chain variable domains that specifically bind to the fourth target, independent of the pair of antibody light chain variable domains and antibody heavy chain variable domains that specifically bind to the second target, is selected from the group consisting of Fv, scFc, Fab, scFab, dsscFab, CrossFab, bispecific Fab, sdAb and VHH.

[0861] In one embodiment of all aspects of the invention the first and second polypeptides further comprise an immunoglobulin G CH2 domain located N-terminally to the CH3 domain.

[0862] In one embodiment of all aspects of the invention the human immunoglobulin G is human IgG1 or human IgG2 or IgG3 or human IgG4.

[0863] B) Compositions for use in the methods of the present invention

[0864] The on-cell exchange reaction / method that can be performed with a composition comprising two multimeric molecules according to the present invention comprises the following steps

[0865] - bringing a first (starting) multimeric polypeptide (which comprises a first polypeptide and a second polypeptide) and a second (starting) multimeric polypeptide (which comprises a third polypeptide and a fourth polypeptide) into proximity on the cell surface, whereby the second and third polypeptides cross-exchange to form a third multimeric polypeptide and a fourth multimeric polypeptide,

[0866] in

[0867] i) the second polypeptide comprises a (first interfering) mutation resulting in the first multimeric polypeptide being destabilized compared to a (multimeric) polypeptide that is identical to the first multimeric polypeptide except for said mutation in the second polypeptide,

[0868] ii) the third polypeptide comprises a (second interfering) mutation resulting in the second multimeric polypeptide being destabilized compared to a (multimeric) polypeptide that is identical to the second multimeric polypeptide except for said mutation in the third polypeptide,

[0869] iii) the (first interfering) mutation in the second polypeptide and the (second interfering) mutation in the third polypeptide result in a stabilization of the third (exchanged) multimeric polypeptide comprising the second polypeptide and the third polypeptide compared to the first (starting) multimeric polypeptide and / or the second (starting) multimeric polypeptide,

[0870] iv) the fourth (exchanged) multimeric polypeptide is stable compared to the first (starting) multimeric polypeptide and / or the second (starting) multimeric polypeptide,

[0871] v) the first multimeric polypeptide and the second multimeric polypeptide each comprise only a portion of one or both new binding sites that is non-functional (i.e., unable to bind to its target), and

[0872] vi) the third and / or fourth multimeric polypeptide comprises the one or two new binding sites in a functional form, i.e. a form allowing specific binding to the respective target, wherein these functional forms of the binding sites have been generated / activated by performing a polypeptide exchange between the first and the second multimeric polypeptide, i.e. bringing together non-functional parts of the one or two new binding sites to form one or two new functional binding sites.

[0873] One aspect of the present invention is a composition comprising a first multimeric polypeptide according to the present invention and a second multimeric polypeptide according to the present invention, wherein

[0874] The first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide comprise a T366W mutation or a T366S / L368A / Y407V mutation,

[0875] and

[0876] The second CH3 domain of the first multimeric polypeptide comprises a D356K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K439E mutation,

[0877] or

[0878] The second CH3 domain of the first multimeric polypeptide comprises an E357K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K370E mutation,

[0879] and

[0880] The first antibody variable domain of the first multimeric polypeptide and the first antibody variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to the first target,

[0881] as well as

[0882] The second antibody variable domain of the first multimeric polypeptide and the second antibody variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to the third target

[0883] and

[0884] The second and fourth targets are independently of each other cell surface antigens.

[0885] In one embodiment of all aspects of the invention the first and / or third target is human CD3.

[0886] In one embodiment of all aspects of the invention, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

[0887] In one embodiment of all aspects of the invention the composition is for use as a medicament.

[0888] C) Fc region variants

[0889] In certain embodiments, according to the present invention, one or more additional amino acid modifications can be introduced into the Fc region of a multimeric polypeptide, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0890] In certain embodiments, the present invention contemplates variant multimeric polypeptides according to the present invention that have certain, but not all, effector functions, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / lack of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the multimeric polypeptides according to the present invention lack FcγR binding (and therefore may lack ADCC activity), but retain FcRn binding ability. The primary cells that mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in US 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA) and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in the animal model disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the antibody lacks CDC activity due to its inability to bind C1q (see, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., SB et al., Int. Immunol. 18 (2006: 1759-1769).

[0891] Multimeric polypeptides comprising an Fc region with reduced effector function according to the present invention include multimeric polypeptides having substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( US 6,737,056 ). Such Fc region mutants include Fc region mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc region mutants having substitutions of residues 265 and 297 to alanine ( US 7,332,581 ).

[0892] Certain multimeric polypeptides according to the invention comprise Fc region variants with improved or reduced binding to FcRs (see, eg, US 6,737,056; WO 2004 / 056312 and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0893] In certain embodiments, a multimeric polypeptide according to the invention comprises an Fc region variant having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0894] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0895] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593, and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc region variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0896] For further examples of Fc region variants, see Duncan, AR and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94 / 29351.

[0897] In one embodiment of all aspects the multimeric polypeptide according to the invention comprises (all positions according to the EU index as in Kabat)

[0898] i) an Fc region of human IgG1 subclass having P329G, L234A and L235A mutations in both Fc region polypeptides, or

[0899] ii) an Fc region of human IgG4 subclass having P329G, S228P and L235E mutations in both Fc region polypeptides, or

[0900] iii) an Fc-region of human IgG1 subclass having P329G, L234A, L235A, I253A, H310A and H435A mutations in both Fc-region polypeptides, or P329G, L234A, L235A, H310A, H433A and Y436A mutations in both Fc-region polypeptides, or

[0901] iv) a heterodimeric Fc region of human IgG1 subclass, wherein the two Fc region polypeptides comprise P329G, L234A and L235A mutations, and

[0902] a) one Fc region polypeptide comprises a T366W mutation and the other Fc region polypeptide comprises T366S, L368A and Y407V mutations, or

[0903] b) one Fc region polypeptide comprises T366W and Y349C mutations and the other Fc region polypeptide comprises T366S, L368A, Y407V and S354C mutations, or

[0904] c) one Fc region polypeptide comprises T366W and S354C mutations, and the other Fc region polypeptide comprises T366S, L368A, Y407V, and Y349C mutations,

[0905] or

[0906] v) a heterodimeric Fc region of human IgG4 subclass, wherein the two Fc region polypeptides comprise P329G, S228P and L235E mutations, and

[0907] a) one Fc region polypeptide comprises a T366W mutation and the other Fc region polypeptide comprises T366S, L368A and Y407V mutations, or

[0908] b) one Fc region polypeptide comprises T366W and Y349C mutations and the other Fc region polypeptide comprises T366S, L368A, Y407V and S354C mutations, or

[0909] c) one Fc region polypeptide comprises T366W and S354C mutations, and the other Fc region polypeptide comprises T366S, L368A, Y407V, and Y349C mutations,

[0910] or

[0911] vi) A combination of one of i), ii) and iii) with one of iv) and v).

[0912] In one embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention comprising a CH3 domain comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). In one embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention comprising a CH3 domain comprises an additional C-terminal glycine residue (G446, numbering according to the Kabat EU index).

[0913] The multimeric polypeptide according to the invention comprises in one embodiment an Fc region of human IgG1 subclass having PVA236, L234A / L235A mutations and / or GLPSS331 (numbering according to the Kabat EU index), or is of the IgG4 subclass. In another embodiment, the multimeric polypeptide according to the invention has the following features: comprising an Fc region of any IgG class, in one embodiment IgG1 or IgG4 subclass, comprising at least one mutation in E233, L234, L235, G236, D270, N297, E318, K320, K322, A327, A330, P331 and / or P329 (numbering according to the EU index of Kabat). In one embodiment, the multimeric polypeptide according to the invention comprises an Fc region of human IgG4 subclass containing an S228P mutation, or an S228P and L235E mutation (Angal, S. et al., Mol. Immunol. 30 (1993) 105-108) (numbering according to the EU index of Kabat).

[0914] The C-terminus of the Fc region polypeptide contained in the multimeric polypeptide according to the present invention can be a complete C-terminus ending with the amino acid residue PGK. The C-terminus can be a shortened C-terminus in which one or both of the C-terminal amino acid residues have been deleted. In a preferred embodiment, the C-terminus is a shortened C-terminus ending with the amino acid residue PG.

[0915] D) Heterodimerization

[0916] Several CH3 modification methods for supporting heterodimerization have been described in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291, which are incorporated herein by reference.

[0917] Typically, in methods known in the art, the CH3 domain of a first heavy chain and the CH3 domain of a second heavy chain are engineered in a complementary manner, such that a heavy chain comprising one engineered CH3 domain no longer homodimerizes with another heavy chain of the same structure (e.g., a first heavy chain engineered with a CH3 domain no longer homodimerizes with another first heavy chain engineered with a CH3 domain; a second heavy chain engineered with a CH3 domain no longer homodimerizes with another second heavy chain engineered with a CH3 domain). Thus, a heavy chain comprising one engineered CH3 domain will heterodimerize with another heavy chain comprising a CH3 domain engineered in a complementary manner. For this embodiment, the CH3 domain of the first heavy chain Fc region polypeptide and the CH3 domain of the second heavy chain Fc region polypeptide are engineered in a complementary manner by amino acid substitution, such that the first heavy chain Fc region polypeptide and the second heavy chain Fc region polypeptide can heterodimerize, while the first heavy chain Fc region polypeptide and the second heavy chain Fc region polypeptide no longer homodimerize (e.g., due to steric hindrance).

[0918] The different methods known in the art for supporting heavy chain heterodimerization listed and enumerated above can be considered as different alternatives for providing the heterodimeric / multimeric polypeptides (eg 2 / 3-IgG) reported herein.

[0919] The CH3 domains of the multimeric polypeptides according to the present invention can be modified using the "knob-into-hole" technique, which is described in detail with several examples in, for example, WO 96 / 027011, Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to enhance heterodimerization of the two heavy chain Fc region polypeptides comprising these two CH3 domains. Each of the two CH3 domains (of the two heavy chain Fc region polypeptides) can be a "knob," while the other can be a "hole." Disulfide bonds can additionally be introduced to further stabilize the heterodimers (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and to increase the yield in the exchange reaction according to the invention.

[0920] In a preferred embodiment, the multimeric polypeptide according to the present invention comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and Y407V mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index). Additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681), for example by introducing a Y349C mutation in one CH3 domain of the knob chain and an E356C mutation or an S354C mutation in one CH3 domain of the hole chain (in the exchange reaction according to the present invention, two multimers are used as starting material, only one of the CH3 domains of said multimers comprising this additional cysteine residue, whereby this additional disulfide bond can only be formed in the exchanged product). Therefore, in another preferred embodiment, the multimeric polypeptide according to the present invention comprises Y349C and T366W mutations in one CH3 domain of the first multimer and E356C, T366S, L368A and Y407V mutations in the corresponding complementary CH3 domain of the second multimer; or the multimeric polypeptide according to the present invention comprises Y349C and T366W mutations in one CH3 domain of the first multimer and S354C, T366S, L368A and Y407V mutations in the corresponding complementary CH3 domain of the second multimer (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the corresponding CH3 domain form an interchain disulfide bridge) (numbering according to the Kabat EU index).

[0921] However, other knob-into-hole techniques as described in EP 1 870 459 A1 may alternatively or additionally be used. In one embodiment, the multimeric polypeptide according to the invention comprises R409D and K370E mutations in the CH3 domain of the "knob chain" and D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0922] In one embodiment, the multimeric polypeptide according to the invention comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A and Y407V mutations in the CH3 domain of the "hole chain", and additionally comprises R409D and K370E mutations in the CH3 domain of the "knob chain" and D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0923] In one embodiment, the multimeric polypeptide according to the invention comprises Y349C and T366W mutations in one of the CH3 domains and S354C, T366S, L368A and Y407V mutations in the complementary CH3 domain; or the multimeric polypeptide according to the invention comprises Y349C and T366W mutations in one of the CH3 domains and S354C, T366S, L368A and Y407V mutations in the complementary CH3 domain, and in addition, R409D and K370E mutations in the CH3 domain of the "knob chain" and D399K and E357K mutations in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0924] In addition to the "knobs-in-hole technology", other technologies for modifying the heavy chain CH3 domain of the multimeric polypeptides according to the present invention to achieve heterodimerization are known in the art. These technologies, in particular those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291, can be considered in combination with the multimeric polypeptides according to the present invention as an alternative to the "knobs-in-hole technology".

[0925] In one embodiment of the multimeric polypeptide according to the invention, the method described in EP 1 870 459 A1 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multimeric polypeptide according to the invention. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the first and second heavy chains.

[0926] Thus, this embodiment relates to a multimeric polypeptide according to the present invention, wherein, in the multimeric tertiary structure, the CH3 domain of a first heavy chain Fc region polypeptide and the CH3 domain of a second heavy chain Fc region polypeptide form an interface located between the respective CH3 domains, wherein the amino acid sequences of the CH3 domain of the first heavy chain Fc region polypeptide and the CH3 domain of the second heavy chain Fc region polypeptide each comprise a set of amino acids located within the interface in the tertiary structure of the multimeric polypeptide according to the method of the present invention, wherein a first amino acid from the set of amino acids located within the interface in the CH3 domain of one heavy chain Fc region polypeptide is replaced with a positively charged amino acid, and a second amino acid from the set of amino acids located within the interface in the CH3 domain of the other heavy chain Fc region polypeptide is replaced with a negatively charged amino acid. The multimeric polypeptide according to this embodiment is also referred to herein as a "CH3 (+ / -) engineered multimeric polypeptide" (wherein the abbreviation "+ / -" represents the oppositely charged amino acids introduced into the respective CH3 domains).

[0927] In one embodiment of the CH3(+ / -) engineered multimeric polypeptide according to the present invention, the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.

[0928] In one embodiment of the CH3(+ / -) engineered multimeric polypeptide according to the present invention, the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.

[0929] In one embodiment of the CH3(+ / -) engineered multimeric polypeptide according to the present invention, the positively charged amino acid is K and the negatively charged amino acid is E.

[0930] In one embodiment of the CH3(+ / -) engineered 2 / 3 IgG reported herein, in the CH3 domain of one heavy chain, amino acid R at position 409 is replaced by D and amino acid K at position 370 is replaced by E, and in the CH3 domain of the other heavy chain, amino acid D at position 399 is replaced by K and amino acid E at position 357 is replaced by K (numbering according to the Kabat EU index).

[0931] In one embodiment of the multimeric polypeptide according to the present invention, the method described in WO 2013 / 157953 is used to support heterodimerization of a first heavy chain Fc region polypeptide and a second heavy chain Fc region polypeptide of the multimeric polypeptide. In one embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by K, and in the CH3 domain of another heavy chain Fc region polypeptide, the amino acid L at position 351 is replaced by D (numbering according to the Kabat EU index). In another embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by K, the amino acid L at position 351 is replaced by K, and in the CH3 domain of another heavy chain Fc region polypeptide, the amino acid L at position 351 is replaced by D (numbering according to the Kabat EU index).

[0932] In another embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by K, the amino acid L at position 351 is replaced by K, and in the CH3 domain of the other heavy chain Fc region polypeptide, the amino acid L at position 351 is replaced by D (numbering according to the Kabat EU index). In addition, at least one of the following substitutions is contained in the CH3 domain of the other heavy chain Fc region polypeptide: the amino acid Y at position 349 is replaced by E, the amino acid Y at position 349 is replaced by D, and the amino acid L at position 368 is replaced by E (numbering according to the Kabat EU index). In one embodiment, the amino acid L at position 368 is replaced by E (numbering according to the Kabat EU index).

[0933] In one embodiment of the multimeric polypeptide according to the present invention, the method described in WO 2012 / 058768 is used to support heterodimerization of a first Fc region polypeptide and a second Fc region polypeptide according to the multimeric polypeptide according to the present invention. In one embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid L at position 351 is replaced by Y, the amino acid Y at position 407 is replaced by A, and in the CH3 domain of another heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by A, and the amino acid K at position 409 is replaced by F (numbering according to the Kabat EU index). In another embodiment, in addition to the above replacements, in the CH3 domain of another heavy chain Fc region polypeptide, there is at least one amino acid replacement at positions 411 (formerly T), 399 (formerly D), 400 (formerly S), 405 (formerly F), 390 (formerly N) and 392 (formerly K) (numbering according to the Kabat EU index). Preferred replacements are:

[0934] - replacing the amino acid T at position 411 with an amino acid selected from the group consisting of N, R, Q, K, D, E and W (numbering according to the Kabat EU index),

[0935] - replacing the amino acid D at position 399 (numbering according to the Kabat EU index) with an amino acid selected from the group consisting of R, W, Y and K,

[0936] - replacing the amino acid S at position 400 (numbering according to the Kabat EU index) with an amino acid selected from the group consisting of E, D, R and K,

[0937] - substitution of amino acid F at position 405 with an amino acid selected from the group consisting of I, M, T, S, V and W (numbering according to the Kabat EU index),

[0938] - substitution of amino acid N at position 390 (numbering according to the Kabat EU index) with an amino acid selected from the group consisting of R, K and D, and

[0939] - Substitution of amino acid K at position 392 with an amino acid selected from the group consisting of V, M, R, L, F and E (numbering according to the Kabat EU index).

[0940] In another embodiment of the multimeric polypeptide according to the present invention (engineered according to WO 2012 / 058768), in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid L at position 351 is replaced by Y, the amino acid Y at position 407 is replaced by A, and in the CH3 domain of the other heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by V, and the amino acid K at position 409 is replaced by F (numbering according to the Kabat EU index). In another embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc region polypeptide, the amino acid Y at position 407 is replaced by A, and in the CH3 domain of the other heavy chain Fc region polypeptide, the amino acid T at position 366 is replaced by A, and the amino acid K at position 409 is replaced by F (numbering according to the Kabat EU index). In the above embodiment, in the CH3 domain of the other heavy chain Fc region polypeptide, the amino acid K at position 392 is replaced by E, the amino acid T at position 411 is replaced by E, the amino acid D at position 399 is replaced by R, and the amino acid S at position 400 is replaced by R (numbering according to the Kabat EU index).

[0941] In one embodiment of the multimeric polypeptide according to the invention, the method described in WO 2011 / 143545 is used to support heterodimerization of a first Fc-region polypeptide and a second Fc-region polypeptide of the multimeric polypeptide according to the invention. In one embodiment of the multimeric polypeptide according to the invention, amino acid alterations are introduced at positions 368 and / or 409 (numbering according to the Kabat EU index) in the CH3 domains of the two heavy chain Fc-region polypeptides.

[0942] In one embodiment of the multimeric polypeptide according to the present invention, the method described in WO 2011 / 090762 is used to support heterodimerization of a first Fc-region polypeptide and a second Fc-region polypeptide of the multimeric polypeptide according to the present invention. WO 2011 / 090762 relates to amino acid modifications based on the "knob-in-hole" technique. In one embodiment of the CH3 (KiH) engineered multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc-region polypeptide, the amino acid T at position 366 is replaced by W, and in the CH3 domain of the other heavy chain Fc-region polypeptide, the amino acid Y at position 407 is replaced by A (numbering according to the Kabat EU index). In one embodiment of the CH3 (KiH) engineered multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc-region polypeptide, the amino acid T at position 366 is replaced by Y, and in the CH3 domain of the other heavy chain Fc-region polypeptide, the amino acid Y at position 407 is replaced by T (numbering according to the Kabat EU index).

[0943] In one embodiment of the multimeric polypeptide of the IgG2 isotype according to the invention, the method described in WO 2011 / 090762 is used to support heterodimerization of the first heavy chain Fc-region polypeptide and the second heavy chain Fc-region polypeptide of the multimeric polypeptide according to the invention.

[0944] In one embodiment of the multimeric polypeptide according to the present invention, the method described in WO 2007 / 147901 is used to support heterodimerization of a first Fc-region polypeptide and a second Fc-region polypeptide of the multimeric polypeptide according to the present invention. In one embodiment of the multimeric polypeptide according to the present invention, in the CH3 domain of one heavy chain Fc-region polypeptide, amino acid K at position 253 is replaced by E, amino acid D at position 282 is replaced by K, and amino acid K at position 322 is replaced by D, and in the CH3 domain of the other heavy chain Fc-region polypeptide, amino acid D at position 239 is replaced by K, amino acid E at position 240 is replaced by K, and amino acid K at position 292 is replaced by D (numbering according to the Kabat EU index).

[0945] In one embodiment of the multimeric polypeptide according to the invention, the method described in WO 2007 / 110205 is used to support heterodimerization of the first polypeptide and the second polypeptide of the multimeric polypeptide according to the invention.

[0946] In one embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention has the structure of the constant domain of an IgG type antibody. In another embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention is characterized in that the multimeric polypeptide comprises an Fc region of the human IgG1 subclass, or an Fc region of the human IgG1 subclass with the mutations L234A and L235A and optionally P329G. In another embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention is characterized in that the multimeric polypeptide comprises an Fc region of the human IgG2 subclass. In another embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention is characterized in that the multimeric polypeptide comprises an Fc region of the human IgG3 subclass. In another embodiment of all aspects as reported herein, the multimeric polypeptide according to the invention is characterized in that the multimeric polypeptide comprises an Fc region of the human IgG4 subclass, or an Fc region of the human IgG4 subclass with the additional mutations S228P and L235E and optionally P329G.

[0947] In one embodiment of all aspects the multimeric polypeptide according to the invention comprises a first Fc-region polypeptide and a second Fc-region polypeptide, wherein

[0948] a) the first and second Fc-region polypeptides comprise a Y436A mutation, or

[0949] ii) the first and second Fc region polypeptides comprise I253A, H310A and H435A mutations, or

[0950] iii) the first and second Fc region polypeptides comprise H310A, H433A and Y436A mutations, or

[0951] iv) the first and second Fc region polypeptides comprise L251D, L314D and L432D mutations, or

[0952] v) the first Fc region polypeptide comprises a Y436A mutation, and the second Fc region polypeptide comprises

[0953] a) I253A, H310A and H435A mutations, or

[0954] b) H310A, H433A and Y436A mutations, or

[0955] c) L251D, L314D and L432D mutations,

[0956] or

[0957] vi) the first Fc region polypeptide comprises I253A, H310A and H435A mutations, and the second Fc region polypeptide comprises

[0958] a) H310A, H433A and Y436A mutations, or

[0959] b) L251D, L314D and L432D mutations,

[0960] or

[0961] vii) the first Fc region polypeptide comprises H310A, H433A and Y436A mutations, and the second Fc region polypeptide comprises

[0962] a) L251D, L314D and L432D mutations.

[0963] V. Recombinant Methods and Compositions

[0964] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, there is provided an isolated nucleic acid encoding a 2 / 3-IgG without a heavy chain-heavy chain disulfide bond or a 2 / 3-BiFab without a heavy chain-heavy chain disulfide bond. In one embodiment, there is provided one or more vectors (e.g., expression vectors) comprising such nucleic acids. In one embodiment, there is provided a host cell comprising such nucleic acid. In one embodiment, the host cell is a eukaryotic cell, such as a human embryonic kidney (HEK) cell, or a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method for preparing 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is provided, wherein the method comprises: culturing the host cell provided above containing the nucleic acid encoding 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds under conditions suitable for antibody expression, and optionally recovering the 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds from the host cell (or host cell culture).

[0965] In order to recombinantly produce 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, nucleic acids encoding 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells.

[0966] Suitable host cells for cloning or expressing vectors encoding 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds include prokaryotic or eukaryotic cells described herein.

[0967] For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0968] In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are suitable cloning or expression hosts for vectors encoding 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds with a partially or fully human glycosylation pattern. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0969] Suitable host cells for expressing glycosylated 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0970] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0971] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Examples of other useful mammalian host cell lines include: monkey kidney CV1 cell line transformed with SV40 (COS-7); human embryonic kidney cell line (293 or 293 cell line, described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, such as described in, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT) 060562); TRI cells, as described, for example, in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0972] VI. Methods and Compositions for Diagnosis and Detection

[0973] In certain embodiments, any of the 2 / 3-IgGs without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFabs without heavy chain-heavy chain disulfide bonds provided herein can be used to detect the presence of their target in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues.

[0974] In one embodiment, a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is provided for use in a diagnostic or detection method.

[0975] In certain embodiments, a labeled 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is provided. Labels include, but are not limited to, directly detectable markers or moieties (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive markers), as well as moieties that are indirectly detected, such as enzymes or ligands, for example, by enzyme reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 13 II; fluorophores such as rare earth chelates or fluorescein and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (US 4,737,456); luciferin; 2,3-dihydrophthalazinediones; horseradish oxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; carbohydrate oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase; coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase or microperoxidase; biotin / avidin; spin labels; phage labels; stable free radicals, etc.

[0976] VII. Immunoconjugates

[0977] The present invention also provides immunoconjugates comprising a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds as reported herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g. protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioactive isotopes.

[0978] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is conjugated to one or more drugs, including but not limited to: a maytansinoid (see US 5,208,020; US 5,416,064 and EP 0 425 235 B1); an auristatin, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see US 5,635,483; US 5,780,588 and US 7,498,298); dolastatin; a calicheamicin or its derivatives (see US 5,712,374; US 5,714,586; US 5,739,116; US 5,767,285; US 5,770,701; US 5,770,710; US 5,773,001 and US 5,877,296; Hinman, LM et al., Cancer Res. 53 (1993) 3336-3342; and Lode, HN et al., Cancer Res. 58 (1998) 2925-2928); anthracyclines, such as daunomycin or doxorubicin (see Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523; Jeffrey, SC et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362; Torgov, MY et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. et al., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, GM et al., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, HD et al., J. Med. Chem. 45 (200294336-4343; and US 6,630,579); methotrexate; vindesine; taxane compounds (taxanes), such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecenes and CC1065.

[0979] In another embodiment, the immunoconjugate comprises a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha sarcina, Aleurites fordii proteins, dianthus proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and trichothecenes. See, e.g., WO 93 / 21232 published October 28, 1993.

[0980] In another embodiment, the immunoconjugate comprises a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds as described herein conjugated to a radioactive atom to form a radioconjugate. Various radioactive isotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as TC for scintigraphic studies. 99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0981] Conjugates of 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds and cytotoxic agents can be prepared using various bifunctional coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imidothiocyanate (I ... Bifunctional derivatives of esters (such as dimethyl adipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta, ES et al., Science 238 (1987) 1098-1104. C14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. Such a linker may be a "cleavable linker" that facilitates intracellular release of the cytotoxic drug. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52 (1992) 127-131; US Pat. No. 5,208,020) may be used.

[0982] The immunoconjugates or ADCs herein specifically encompass, but are not limited to, such conjugates prepared using cross-linkers including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0983] The conjugate of 2 / 3-IgG without a disulfide bond between heavy chain and heavy chain or 2 / 3-BiFab without a disulfide bond between heavy chain and heavy chain and one or more small molecule toxins as reported herein is also encompassed herein, and the small molecule toxins are such as calicheamicin, maytansine (US 5,208,020), trichothecenes and CC1065. In one embodiment of the invention, 2 / 3-IgG without a disulfide bond between heavy chain and heavy chain or 2 / 3-BiFab without a disulfide bond between heavy chain and heavy chain are conjugated to one or more maytansine molecules (for example, each antagonist molecule is about 1 to 10 maytansine molecules). Maytansine (maytansine) can be, for example, converted to May-SS-Me, subsequently reduced to May-SH3, and reacted with modified antagonist (Chari et al. Cancer Research 52:127-131 (1992)), thereby producing maytansine sample-antibody conjugates.

[0984] Alternatively, 2 / 3-IgG without a heavy chain-heavy chain disulfide bond or 2 / 3-BiFab without a heavy chain-heavy chain disulfide bond as reported herein is conjugated to one or more calicheamicin molecules. The calicheamicin antibiotic family can cause double-stranded DNA breaks at sub-picomolar concentrations. Available structural analogs of calicheamicin include, but are not limited to, γ 1I, α 2I, α 3I, N-acetyl-γ 1I, PSAG, and θ 1I (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al. Cancer Research 58:2925-2928 (1998)).

[0985] The present invention further encompasses the conjugation of a 2 / 3-IgG without heavy chain-heavy chain inter-disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain inter-disulfide bonds as reported herein to a compound having nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0986] Alternatively, a fusion protein comprising a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds as reported herein and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis.

[0987] VIII. Pharmaceutical Preparations

[0988] Pharmaceutical preparations of 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds as described herein, or a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, can be prepared by mixing the 2 / 3-IgG(s) or 2 / 3-BiFab(s) having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)) in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl benzoates such as methyl or propyl benzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 10 residues) polyols. Peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides; disaccharides; and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronase glycoprotein, such as rhuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs (including rhuPH20) and methods of use are described in US 2005 / 0260186 and US 2006 / 0104968. In one aspect, sHASEGP is used in combination with one or more additional glycosaminoglycanases (such as chondroitinases).

[0989] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0990] Lyophilized formulations suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted with an appropriate diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the mammal to be treated.

[0991] Depending on the needs of the specific indication being treated, the formulations of the present invention may also contain more than one active ingredient, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in the composition in an amount effective for the intended purpose.

[0992] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. This technology is disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0993] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrix being in the form of a shaped article, such as a film, or a microcapsule. Examples of sustained-release matrices include polyesters; hydrogels (e.g., poly(2-hydroxyethyl-methacrylate); or poly(vinyl alcohol)); polylactides (US 3,773,919); copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid; non-degradable ethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-co-glycolic acid and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0994] The preparations for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtering through a sterile filtration membrane. In one embodiment, the preparation is isotonic.

[0995] IX. Methods of Treatment and Compositions

[0996] Any of the 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds provided herein, or a composition comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds can be used in a method of treatment.

[0997] In one aspect, a composition comprising a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, or comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is provided for use as a medicament. In certain embodiments, a composition comprising a 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or a 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds, or comprising two different 2 / 3-IgG without heavy chain-heavy chain disulfide bonds or two different 2 / 3-BiFab without heavy chain-heavy chain disulfide bonds is provided for use in a method of treatment. According to any of the above embodiments, the "subject" is preferably a human. [0998...

Claims

1. A composition comprising: a) a first multimeric polypeptide consisting of a first polypeptide and a second polypeptide, wherein The first polypeptide comprises From N-terminus to C-terminus, Fab, which comprises a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to cell surface antigens, a human IgG1 CH1 domain and a light chain CL domain, The hinge region of SEQ ID NO: 66, wherein the hinge region is connected to the human IgG1 CH1 domain or the CL domain, a first antibody variable domain selected from a light chain variable domain and a heavy chain variable domain of a pair of antibodies that specifically bind to a first target, and a first CH3 domain from a human IgG1 CH3 domain, The second polypeptide comprises In the N-terminal to C-terminal direction, a) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a third target, and b) a second CH3 domain from a human IgG1 CH3 domain, wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain; or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain, and wherein the second CH3 domain comprises an interfering mutation, wherein the interfering mutation is D356K or E357K, wherein all numbering is according to the Kabat EU index, such that the first CH3 domain comprises a) amino acid residue K at position 439, if the interfering mutation is D356K, or b) amino acid residue K at position 370, if the interfering mutation is E357K, and b) a second multimeric polypeptide consisting of the first polypeptide and the second polypeptide, wherein: The first polypeptide comprises From N-terminus to C-terminus, Fab, which comprises a pair of antibody light chain variable domains and heavy chain variable domains that specifically bind to cell surface antigens, a human IgG1 CH1 domain and a CL domain, The hinge region of SEQ ID NO: 66, wherein the hinge region is connected to the human IgG1 CH1 domain or the CL domain, a first antibody variable domain selected from a light chain variable domain and a heavy chain variable domain of a pair of antibodies that specifically bind to a first target, and a first CH3 domain from a human IgG1 CH3 domain, The second polypeptide comprises In the N-terminal to C-terminal direction, a) a second antibody variable domain selected from a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically binds to a third target, and b) a second CH3 domain from a human IgG1 CH3 domain, wherein if the first antibody variable domain is an antibody heavy chain variable domain, the second antibody variable domain is an antibody light chain variable domain; or if the first antibody variable domain is an antibody light chain variable domain, the second antibody variable domain is an antibody heavy chain variable domain, and wherein the second CH3 domain comprises an interfering mutation, wherein the interfering mutation is K370E or K439E, wherein all numbering is according to the Kabat EU index, such that the first CH3 domain comprises a) amino acid residue E at position 357, if the interfering mutation is K370E, or b) amino acid residue D at position 356, if the interfering mutation is K439E; The first multimeric polypeptide and the second multimeric polypeptide are characterized by: - the second CH3 domain of the first multimeric polypeptide comprises a D356K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K439E mutation, or The second CH3 domain of the first multimeric polypeptide comprises an E357K mutation, and the second CH3 domain of the second multimeric polypeptide comprises a K370E mutation; and - the first antibody variable domain of the first multimeric polypeptide and the first antibody variable domain of the second multimeric polypeptide are a pair of antibody light chain variable domain and antibody heavy chain variable domain that specifically bind to the first target, and The second antibody variable domain of the first multimeric polypeptide and the second antibody variable domain of the second multimeric polypeptide are a pair of an antibody light chain variable domain and an antibody heavy chain variable domain that specifically bind to a third target.

2. The composition according to claim 1, wherein In the first multimeric polypeptide and the second multimeric polypeptide, the first CH3 domain and the second CH3 domain further comprise a mutation that promotes heterodimer formation between the first CH3 domain and the second CH3 domain, and the mutation is different from the interfering mutation.

3. The composition of claim 1 , wherein the first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation, and wherein the second CH3 domain of the first multimeric polypeptide and the first CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation.

4. The composition of claim 2, wherein the first CH3 domain of the first multimeric polypeptide and the second CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation, and wherein the second CH3 domain of the first multimeric polypeptide and the first CH3 domain of the second multimeric polypeptide comprise the same mutation to promote heterodimer formation.

5. The composition according to claim 2, wherein The first CH3 domain of the first polypeptide comprises a) T366W mutation, or b) T366S / L368A / Y407V mutations, as well as The second CH3 domain of the second polypeptide comprises a) T366S / L368A / Y407V mutations, if the first CH3 domain contains a T366W mutation, or b) T366W mutation, if the first CH3 domain comprises T366S / L368A / Y407V mutations.

6. The composition according to any one of claims 1 to 4, wherein the first polypeptide and the second polypeptide are non-covalent dimers.

7. The composition according to any one of claims 1 to 4, wherein the first antibody variable domain and the second antibody variable domain form a non-functional binding site.

8. The composition according to any one of claims 1 to 4, wherein the first and second polypeptides comprise the amino acid sequence DKTHTSPPS (SEQ ID NO: 66) at the N-terminus of the first and second antibody variable domains, respectively.

9. The composition according to any one of claims 1 to 4, wherein the first, second and third targets are different.

10. The composition according to any one of claims 1 to 4, wherein the first and second polypeptides each further comprise an immunoglobulin G CH2 domain directly N-terminal to the CH3 domain.

11. The composition according to any one of claims 1 to 4, wherein the first target and / or the third target is human CD3.

12. The composition according to any one of claims 1 to 4, wherein the composition is a pharmaceutical composition and optionally further comprises a pharmaceutically acceptable excipient.

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