Chimeric heavy chain constant domains with reduced binding to FC gamma receptors and uses thereof

Abstract

The present disclosure relates to chimeric heavy chain constant domains with reduced effector function. Also disclosed are recombinant polypeptides comprising such chimeric heavy chain constant domains, including antibodies such as multispecific antibodies, fusion proteins, and other recombinant proteins. Also disclosed are nucleic acids encoding such recombinant polypeptides, as well as cells expressing such recombinant polypeptides and pharmaceutical compositions comprising such recombinant polypeptides.

Description

1. Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 333,293, filed on April 21, 2022, the contents of which are incorporated herein by reference in their entirety. 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The copy created on April 12, 2023 is named RGN-013WO_SL.xml and is 58,069 bytes in size. 3. Background technology

[0005] IgG class antibodies are attractive therapeutic agents. IgG exists in humans in the form of four subclasses: IgG1, IgG2, IgG3 and IgG4. The heavy chain constant (CH) region of IgG includes three domains CH1, CH2, CH3 and a hinge linking CH1 and CH2. Although the effect of each subclass seems to vary from species to species, the heavy chain constant domain is responsible for various biological effector functions. Human IgG subclasses mediate several cellular immune responses, such as cell killing, phagocytosis and opsonization, by interacting with Fcγ (FcγR). Such interactions involve at least functional CH2 and CH3 domains of the heavy chain constant region and the combination of FcγRs on the surface of effector cells (such as natural killer cells and activated macrophages). The interaction of the Fc region (e.g., CH2 and CH3 domains) with various complement components can also trigger complement-mediated lysis.

[0006] Effector functions are useful in some antibody therapies (such as the treatment of some cancers or pathogens), where the effector functions are primarily or at least partially responsible for killing cancer cells or pathogens. However, other antibody therapies are mediated entirely or primarily by effector-independent mechanisms, such as inhibiting receptor-ligand interactions or agonizing receptors. In such therapies, antibody effector functions have little or no useful purpose, but may cause undesirable effects, including inflammation. In such cases, the Fc receptor binding properties of antibodies are engineered to suppress some or all of the available effector mechanisms, without significantly affecting the pharmacokinetic properties, immunogenicity, and variable region specificity and affinity of the antibody, which may be advantageous. Several groups have performed such antibody engineering.

[0007] The IgG heavy chain constant region has been mutated at various positions to test the effect of amino acids on IgG / FcγR interactions (see, e.g., Canfield and Morrison, 1991, J Exp Med 73: 1483-1491; Chappel et al., 1993, JSC 268 (33): 25124-31; and Armour et al., Eur J Immunol 29: 2613-24). Several amino acid residues in the hinge region and the CH2 domain of the heavy chain constant region have been proposed to mediate binding to Fcγ receptors (see Sarmay et al., 1992, Mol Immunol 29: 633-9; Greenwood et al., Eur J Immunol 23 (5): 1098, Morgan et al., 1995, Immunology 86: 319; Stevenson, 1997, Chem Immunol 65: 57-72). Glycosylation of a site (N297) in the CH2 domain and changes in its carbohydrate composition also strongly affect IgG / FcγR interactions (Stevenson, 1997, Chem Immunol 65:57-72; Siberil et al., 2006, Immunol Ltrs 106:111-118). WO2014121087 describes a chimeric IgG comprising an IgG1 upper hinge, an IgG1 lower hinge, an IgG4 CH2, and an IgG1 CH3, wherein the amino acids in the lower hinge region are substituted with corresponding amino acids from a human IgG2 isotype to reduce FcγR binding without unacceptable conformational changes and consequent immunogenicity. WO2016161010A2 describes an engineered IgG heavy chain constant domain with a modified hinge region, wherein amino acid positions 233 to 236 (EU numbering) are modified to Gly, Gly, Gly, and unoccupied; Gly, Gly, unoccupied, and unoccupied; Gly, unoccupied, unoccupied, and unoccupied; or all unoccupied, and the positions are numbered according to EU numbering (such as WO2016161010A2). Figure 1 shown).

[0008] Alternatives with those chimeric heavy chain constant domains known in the art are valuable. Having alternative chimeric heavy chain constant regions based on different IgG subclasses allows, for example, optimization of antibody activity and improved expression and production. Therefore, heavy chain constant regions with reduced binding to FcγRs and therapeutic polypeptides (e.g., recombinant antibodies) comprising the same are sought, which can be used to treat diseases or disorders in which effector functions are to be minimized. 4. Summary of the invention

[0009] The present disclosure relates to chimeric heavy chain constant domains with reduced Fc receptor and / or effector function.

[0010] The chimeric heavy chain constant domains of the present disclosure are based on IgG1 heavy chain constant domains in which the hinge region is modified. Unexpectedly, incorporation of the chimeric constant domains of the present disclosure into exemplary antibodies improves the expression and activity of the antibodies in addition to reducing Fc receptor and effector functions (see, e.g., Section 8.2 (Example 1), Section 8.5 (Example 4), Section 8.6 (Example 5), and Section 8.7 (Example 6)).

[0011] Therefore, the present disclosure provides a recombinant protein comprising a chimeric heavy chain constant domain of the present disclosure, for example, a fusion protein. The chimeric heavy chain constant domain can be advantageously used as a dimerization portion in a variety of engineered proteins (e.g., recombinant antibodies, immunocytokines, and soluble receptors). In addition to the chimeric heavy chain constant domain of the present disclosure, the recombinant polypeptide may include one or more target binding regions (e.g., one or more Fab portions, one or more scFv portions, or a combination thereof) and / or one or more linker portions that separate one or more portions in the recombinant protein. In some embodiments, the recombinant protein is a recombinant antibody, such as a recombinant multispecific antibody.

[0012] Exemplary chimeric heavy chain constant domains are disclosed in Section 6.2.1. Exemplary recombinant proteins comprising constant domains are disclosed in Section 6.2 (Examples 1 to 105 and 110 to 214 numbered as Group A, Examples 1 to 45 numbered as Group C, and Examples 1 to 24 numbered as Group D). Where the recombinant protein is an antibody, exemplary target binding domains for incorporation into antibodies are disclosed in Section 6.2.2, and exemplary linkers that can be used to connect the constant domain to the target binding domain or different components of the target binding domain are described in Section 6.2.5.

[0013] The disclosure further provides nucleic acids encoding recombinant proteins of the disclosure. The nucleic acids may be in the form of a single nucleic acid (e.g., a vector encoding two or more polypeptide chains) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains). The disclosure further provides host cells and cell lines engineered to express nucleic acids and recombinant proteins of the disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 6.3 (Examples 108, 109, 216, and 217, numbered as Group A, Examples 48 to 51, and Examples 25 to 27, numbered as Group D).

[0014] Methods of producing recombinant polypeptides and methods of using the constant domains of the present disclosure to increase the expression and / or activity of recombinant proteins are described in Section 6.3 (Examples 218 to 219 numbered as Group A, Examples 1 to 69 numbered as Group B, Examples 52 numbered as Group C, and Examples 28 to 41 numbered as Group D). In certain embodiments, the increased expression of the recombinant proteins of the present disclosure is demonstrated by increased protein yield and / or production, such as an increase in the total amount of protein obtained from an expression system (such as the expression system described in Section 8.1.6). In certain embodiments, the increased activity of the recombinant proteins of the present disclosure is demonstrated by increased target binding and / or signal modulation.

[0015] The present disclosure also provides compositions, such as populations of proteins and pharmaceutical compositions comprising the recombinant proteins of the present disclosure. Exemplary compositions are described in Section 6.4 (Examples 106, 107, 214 and 215 numbered as Group A, Examples 70 to 72 numbered as Group B, Examples 46, 47 and 53 to 55 numbered as Group C, and Examples 42 to 46 numbered as Group D). 5. Description of the drawings

[0016] Figure 1 The wild-type sequence of the heavy chain constant region of human IgG1 is depicted (human IGHG1 heavy chain constant region; UniProt accession number P01857). CH1 = amino acids 1 to 98; upper hinge = amino acids 99 to 108; core hinge = 109 to 112; lower hinge = 113 to 121; CH2 = 120 to 223; CH3 = 224 to 330. The indicated amino acid numbers are relative to the depicted sequence. The upper hinge region, core hinge region, and lower hinge region are marked with boxes. As shown in the figure, the last two amino acids of the lower hinge correspond to the first two amino acids of the CH2 domain. Figure 1 SEQ ID NO: 48 is disclosed.

[0017] Figure 2 The wild-type sequence of the heavy chain constant region of human IgG2 is depicted (human IGHG2 heavy chain constant region; UniProt accession number P01859). CH1 = amino acids 1 to 98; upper hinge = amino acids 99 to 105; core hinge = 106 to 109; lower hinge = 110 to 117; CH2 = 116 to 219; CH3 = 220 to 326. The indicated amino acid numbers are relative to the depicted sequence. As shown in the figure, the last two amino acids of the lower hinge correspond to the first two amino acids of the CH2 domain. Figure 2 SEQ ID NO: 49 is disclosed.

[0018] Figure 3The wild-type sequence of the heavy chain constant region of human IgG4 is depicted (human IGHG4 heavy chain constant region; UniProt accession number P01861). CH1 = amino acids 1 to 98; upper hinge = amino acids 99 to 105; core hinge = 106 to 109; lower hinge = 110 to 118; CH2 = 117 to 220; CH3 = 221 to 227. The indicated amino acid numbers are relative to the depicted sequence. As shown in the figure, the last two amino acids of the lower hinge correspond to the first two amino acids of the CH2 domain. Figure 3 SEQ ID NO: 50 is disclosed.

[0019] Figure 4 Depicted is the amino acid sequence alignment of the upper hinge, core hinge, lower hinge, CH2 and CH3 of the indicated chimeric IgG heavy chain constant domain constructs. The indicated amino acid numbering is EU numbering. The shaded cells of the lower hinge represent amino acids that also correspond to the first two amino acids of the CH2 domain. Figure 4 SEQ ID NO: 51 (lines 3 and 4), SEQ ID NO: 52 (line 5), SEQ ID NO: 53 (line 6), SEQ ID NO: 54 (line 7) and SEQ ID NO: 55 (line 8) are disclosed.

[0020] Figure 5 Depicted are representative data illustrating antibody titers of the depicted alternative formats of antibodies comprising heterodimers of IgG4 S108P / IgG4 S108P Star (H315R, Y316F) or IgG1 PVA / IgG1 PVA Star (H315R, Y316F) after stable expression in Chinese hamster ovary (CHO) cells and having the depicted 2+1N-scFv format. Different lengths of linkers between the Fab and scFv were tested.

[0021] FIG. 6A to FIG. 6G Depicted are representative enzyme-linked immunosorbent assay (ELISA) data demonstrating that the indicated controls and antibodies interact with hFCRγ1 ( Fig. 6A ); hFCRγ2A(H131)( Figure 6B ); hFCRγ2A(R131)( Figure 6C ); hFCRγ2B( Fig.6D ); hFCRγ3A(V158)( Fig. 6E ); hFCRγ3A(F158)( Fig. 6F ); and hFCRγ3B ( Figure 7 Descriptions of control and test antibodies are provided in Table 3.

[0022] Figure 7Depicted are representative results from alternative antibody-dependent cell-mediated cytotoxicity (ADCC) assays in which the indicated 2+1 N-scFv alternative format antibodies (AF1) with different Fc regions were tested as well as controls.

[0023] Figure 8 Depicted are representative results from an alternative ADCC assay in which the indicated 2+1 N-Fab alternative format antibodies (AF2) with different Fc regions were tested as well as controls.

[0024] Fig. 9 Depicted are representative results from a luciferase reporter assay demonstrating that 2+1 N-scFv format antibodies (AF1) with different Fc regions as well as controls elicit activation of HEK293.SREluc.hFGFR1c.hKLB cells.

[0025] Fig.10 Depicted are representative results from a luciferase reporter assay demonstrating that 2+1 N-Fab format antibodies with different Fc regions (AF2 and AF3) as well as controls elicit activation of HEK293.SREluc.hFGFR1c.hKLB cells.

[0026] Fig.11 Depicted are representative results from a phospho-ERK activation assay demonstrating that antibodies in the form of 2+1 N-scFv (AF1) or 2+1 N-Fab (AF3) with IgG4 S108P or IgG1 PVA Fc regions or His.hFGF21 elicited activation in primary human adipocytes.

[0027] Fig.12 Depicted are representative flow cytometric binding assay results demonstrating that bispecific antibodies with IgG1 PVA Fc regions bind to cell surface targets with higher maximal MFI signals than bispecific antibodies with IgG4 S108P Fc regions.

[0028] FIG. 13A to FIG. 13E Negative staining EM 2D class averages of IgG-CD40 complexes are depicted. Fig.13A is an exemplary negative stain EM 2D image showing features corresponding to Fc, Fab and CD40 as well as the Fab-Fab angle. Fig. 13B yes Fig.13A and its components. Fig. 13C The 2D class averages of the IgG1-CD40 complex are shown. Fig.13D The 2D class averages of the IgG1-PVA-CD40 complex are shown, and Fig.13E The 2D class averages of IgG2-CD40 complexes are shown. 6. Specific implementation methods

[0029] 6.1. Definitions

[0030] About : Throughout the specification, the terms "about", "approximately", etc. are used in front of a number to indicate that the number is not necessarily exact (e.g., to take into account fractions, variations in measurement accuracy and / or precision, timing, etc.). It should be understood that disclosing "about X" or "approximately X" where X is a number also discloses "X". Thus, for example, disclosing an embodiment in which a sequence has "about X% sequence identity" to another sequence also discloses an embodiment in which the sequence has "X% sequence identity" to the other sequence.

[0031] and and or : Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, including feature selection among alternatives (A or B, where the selection of A is mutually exclusive with B) and feature selection in conjunctions (A or B, where both A and B are selected). In some places in this text, the term "and / or" is used for the same purpose, which should not be interpreted as implying the use of "or" involving mutually exclusive alternatives.

[0032] Antibody : The term "antibody" as used herein includes any form of antibody having at least one antigen binding fragment, including monovalent fragments (e.g., scFv), bivalent tetrameric molecules of two heavy chains and two light chains, and higher order complexes of any of them. Antibodies can be monospecific, in which case all binding regions have the same specificity, or multispecific, in which the binding site has at least two specificities (e.g., bispecific). The term "antibody" encompasses monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, etc.

[0033] Association : The term "association" in the context of a chimeric constant domain, a recombinant polypeptide comprising a chimeric constant domain, or a component of a recombinant polypeptide (e.g., an antigen binding domain) refers to a functional relationship between two or more polypeptide chains or polypeptide chain portions. Specifically, the term "association" means that two or more polypeptides associate with each other, such as non-covalently by molecular interactions or covalently by one or more disulfide bridges or chemical crosslinks, so as to produce, for example, a functional antigen binding domain or Fc region. Examples of associations that may exist within a recombinant polypeptide of the present disclosure or between a recombinant polypeptide of the present disclosure and one or more additional polypeptides include, but are not limited to, associations between homodimeric or heterodimeric chimeric heavy chain constant domains of the present disclosure, associations between VH and VL regions in Fab or scFv, associations between CH1 and CL in Fab, and associations between CH3 and CH3 in a domain-substituted Fab.

[0034] Two-price : The term "bivalent" as used herein in relation to an antibody comprising a recombinant polypeptide and / or a chimeric constant domain of the present disclosure means that the antibody has two antigen-binding portions (e.g., two antigen-binding fragments of an antibody, or a first antigen-binding fragment of a first antibody and a second antigen-binding fragment of a second antibody). An antibody comprising a recombinant polypeptide and / or a chimeric constant domain of the present disclosure may be bivalent for one type of portion (e.g., two antigen-binding fragments of a first antibody) and monovalent for another type of portion (e.g., a single antigen-binding fragment of a second antibody).

[0035] Chimeric heavy chain constant domains and chimeric constant domains : The terms "chimeric heavy chain constant domain" and "chimeric constant domain" when used in relation to the constant domains of the present disclosure are used interchangeably to refer to an IgG1 constant domain comprising an IgG1 upper hinge domain, an IgG1 core hinge domain, and an IgG1 lower hinge domain having a substitution / deletion mutation ELLG (SEQ ID NO: 23) → PVA- (or "PVA-deletion") at amino acid positions 233 to 236 (EU numbering), an IgG1 CH2 domain, and an IgG1 CH3 domain. In some embodiments, the chimeric constant domain further comprises an IgG1 CH1 domain. In some embodiments, the chimeric constant domain may be further modified to, for example, further alter effector function and / or provide heterodimerization. The chimeric constant domains of the present disclosure are further described in Section 6.2.

[0036] The chimeric constant domain can promote the association between two recombinant polypeptide chains to form a dimer. The two chimeric constant domains in the dimer can be the same or different. Therefore, the resulting dimer can be a homodimer or a heterodimer.

[0037] Chimeric constant domains comprising an IgG1 lower hinge domain having the substitution / deletion mutation ELLG (SEQ ID NO: 23)→PVA- (or "PVA-deleted") are sometimes referred to as having the "IgG1 PVA" isotype or similar terminology.

[0038] Dimerization part : The term "dimerization moiety" refers to a polypeptide chain or amino acid sequence that can promote the association between two polypeptide chains to form a dimer. A first dimerization moiety can associate with the same second dimerization moiety, or can associate with a second dimerization moiety that is different from the first dimerization moiety. In some embodiments, the dimerization moiety is a recombinant constant domain of the present disclosure, wherein the two recombinant constant domains associate to form an Fc region. Therefore, the Fc region can be a homodimer or a heterodimer.

[0039] EC50: The term "EC50" refers to the half-maximal effective concentration of a molecule, such as an antibody comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure, which induces a response halfway between baseline and maximum after a specific exposure time. EC50 essentially represents the concentration of the antibody at which 50% of the maximum effect of the antibody is observed. In certain embodiments, the EC50 value is equal to the concentration of the antibody that gives half-maximal activation in a luciferase reporter assay.

[0040] Fab : The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising a variable heavy (VH) domain of an antibody at the N-terminus of a first constant domain (referred to herein as C1), and the second polypeptide chain comprising a variable light (VL) domain of an antibody at the N-terminus of a second constant domain (referred to herein as C2), which is capable of pairing with the first constant domain. In natural antibodies, VH is at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is at the N-terminus of the light chain constant domain (CL). The Fab of the present disclosure may be arranged according to the natural orientation or include domain substitutions or exchanges that are conducive to correct VH and VL pairing. For example, it is possible to replace the CH1 and CL domain pairs in Fab with a CH3 domain pair to promote correct modified Fab chain pairing in heterodimer molecules. It is also possible to reverse CH1 and CL so that CH1 is connected to VL and CL is connected to VH, and this configuration is generally referred to as Crossmab.

[0041] Fc domain and Fc region : The term "Fc domain" refers to a portion of an immunoglobulin heavy chain that is paired with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, for example via knob-in-hole interactions, and / or to allow purification, for example via star mutations. The chimeric constant domains of the present disclosure comprise Fc domains.

[0042] Hinge region or hinge domain: The terms "hinge region" and "hinge domain" as used herein refer to the consecutive amino acid residues that connect the C-terminus of the CH1 domain of an immunoglobulin to the N-terminus of the CH2 domain. In human IgG1, IgG2, and IgG4, the hinge region extends from residue 216 to 238 according to EU numbering, wherein residues 237 and 238 overlap with the CH2 domain. Residues 216 to 225 (EU numbering) form the upper hinge, residues 226 to 229 (EU numbering) form the middle (or core) hinge, and residues 230-238 (EU numbering) form the lower hinge. The upper hinge and middle hinge of IgG1, IgG2, and IgG4 are 12-15 consecutive amino acids encoded by different hinge exons. The lower hinge includes several N-terminal amino acids of the CH2 domain (encoded by the CH2 exon) (Brekke et al., 1995, Immunology Today 16 (2): 85-90). See, e.g. Figure 4 .

[0043] Host cells or recombinant host cells : The terms "host cell" and "recombinant host cell" as used herein refer to cells that have been genetically engineered, for example, by the introduction of heterologous nucleic acids. It should be understood that such terms refer not only to specific subject cells, but also to the progeny of such cells. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may actually be different from the parental cells, but are still included in the scope of the term "host cell" as used herein. The host cell may transiently carry the heterologous nucleic acid, for example, on an extrachromosomal heterologous expression vector, or stably carry the heterologous nucleic acid, for example, by integrating the heterologous nucleic acid into the host cell genome. For expression of the recombinant polypeptides of the present disclosure, the host cell may be a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293 (and derivatives, such as Expi293 adapted to higher density growth), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants thereof. Engineered variants include, for example, modified glycan profiles and / or site-specific integration site derivatives.

[0044] unit price: The term "monovalent" as used herein in relation to an antibody comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure means that the antibody has one antigen-binding portion that is specific for a first target molecule. An antibody comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure may be monovalent for one type of portion (e.g., a single antigen-binding fragment of a first antibody) and may be bivalent for another type of portion (e.g., two antigen-binding fragments of a second antibody).

[0045] Multi-price : The term "multivalent" as used herein in relation to an antibody comprising a recombinant polypeptide and / or a chimeric constant domain of the present disclosure means that the antibody has two or more antigen-binding portions (e.g., two antigen-binding fragments of an antibody, or a first antigen-binding fragment of a first antibody and a second antigen-binding fragment of a second antibody). An antibody comprising a recombinant polypeptide and / or a chimeric constant domain of the present disclosure may be multivalent for one type of portion (e.g., two or more antigen-binding fragments of a first antibody) and monovalent for another type of portion (e.g., a single antigen-binding fragment of a second antibody).

[0046] Operably Linked : As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are linked to produce a functional polypeptide, or two or more nucleic acid sequences are linked, for example, to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0047] Recombinant Peptide : The term "recombinant polypeptide" refers to a polypeptide comprising a chimeric constant domain of the present disclosure. Generally, a recombinant polypeptide comprises a chimeric constant domain of the present disclosure and at least one antigen binding portion.

[0048] In the context of the present disclosure, the term "recombinant polypeptide" sometimes refers to the core component of the molecule, i.e., the chimeric constant domain, and sometimes refers to the antigen binding portion. It should be understood that, unless the context indicates otherwise, the term "recombinant polypeptide" also extends to polypeptides comprising additional features, such as one or more stabilizing parts, one or more linker parts, and any combination of the foregoing.

[0049] Single-chain Fv or scFv : The term "single-chain Fv" or "scFv" as used herein refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0050] Specific (or selective) binding: As used herein, the term "specific (or selective) binding" refers to an antigen binding portion, such as an antibody or its antigen binding domain ("ABD"), that forms a relatively stable complex with a target molecule under physiological conditions. Specific binding is characterized by K D About 5x10 -2 M or smaller (e.g., less than 5x10 -2 , less than 10 -2 M, less than 5x10 -3 M, less than 10 -3 M, less than 5x10 -4 M, less than 10 -4 M, less than 5x10 -5 M, less than 10 -5 M, less than 5x10 -6 M, less than 10 -6 M, less than 5x10 -7 M, less than 10 -7 M, less than 5x10 -8 M, less than 10 -8 M, less than 5x10 -9 M, less than 10 -9 M or less than 10 -10 M). Methods for determining the binding affinity of an antibody or antibody fragment for a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assays), fluorescence activated cell sorting (FACS) binding assays, etc. However, an antigen binding portion that specifically binds to a target molecule from one species may have cross-reactivity with target molecules from one or more other species.

[0051] Target binding domain : The term "target binding domain" as used herein refers to a polypeptide sequence or group of associated polypeptide sequences that can specifically, non-covalently and reversibly bind to a target molecule. The term includes "antigen binding domain" and "antigen binding fragment", which refers to a portion of an antibody that can specifically, non-covalently and reversibly bind to a target molecule. "Target binding domain" also encompasses target binding portions of receptors (e.g., TNF receptors) that can specifically, non-covalently and reversibly bind to a target molecule (e.g., TNF). The target binding domain can be incorporated into a recombinant polypeptide of the present disclosure. In some embodiments, a recombinant polypeptide comprising one or more target binding domains may be included in an antibody or fusion protein. Such antibodies are described in Section 6.2.2 below. Fusion proteins are described in Section 6.2.3 below.

[0052] Target molecule: As used herein, the term "target molecule" refers to any biological molecule (e.g., protein, carbohydrate, lipid, or a combination thereof) expressed on the cell surface or in the extracellular matrix, which can be specifically bound by the antigen binding portion of an antibody comprising a recombinant polypeptide of the present disclosure.

[0053] Universal light chain : The term "universal light chain" in the context of an antigen binding moiety as used herein refers to a light chain polypeptide that can pair with a heavy chain region of an antigen binding moiety and can also pair with other heavy chain regions. Universal light chains are also referred to as "shared light chains".

[0054] VH : The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of a scFv or Fab.

[0055] V L : The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of a scFv or Fab.

[0056] 6.2. Recombinant Peptides

[0057] The present disclosure provides a recombinant polypeptide comprising a chimeric constant domain based on an IgG1 heavy chain constant domain, wherein the hinge region is modified to reduce Fc receptors and / or effector functions. The recombinant polypeptide of the present disclosure can serve as a dimerization portion, capable of promoting the association between two polypeptide chains to form a dimer. In some embodiments, the recombinant polypeptide of the present disclosure can dimerize to form an antibody. Antibodies comprising recombinant polypeptides of the present disclosure are described in Section 6.2.2 below. In other embodiments, recombinant polypeptides of the present disclosure can dimerize to form a fusion protein. Fusion proteins comprising recombinant polypeptides of the present disclosure are described in Section 6.2.3 below. In addition to the chimeric constant domains of the present disclosure, the recombinant polypeptide of the present disclosure contained in the antibody or fusion protein of the present disclosure may include one or more target binding domains (e.g., one or more Fab portions, one or more scFv portions, one or more target receptors or their binding fragments) and / or one or more joint portions separating one or more parts in the recombinant polypeptide. The target binding domain is described in Section 6.2.4. The joints that can be used for recombinant polypeptides of the present disclosure are described in Section 6.2.5.

[0058] 6.2.1. Chimeric constant domains

[0059] The present disclosure provides a chimeric constant domain based on an IgG1 heavy chain constant domain. The chimeric constant domain of the present disclosure comprises an IgG1 upper hinge domain, an IgG1 lower hinge domain having a substitution / deletion mutation ELLG (SEQ ID NO: 23) → PVA- at amino acid positions 233 to 236 (EU numbering), an IgG1 CH2 domain, and an IgG1 CH3 domain. In some embodiments, the chimeric constant domain further comprises an IgG1 CH1 domain or a fragment thereof.

[0060] The IgG heavy chain constant region has been mutated at various positions to test the effect of amino acids on IgG / FcγR interactions (see, e.g., Canfield and Morrison, 1991, J Exp Med 73: 1483-1491; Chappel et al., 1993, JSC 268 (33): 25124-31; and Armour et al., Eur J Immunol 29: 2613-24). Several amino acid residues in the hinge region and the CH2 domain of the heavy chain constant region have been proposed to mediate binding to Fcγ receptors (see Sarmay et al., 1992, Mol Immunol 29: 633-9; Greenwood et al., Eur J Immunol 23 (5): 1098, Morgan et al., 1995, Immunology 86: 319; Stevenson, 1997, Chem Immunol 65: 57-72). Glycosylation of a site in the CH2 domain (N297) and changes in its carbohydrate composition also strongly influence IgG / FcγR interactions (Stevenson, 1997, Chem Immunol 65:57-72; Siberil et al., 2006, Immunol Ltrs 106:111-118).

[0061] Alanine residues are usually preferred substituents for replacing natural amino acids with non-natural amino acids to reduce function, because alanine has a side chain without any functional group. For example, the well-known alanine scanning mutagenesis technique systematically replaces each native residue in a protein or protein domain with alanine to identify which native residues contribute primarily to function. Replacing amino acids with functional groups with alanine eliminates the functional group and its contribution to binding to any receptor, but the presence of the alanine side chain substantially retains the conformation, reducing the possibility of immunogenicity or other complications due to conformational changes. Alternative strategies replace the amino acids in the lower hinge region of a chimeric IgG comprising an IgG1 upper hinge, an IgG1 lower hinge, IgG4 CH2, and IgG1 CH3 with the corresponding amino acids from the human IgG2 isotype, so as to reduce FcγR binding without unacceptable conformational changes, and therewith reduce immunogenicity (see WO14 / 121087). In yet another alternative strategy, amino acid positions 233 to 236 (EU numbering) of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, and the positions are numbered according to EU numbering (e.g., WO2016161010A2 Figure 1 shown).

[0062] An IgG1 heavy chain constant region having a hinge region modified to reduce Fc receptor and / or effector function is provided. The modification occurs by replacing / deleting ELLG (SEQ ID NO: 23) with PVA- at amino acid positions 233 to 236 (EU numbering) (wherein amino acid 236 is deleted). In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0063] As shown in this example, this substitution / deletion mutation (ELLG (SEQ ID NO:23)→PVA- at amino acid positions 233 to 236 (EU numbering)) in an IgG1 background was found to maximize hinge flexibility while reducing FcR engagement and minimizing immunogenicity, thereby providing alternative chimeric heavy chain constant regions for inclusion in, for example, therapeutic antibodies and Fc fusion proteins. It is valuable to have alternative chimeric heavy chain constant regions to those known in the art. Having alternative chimeric heavy chain constant regions based on different IgG subclasses allows, for example, optimization of antibody activity. Such modified immunoglobulin constant regions can be incorporated into virtually any form of antibody or Fc fusion protein. Such antibodies or fusion proteins can be used in therapeutic methods, particularly therapeutic methods in which the mechanism of action of the antibody or Fc fusion protein is primarily or entirely independent of effector function, such as in cases where the antibody inhibits receptor-ligand interactions or causes receptor agonism.

[0064] Excluding the ELLG (SEQ ID NO:23)→PVA-substitution / deletion at amino acid positions 233 to 236 (EU numbering), the chimeric constant domain is considered to be of the IgG1 isotype if it differs from IgG1 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, deletions or insertions, with the following exceptions: the CH1 domain can optionally be omitted entirely, as can the upper hinge region. The CH1, CH2 and CH3 domains are each considered to be of the IgG1 isotype if they differ from the CH1, CH2 and CH3 regions of the IgG1 wild-type sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or insertions. The substitution, deletion and / or insertion (excluding ELLG (SEQ ID NO: 23) → PVA-substitution / deletion at amino acid positions 233 to 236 (EU numbering)) can be any substitution, deletion and / or insertion. In some embodiments, the substitution, deletion and / or insertion does not produce a sequence identical to, for example, CH1, CH2 or CH3 of another IgG isotype (e.g., IgG2, IgG3 or IgG4). For example, in some embodiments, if the chimeric constant domain of the present disclosure comprises one or more of H268Q, K274Q, Y296F, A327G, A330S and P331S (EU numbering), they do not appear at the same time. The wild-type sequences of the heavy chain constant domains of IgG1, IgG2 and IgG4 are depicted in Figure 1 , Figure 2 and Figure 3 , the CH1, hinge, CH2 and CH3 regions are depicted. Exemplary mutations that may be included in the chimeric constant domains of the present disclosure are provided below.

[0065] The sequence of the wild-type IgG1 heavy chain constant region (amino acids 216 to 447; EU numbering) includes:

[0066] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0067] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT

[0068] ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID

[0069] NO:1), wherein the upper hinge region comprises EPKSCDKTHT (SEQ ID NO:24) (amino acids 216 to 225 (EU numbering); amino acids 1 to 10 of the wild-type IgG1 constant domain sequence); the core hinge comprises CPPC (SEQ ID NO:25) (amino acids 226 to 229 (EU numbering); amino acids 11 to 14 of the wild-type IgG1 constant domain sequence); the lower hinge region comprises PAPELLG (SEQ ID NO:26) (amino acids 230 to 236 (EU numbering); amino acids 15 to 21 of the wild-type IgG1 constant domain sequence), and CH2 comprises

[0070] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 27) (amino acids 237-340 (EU numbering); amino acids 22 to 125 of the wild-type IgG1 constant domain sequence), and CH3 comprises

[0071] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28) (amino acids 341 to 447 (EU numbering); amino acids 126 to 232 of the wild-type IgG1 constant sequence). Those skilled in the art will recognize that amino acids 237 to 238 (EU numbering) represent both the C-terminus of the lower hinge and the N-terminus of the CH2 region. However, for the purposes of this disclosure, amino acids 237 to 238 (EU numbering) are shown as part of the lower hinge.

[0072] In some embodiments, the sequence of the chimeric constant region of the present disclosure, also referred to herein as IgG1 PVA (amino acids 216 to 447; EU numbering)) comprises:

[0073] EPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0074] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

[0075] SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:2), wherein the upper hinge region comprises EPKSCDKTHT (SEQ ID NO:56) (amino acids 216 to 225 (EU numbering); amino acids 1 to 10 of the IgG1 PVA constant domain sequence); the core hinge comprises CPPC (SEQ ID NO:57) (amino acids 226 to 229 (EU numbering); amino acids 11 to 14 of the IgG1 PVA constant domain sequence); the lower hinge region comprises PAPPVA (SEQ ID NO:29) (amino acids 230 to 236 (EU numbering); amino acids 15 to 20 of the IgG1 PVA constant domain sequence), and CH2 comprises

[0076] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:58) (amino acids 237 to 340 (EU numbering); amino acids 21 to 124 of the IgG1 PVA sequence), and CH3 contains GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:59) (amino acids 341-447 (EU numbering); amino acids 125 to 231 of the IgG1 PVA sequence).

[0077] In some embodiments, the chimeric constant domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, or 100% sequence identity to:

[0078] EPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0079] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

[0080] SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2).

[0081] The chimeric constant domains may also include knob mutations, hole mutations, star mutations, disulfide bridge formation mutations, etc. to facilitate heterodimerization and / or purification.

[0082] In some embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 3 (IgG1 PVA).

[0083] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 15 (IgG1 PVA with knob mutation T366W, EU numbering).

[0084] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 16 (IgG1 PVA with hole mutations T366S, L368A, Y407V, EU numbering).

[0085] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 17 (IgG1 PVA with knob mutation T366W and star mutations H435R, Y436F, EU numbering).

[0086] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 18 (IgG1 PVA with hole mutations T366S, L368A, Y407V and star mutations H435R, Y436F, EU numbering).

[0087] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO: 19 (IgG1 PVA with knob mutation T366W and Cys mutation S354C, EU numbering).

[0088] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO:20 (IgG1 PVA with hole mutations T366S, L368A, Y407V, and Cys mutation S354C, EU numbering).

[0089] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO:21 (IgG1 PVA with knob mutation T366W, star mutation H435R, Y436F, and Cys mutation S354C, EU numbering).

[0090] In other embodiments, the chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the chimeric constant domain of SEQ ID NO:22 (IgG1 PVA with hole mutations T366S, L368A, Y407V, star mutations H435R, Y436F, and Cys mutation S354C, EU numbering).

[0091] In some embodiments, the chimeric constant domain comprises a CH1 domain or a fragment thereof at the N-terminus of the chimeric constant domain. In some embodiments, the CH1 domain comprises

[0092] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30) or a fragment or variant thereof, e.g., a variant having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or insertions. In some embodiments, the chimeric constant domain comprises a CH1 fragment. In certain embodiments, the CH1 fragment comprises or consists of DKKV (SEQ ID NO: 31). In other embodiments, the CH1 fragment comprises or consists of DKRV (SEQ ID NO: 32).

[0093] 6.2.1.1. Modified chimeric constant domains

[0094] In some embodiments, the chimeric constant domains are modified to further alter effector function and / or provide heterodimerization. Exemplary modifications are described in Sections 0 and 0.

[0095] 6.2.1.1.1. Chimeric constant domains with further altered effector functions

[0096] In some embodiments, the chimeric constant domain comprises one or more amino acid substitutions in addition to the ELLG (SEQ ID NO:23)→PVA-substitution / deletion at amino acid positions 233 to 236 (EU numbering), wherein the additional amino acid substitutions further reduce binding to an Fc receptor and / or effector function.

[0097] In some embodiments, the Fc receptor is an Fcγ receptor. In certain embodiments, the Fc receptor is a human Fc receptor. In a specific embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is selected from one or more of the groups of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0098] In one embodiment, the chimeric constant domain comprises one or more amino acid substitutions at one or more of positions L234, L235, G237, D265, N297, P329, A330, P331, and P329 (EU numbering).

[0099] In some embodiments, the chimeric constant domain comprises an amino acid substitution at position P329 (EU numbering). In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (EU numbering). In one embodiment, the chimeric constant domain comprises an amino acid substitution at position P329 and another amino acid substitution at position N297 and / or P331 (EU numbering). In a more specific embodiment, the other amino acid substitution is N297A or N297D and / or P331S.

[0100] In some embodiments, the chimeric constant domain comprises amino acid substitutions at positions G237, A330, and P331 (EU numbering). In more specific embodiments, the amino acid substitutions are G237A, A330S, and P331S (EU numbering).

[0101] In some embodiments, the chimeric constant domain comprises D265A and N297A mutations (EU numbering) to reduce effector function.

[0102] When incorporated into an antibody or fusion protein, the same one or more additional amino acid substitutions may be present in each of the two chimeric constant domains contained in the antibody or fusion protein. For example, in a specific embodiment, each chimeric constant domain comprises amino acid substitutions G237A, A330S and P331S (EU numbering), i.e., in each of the first chimeric constant domain and the second chimeric constant domain, the glycine residue at position 237 is replaced by an alanine residue (G237A), the alanine residue at position 330 is replaced by a serine residue (A330S), and the proline residue at position 331 is replaced by a serine residue (P331S) (EU numbering).

[0103] 6.2.1.1.2. Chimeric Constant Domain Heterodimerization Variants

[0104] In certain embodiments, the recombinant polypeptide of the present disclosure is dimerized via a chimeric constant domain. In certain embodiments, the resulting dimer forms an antibody or Fc fusion protein or forms a part of an antibody or Fc fusion protein. In certain embodiments, the first recombinant polypeptide dimerizes with the same second recombinant polypeptide to form a homodimer. In other embodiments, the first recombinant polypeptide dimerizes with a second recombinant polypeptide that is not identical to the first recombinant polypeptide to form a heterodimer. For example, the first recombinant polypeptide may include a first target binding domain that is specific to the first target molecule, and the second recombinant polypeptide may include a second target binding domain that is specific to the second target molecule. When the first recombinant polypeptide and the second recombinant polypeptide dimerize via their respective chimeric constant domains, the result is a heterodimer. The insufficient heterodimerization of the recombinant polypeptide (e.g., the heterodimerization of the chimeric constant domains of each recombinant polypeptide to form an Fc region) may be an obstacle to increasing the yield of the desired heterodimer molecule, and poses a challenge to purification. A variety of methods available in the art can be used to enhance dimerization of the recombinant polypeptides of the present disclosure, such as disclosed in EP 1870459A1; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0105] The present disclosure provides heterodimers comprising recombinant polypeptides of the present disclosure. In some embodiments, the heterodimer is an antibody. In other embodiments, the heterodimer is an Fc fusion protein. The heterodimerization of the recombinant polypeptide via the CH3 domain of the chimeric constant domain can produce the desired heterodimer (e.g., antibody or fusion protein), while the homodimerization of the same chimeric constant domain will reduce the yield. Therefore, in some embodiments, the recombinant polypeptide that associates to form an antibody or fusion protein of the present disclosure will include a chimeric constant domain containing a modified CH3 domain that is conducive to heterodimer association relative to an unmodified constant domain.

[0106] In a specific embodiment, the modification that promotes the formation of heterodimers is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the chimeric constant domains and a "hole" modification in the other chimeric constant domain. Knob-hole technology is described in the following literature: for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size to the protuberances are created in the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (eg, alanine or threonine).

[0107] Therefore, in some embodiments, the amino acid residues in the CH3 domain of the first chimeric constant domain are replaced by amino acid residues with a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first chimeric constant domain, which can be positioned in the cavity in the CH3 domain of the second chimeric constant domain, and the amino acid residues in the CH3 domain of the second chimeric constant domain are replaced by amino acid residues with a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second chimeric constant domain, and the protrusion in the CH3 domain of the first chimeric constant domain can be positioned in the cavity. Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). Protrusions and cavities can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutation formation or by peptide synthesis.

[0108] In a specific embodiment, in the first chimeric constant domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second chimeric constant domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (EU numbering). In a further embodiment, in the first chimeric constant domain, additionally, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (specifically, the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second chimeric constant domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (EU numbering). In a specific embodiment, the first chimeric constant domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (EU numbering).

[0109] In some embodiments, electrostatic manipulation (eg, as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote the association of the first chimeric constant domain and the second chimeric constant domain.

[0110] As an alternative to or in addition to using a chimeric constant domain modified to promote heterodimerization, the chimeric constant domain can be modified to allow a purification strategy that enables the selection of chimeric constant domain heterodimers. In one such embodiment, a recombinant polypeptide comprises a modified chimeric constant domain that eliminates its binding to protein A, thereby enabling a purification method for producing heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Therefore, an antibody or fusion protein may comprise a first CH3 domain and a second CH3 domain, wherein the first and second CH3 domains are different from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the antibody or fusion protein to protein A compared to a corresponding antibody or fusion protein lacking amino acid differences. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as H435R modification (EU numbering). The second CH3 may further include Y436F modification (EU numbering). Such modifications are referred to herein as "star" mutations.

[0111] In some embodiments, the chimeric constant domain may contain one or more mutations (eg, knob and hole mutations) to promote heterodimerization, and star mutations to promote purification.

[0112] 6.2.2. Antibodies

[0113] In some embodiments, recombinant polypeptides comprising chimeric constant domains dimerize to form antibodies. The recombinant polypeptides can be used for any type of engineered antibodies, including chimeric antibodies, humanized antibodies, veneered antibodies, or human antibodies. The antibodies can be monoclonal antibodies or genetically engineered polyclonal antibodies.

[0114] The antibodies of the present disclosure generally comprise or consist of recombinant polypeptides of the present disclosure, each of which comprises a chimeric constant domain and one or more antigen binding domains and optionally one or more linkers separating the one or more domains in the recombinant polypeptide.

[0115] The antibody may be composed of one or more recombinant polypeptides of the present disclosure. In some embodiments, the antibody is composed of two recombinant polypeptides optionally associated with one or more additional polypeptide chains (e.g., polypeptide chains of light chains comprising antigen binding domains, such as in the case of Fab). The two recombinant polypeptides may be identical to form homodimers, or different to form heterodimers. The two recombinant polypeptides are typically configured to dimerize together via the chimeric constant domains of each recombinant polypeptide. In some embodiments, one or more additional polypeptide chains are associated with the dimerized recombinant polypeptide. Therefore, the antibody may comprise two, three, four or more polypeptide chains.

[0116] When two recombinant polypeptides include antigen-binding domains targeting the same epitope on the same target molecule, the antibody is monospecific. When two recombinant polypeptides include antigen-binding domains targeting two or more different epitopes or two or more different target molecules on the same target molecule alone or together, the antibody or fusion protein is multispecific (e.g., bispecific). Most antibodies are multimeric by dimerization via the chimeric constant domains of the recombinant polypeptide. Multispecific antibodies or fusion proteins can be monovalent for the first antigen-binding domain and can be monovalent for the second antigen-binding domain and subsequent antigen-binding domains, or can be monovalent for the first antigen-binding domain, multivalent for the second antigen-binding domain and monovalent or multivalent for any additional (e.g., the third) antigen-binding domain.

[0117] A particularly preferred format of an antibody comprising a constant domain of the present disclosure is an N-terminal scFv format (e.g., as described in WO2021 / 091953A1, the contents of which are incorporated herein by reference in their entirety; examples of N-terminal scFv formats are described herein Figure 5 In some embodiments, the antibody is trispecific and is in the form of a "2+1N-scFv" or "2+1N-Fab" where a conventional bispecific antibody has a scFv or Fab domain, preferably connected to the N-terminus of one of its VH domains via a linker. Suitable linkers are disclosed in Section 6.2.5.

[0118] As disclosed in WO 2021 / 091953 A1, the "2+1N-scFv" format generally comprises: (a) a first polypeptide chain, which comprises (i) a scFv comprising a first antigen binding site ("ABS1") in an N-terminal to C-terminal orientation, which is operably linked to (ii) a first heavy chain region of a first Fab ("Fab1"), which is operably linked to (iii) an Fc domain; (b) a second polypeptide chain, which comprises (i) a second heavy chain region of a second Fab ("Fab2") in an N-terminal to C-terminal orientation, which is operably linked to (ii) an Fc domain; (c) a third polypeptide chain, which comprises a first light chain paired with the first heavy chain region to form Fab1, wherein Fab1 comprises a second antigen binding site ("ABS2"); (d) a fourth polypeptide chain, which comprises a second light chain paired with the second heavy chain region to form Fab2, wherein Fab2 comprises a third antigen binding site ("ABS3"). In some embodiments, ABS1, ABS2, and ABS3 each bind to a different epitope. In some embodiments, both of ABS1, ABS2 and ABS3 specifically bind to different epitopes of the same target molecule. In some embodiments, scFv, Fab1 and Fab2 can specifically bind to their respective targets simultaneously. In some embodiments, at least one of ABS1, ABS2 and ABS3 specifically binds to a target molecule with a first tissue expression profile, and at least one of ABS1, ABS2 and ABS3 specifically binds to a target molecule with a second tissue expression profile, which overlaps but is not identical to the first tissue expression profile. In some embodiments, scFv is linked to the first heavy chain region via a joint. In some embodiments, the antigen binding molecule is trivalent. In some embodiments, scFv is linked to the first heavy chain region via a joint. In some embodiments, the length of the joint is at least 5, 6, 7 or more amino acids. In some embodiments, the length of the joint is at most 30, at most 40 or at most 50 amino acids.

[0119] In some embodiments, relative to antibodies comprising wild-type IgG1 constant heavy chain domains, the effector functions (such as ADCC or ADCP) that rely on Fcγ receptor binding are reduced, for example, by at least 90%, at least 95%, at least 99% or reduced to background levels. Such functions include cell killing or phagocytosis, B cell activation and release of inflammatory mediators, such as cytokines. Some such effects can be quantified by measuring EC50. In certain embodiments, compared to a control antibody with a suitable isotype match of a wild-type constant domain, an antibody comprising a chimeric constant domain of the present invention exhibits less than 20% cytolysis (e.g., % cytotoxicity), less than 10% cytolysis, less than 5% cytolysis, less than 4% cytolysis, less than 3% cytolysis, less than 2% cytolysis or 0% cytolysis or undetectable cytolytic cytotoxic activity, as measured in vitro or in vitro cell killing assays, optionally, measured at an antibody concentration of at least 10 nM.

[0120] In some embodiments, antibodies comprising recombinant polypeptides of the present disclosure exhibit at least 5-fold reduced binding to a human Fc receptor (FcR), at least 10-fold reduced binding, at least 50-fold reduced binding, at least 100-fold reduced binding, at least 500-fold reduced binding, at least 1,000-fold reduced binding, at least 5,000-fold reduced binding, or at least 10,000-fold reduced binding compared to an antibody comprising a wild-type IgG1 constant domain. The FcR can be, for example, FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B. For example, FcR binding can be determined by ELISA as described in Section 8.1.4.

[0121] In some embodiments, the binding affinity of an antibody incorporating a chimeric constant domain of the present disclosure to a target is substantially unaffected by the chimeric constant domain relative to a suitable heavy chain constant domain (e.g., wild-type IgG1). That is, the binding affinity is generally the same within experimental error, or at least within a 2-fold or 3-fold range of a suitable control antibody with an isotype-matched wild-type constant domain. This is also true for functional properties that are independent of FcγR binding, such as the ability to inhibit receptor-ligand binding (e.g., EC50), or the ability to agonize a receptor.

[0122] The immunogenicity of antibodies incorporating recombinant polypeptides of the present invention (and thus chimeric constant domains of the present invention) can be assessed in vitro by dendritic cell maturation or T cell proliferation upon challenge (Gaitonde et al., 2011, Methods Mol Biol 716:267-80) or in vivo by comparing the incidence of reactive antibodies to the administered antibodies between populations compared to isotype-matched controls. In some embodiments, the immunogenicity of antibodies incorporating recombinant polypeptides of the present invention (and thus chimeric constant domains of the present invention) is reduced. In other embodiments, the immunogenicity is not significantly different from an isotype-matched control, or is not 2-fold, 3-fold, or 5-fold worse than an isotype-matched control. Similarly, pharmacokinetic parameters (such as C 最大 , C 平均 The PK parameters (e.g., area under the curve, and half-life) are preferably not significantly different or at least not less than 2-fold, 3-fold, or 5-fold compared to isotype matched controls. Substantial maintenance or improvement of such PK parameters can provide an indication that the antibodies of the present disclosure have not undergone substantial conformational changes that trigger enhanced clearance mechanisms.

[0123] In certain embodiments, compared to an antibody comprising a wild-type IgG1 constant domain (e.g., comprising an IgG1 hinge, IgG1CH2, and IgG1CH3), the antibodies of the present disclosure show increased expression in an expression system. In certain embodiments, compared to an antibody comprising a wild-type IgG1 heavy chain constant domain, the increase is at least 5% increase, at least 10% increase, at least 10% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 40% increase, at least 50% increase, at least 60% increase, at least 70% increase, at least 80% increase, at least 90% increase, or at least 100% increase. In certain aspects, the increase in antibody expression is demonstrated by an increase in secretory proteins (such as antibodies) produced from an in vitro expression system (e.g., an antibody expression system) and / or an increase in total protein production. In certain embodiments, the expression system is a Chinese hamster ovary (CHO) stable expression system. The increase in expression in the CHO stable expression system can be appropriately evaluated as described in section 8.1.6, by stimulating protein production by inducing the culture with doxycycline for multiple days. In other embodiments, the expression system is a HEK293-based expression system, e.g., Expi293F TM Expression system.

[0124] 6.2.3. Fusion proteins

[0125] In some embodiments, recombinant polypeptides comprising chimeric constant domains dimerize to form fusion proteins. Fusion proteins of the present disclosure generally comprise or consist of recombinant polypeptides of the present disclosure, each of which comprises a chimeric constant domain and one or more heterologous polypeptides and optionally one or more linkers separating one or more domains in the recombinant polypeptide.

[0126] The fusion protein may be composed of one or more recombinant polypeptides of the present disclosure. In some embodiments, the fusion protein is composed of two recombinant polypeptides. The two recombinant polypeptides may be identical to form a homodimer, or different to form a heterodimer. The two recombinant polypeptides are generally configured to dimerize together via the chimeric constant domains of each recombinant polypeptide. In some embodiments, one or more additional polypeptide chains are associated with the dimerized recombinant polypeptide. Therefore, the fusion protein may include two, three, four or more polypeptide chains.

[0127] In some embodiments, the recombinant polypeptide comprises a chimeric constant domain and a heterologous polypeptide. In some embodiments, the heterologous polypeptide in the fusion protein is a polypeptide that is non-naturally linked to an immunoglobulin constant domain, including an engineered form of an antigen binding domain. The heterologous polypeptide can be any other protein molecule of interest. Exemplary heterologous polypeptides include, but are not limited to, cytokines, ligands, peptide antigens for pathogens, extracellular receptor domains and functional fragments thereof (e.g., soluble receptors), and non-naturally occurring antigen binding domains (e.g., engineered antigen binding domains such as scFv, domain exchange Fab). Therefore, in some embodiments, the fusion protein can be an antibody. Exemplary receptor proteins whose extracellular domains can bind to chimeric constant domains in recombinant polypeptides are known in the art (see, e.g., Klinkert et al., 1997, J Neuroimmunol 72(2):163-8; Milligan et al., 2004, Curr Pharm Des 10(17):1989-2001; and Schwache and Muller-Newen, 2012, Eur J Cell Biol 91(6-7):428-34).

[0128] In some embodiments, the fusion protein is a homodimer. In other embodiments, the fusion protein is a heterodimer.

[0129] In certain embodiments, the fusion protein is monospecific (e.g., comprising a target binding domain specific for a single target molecule, or comprising a single ligand domain). In other embodiments, the fusion protein is multispecific (e.g., comprising two or more target binding domains, each of which is specific for a different target molecule, or comprising two or more different ligand domains). Most fusion proteins are multimers by dimerization via the chimeric constant domains of the recombinant polypeptide.

[0130] In some embodiments, relative to the fusion protein comprising the wild-type IgG1 constant heavy chain domain, the effector function (such as ADCC or ADCP) that depends on Fcγ receptor binding is reduced by, for example, at least 90%, at least 95%, at least 99% or reduced to background levels. Such functions include cell killing or phagocytosis, B cell activation and release of inflammatory mediators, such as cytokines. Some such effects can be quantified by measuring EC50. In certain embodiments, compared with the control fusion protein with the suitable isotype matching of the wild-type constant domain, the fusion protein comprising the chimeric constant domain of the present disclosure shows less than 20% cytolysis (e.g., % cytotoxicity), less than 10% cytolysis, less than 5% cytolysis, less than 4% cytolysis, less than 3% cytolysis, less than 2% cytolysis or 0% cytolysis or undetectable cytolysis cytotoxic activity, as measured in vitro or in vitro cell killing assays, alternatively, measured at a fusion protein concentration of at least 10 nM.

[0131] In some embodiments, the fusion protein comprising a recombinant polypeptide of the present disclosure exhibits at least 5-fold reduced binding to a human Fc receptor (FcR), at least 10-fold reduced binding, at least 50-fold reduced binding, at least 100-fold reduced binding, at least 500-fold reduced binding, at least 1,000-fold reduced binding, at least 5,000-fold reduced binding, or at least 10,000-fold reduced binding compared to a fusion protein comprising a wild-type IgG1 constant domain. The FcR can be, for example, FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B. For example, FcR binding can be determined by ELISA as described in Section 8.1.4.

[0132] In some embodiments, the binding affinity of the fusion protein incorporating the chimeric constant domain of the present disclosure to the target is substantially unaffected by the chimeric constant domain relative to the appropriate heavy chain constant domain (e.g., wild-type IgG1). That is, the binding affinity is generally the same within the experimental error range, or at least within a 2-fold or 3-fold range of an appropriate control fusion protein with an isotype-matched wild-type constant domain. The same is true for functional properties that are independent of FcγR binding, such as the ability to inhibit receptor-ligand binding (e.g., EC50), or the ability to antagonize receptors.

[0133] In some embodiments, the immunogenicity of the fusion protein incorporating the recombinant polypeptide of the present disclosure (and thus the chimeric constant domain of the present disclosure) is reduced. In other embodiments, the immunogenicity is not significantly different from the isotype-matched control, or is not 2-fold, 3-fold, or 5-fold worse than the isotype-matched control. Similarly, pharmacokinetic parameters (such as C 最大 , C 平均 The PK parameters (e.g., area under the curve, and half-life) are preferably not significantly different or at least not less than 2-fold, 3-fold, or 5-fold compared to isotype matched controls. The substantial maintenance or improvement of such PK parameters can provide an indication that the fusion protein of the present disclosure has not undergone a substantial conformational change that triggers an enhanced clearance mechanism.

[0134] In certain embodiments, compared to a fusion protein comprising a wild-type IgG1 constant domain (e.g., comprising an IgG1 hinge, IgG1CH2, and IgG1CH3), the fusion protein of the present disclosure may show increased expression in an expression system. In certain embodiments, compared to a fusion protein comprising a wild-type IgG1 heavy chain constant domain, the increase is at least 5% increase, at least 10% increase, at least 10% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 40% increase, at least 50% increase, at least 60% increase, at least 70% increase, at least 80% increase, at least 90% increase, or at least 100% increase. In certain aspects, the increase in secretory protein (such as fusion protein) produced by an in vitro expression system (e.g., a fusion protein expression system) and / or an increase in total protein production demonstrates the increase in fusion protein expression. In certain embodiments, the expression system is a Chinese hamster ovary (CHO) stable expression system. Increased expression in a CHO stable expression system can be suitably assessed as described in Section 8.1.6, by inducing the culture with doxycycline for multiple days to stimulate protein production. In other embodiments, the expression system is a HEK293-based expression system, for example, the Expi293F expression system.

[0135] 6.2.4. Target binding domain

[0136] The recombinant polypeptide of the present disclosure may comprise one or more target binding domains (e.g., one, two, three or more target binding domains), each of which specifically binds to a selected target molecule. In some embodiments, the recombinant polypeptide comprises a single target binding domain. The target binding domain may be at the N-terminus of a chimeric constant domain or at the C-terminus of a chimeric constant domain. In some embodiments, a single target binding domain is connected to a chimeric constant domain via a linker.

[0137] In some embodiments, the recombinant polypeptide comprises two or more target binding domains. Two or more target binding domains may be the same or may be different. In some embodiments, two or more target binding domains are the same and bind to the same epitope on the target molecule. In other embodiments, two or more target binding domains are different and either bind to different epitopes on the target molecule or bind to different target molecules. In some embodiments, the recombinant polypeptide comprises three or more target binding domains. In such embodiments, two of the target binding domains may be the same and bind to the same epitope on the first target molecule, while the remaining target binding domains are different and bind to different epitopes on the first target molecule or bind to different target molecules. When the recombinant polypeptide comprises two or more target binding domains, each of the two or more target binding domains may be at the N-terminus of the chimeric constant domain, each of the two or more target binding domains may be at the C-terminus of the chimeric constant domain, or one (or more) target binding domains may be at the N-terminus of the chimeric constant domain, while the other target binding domains are at the C-terminus of the chimeric constant domain. The two or more target binding domains and the chimeric constant domains may be separated by one or more linkers.

[0138] The target binding domain can be specific for any epitope and / or target molecule of interest. The target molecule can be human, mammalian or bacterial. The target can be an antigen such as proteins, glycoproteins and carbohydrates from microbial pathogens (both viral and bacterial) and tumors.

[0139] The target binding domain can be an antigen binding domain or antigen binding fragment of an antibody. In some embodiments, the antigen binding domain is an antigen binding domain of a commercial antibody.

[0140] In certain aspects, the target binding domain contained in the recombinant polypeptide of the present disclosure can be any type of antibody fragment that specifically binds to a selected target molecule or an epitope thereof. Antibody fragments include, but are not limited to, VH (or V H ) fragment, VL (or V L ) fragment, Fab fragment, F(ab') 2Fragments, scFv fragments, Fv fragments, miniantibodies, double antibodies, three antibodies and four antibodies. When the antigen binding domain comprises two independent polypeptide chains (e.g., Fab), the first polypeptide chain (e.g., comprising VH) may be included in the recombinant polypeptide, and the second polypeptide chain (e.g., comprising VL) may be included in a polypeptide capable of binding to the VH contained in the recombinant polypeptide. Exemplary targets of antibody molecules comprising constant domains of the present disclosure are antigens expressed on the cell surface, such as proteins, carbohydrates and lipids. In some embodiments, the target molecule is a protein molecule. Exemplary target molecules include human Klosso β ("KLB"), human fibroblast growth factor receptor 1c isoform ("FGFR1c"), human fibroblast growth factor receptor 3 ("FGFR3"), human CD63 and human amyloid precursor protein 2 (APLP2). Exemplary KLB targeting domains are listed in Tables 2A and 2B of WO 2021 / 091953 A1. Exemplary FGFR1c targeting domains are listed in Tables 3A and 3B of WO 2021 / 091953 A1. Exemplary FGFR3 binding domains are listed in Table 4 of WO 2021 / 091953 A1. Exemplary APLP2 binding domains are listed in Table 5 of WO 2021 / 091953 A1. Exemplary CD3 binding domains are listed in Table 6 of WO 2021 / 091953 A1. The binding domains can be configured in a 2+1 N-Fab format described in WO 2021 / 091953 A1 or in a 2+1 N-Fab format in which the N-terminal scFv of WO 2021 / 091953 is replaced by an N-terminal Fab. The contents of WO 2021 / 091953 A1 are incorporated herein by reference in their entirety.

[0141] In other aspects, the target binding domain contained in the recombinant polypeptide of the present disclosure can be any type of receptor or a target binding portion thereof that specifically binds to a selected target molecule.

[0142] 6.2.4.1.Fab

[0143] Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In antibodies comprising a recombinant polypeptide of the present disclosure, the Fab domain is typically recombinantly expressed, wherein at least a portion of the Fab is contained in the recombinant polypeptide.

[0144] The Fab domain may comprise a constant domain and a variable region sequence from any suitable species, and may therefore be murine, chimeric, human or humanized. In certain embodiments, the variable region sequence and / or constant domain sequence are derived from a known antibody. Examples of known antibodies are provided above.

[0145] The Fab domain generally comprises a CH1 domain connected to a VH domain, which is paired with a CL domain connected to a VL domain. In wild-type immunoglobulins, the VH domain is paired with the VL domain to form the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding module. The disulfide bond between the two constant domains can further stabilize the Fab domain.

[0146] For antibodies comprising recombinant polypeptides of the present disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same antigen binding domain and minimize the abnormal pairing of Fab domains belonging to different antigen binding domains. For example, the Fab heterodimerization strategy shown in Table 1 below can be used:

[0147]

[0148]

[0149] Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is promoted by exchanging the VL and VH domains of the Fab with each other or by exchanging the CH1 and CL domains with each other, as described, for example, in WO 2009 / 080251.

[0150] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain of the Fab and one or more amino acid modifications in the CL domain and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0151] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of the variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13: 1619-1633 provides definitions of framework residues based on the Kabat, Chotia and IMGT numbering schemes.

[0152] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces is widely described in the literature as lock and key fits, knobs and sockets, protrusions and cavities, donors and acceptors, etc., all of which imply the nature of structural and chemical matching between two interacting surfaces.

[0153] In one embodiment, the one or more modifications introduced introduce new hydrogen bonds at the interface of the Fab component. In one embodiment, the one or more modifications introduced introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0154] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0155] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0156] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In an embodiment, 39K, 62E modifications are introduced in the VH domain, H172A, F174G modifications are introduced in the CH1 domain, 1R, 38D, (36F) modifications are introduced in the VL domain, and L135Y, S176W modifications are introduced in the CL domain. In another embodiment, 39Y modifications are introduced in the VH domain, and 38R modifications are introduced in the VL domain.

[0157] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond to increase the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0158] The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with the constant domain of the T cell receptor, replacing the CL domain with the bb domain of the T cell receptor, and pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0159] Instead of or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each Fab VL region of a recombinant polypeptide or antibody of the present disclosure. In various embodiments, the use of a common light chain as described herein reduces the number of inappropriate types of recombinant polypeptides or antibodies compared to the use of original homologous VLs. In various embodiments, the VL domain of a recombinant polypeptide or antibody is identified from a monospecific antibody comprising a common light chain. In various embodiments, the VH region of a recombinant polypeptide or antibody is comprised of a human heavy chain variable gene fragment rearranged in vivo in a mouse B cell, which mouse B cell has previously been engineered to express a limited human light chain library, or a single human light chain homologous to a human heavy chain, and in response to contact with an antigen of interest, an antibody library containing multiple human VHs homologous to one or more of two possible human VLs is produced, wherein the antibody library is specific to the antigen of interest. The common light chain is a light chain derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, and includes somatically mutated (eg, affinity matured) versions. See, eg, US Pat. No. 10,412,940.

[0160] 6.2.4.2.scFv

[0161] Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as a single-chain polypeptide (e.g., a recombinant polypeptide of the present disclosure), and retain the specificity of the intact antibody from which they are derived. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of the scFv are the linkers identified in Section 6.2.5.

[0162] Unless otherwise specified, a scFv as used herein may have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0163] The scFv may comprise VH and VL sequences from any suitable species, such as mouse, human or humanized VH and VL sequences. In some embodiments, the scFv may comprise VH and VL sequences from known antibodies. Examples of known antibodies are provided above.

[0164] To generate a nucleic acid encoding an scFv, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding any linker described in Section 6.2.5 (usually a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4 to Ser) 3 (SEQ ID NO: 13), such that the VH and VL sequences can be expressed as a continuous single-chain protein, with the VL and VH regions joined by a flexible linker (see, e.g., Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 1990, Nature 348: 552-554).

[0165] 6.2.5. Connectors

[0166] In certain aspects, the present disclosure provides recombinant polypeptides, wherein two or more components of the recombinant polypeptide are connected to each other by a peptide linker (sometimes referred to herein as a "linker" for convenience). As an example and not limitation, a linker can be used to connect (a) a target binding domain and a chimeric constant domain; (b) a first target binding domain and a second target binding domain; or (c) different domains within a target binding domain (e.g., VH and VL domains in an scFv).

[0167] The peptide linker can range in length from 2 amino acids to 60 or more amino acids, and in certain aspects, the peptide linker ranges in length from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids.

[0168] In particular aspects, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and alternatively, is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids in length.

[0169] In some of the foregoing embodiments, the length of the linker ranges from 5 amino acids to 50 amino acids, for example, a length range of 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other embodiments of the foregoing, the length of the linker ranges from 6 amino acids to 50 amino acids, for example, a length range of 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In some of the foregoing embodiments, the length of the linker ranges from 7 amino acids to 50 amino acids, for example, a length range of 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0170] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred.

[0171] Examples of flexible linkers that can be used in the recombinant polypeptides of the present disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are or contain repeats of glycine and serine, such as G n S (SEQ ID NO: 33) or SG n (SEQ ID NO: 34), wherein n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the linker is or comprises G 4 S (SEQ ID NO: 11) is a repeating monomer or polymer, for example, (GGGGS) n (SEQ ID NO:35).

[0172] Polyglycine linkers can be appropriately used in the recombinant polypeptides of the present disclosure. In some embodiments, the peptide linker comprises two consecutive glycine (2Gly), three consecutive glycine (3Gly), four consecutive glycine (4Gly) (SEQ ID NO: 36), five consecutive glycine (5Gly) (SEQ ID NO: 37), six consecutive glycine (6Gly) (SEQ ID NO: 38), seven consecutive glycine (7Gly) (SEQ ID NO: 39), eight consecutive glycine (8Gly) (SEQ ID NO: 40) or nine consecutive glycine (9Gly) (SEQ ID NO: 41).

[0173] 6.3. Nucleic Acids and Host Cells

[0174] In another aspect, the present disclosure provides nucleic acids encoding chimeric constant domains, recombinant polypeptides, antibodies and fusion proteins of the present disclosure. In some embodiments, the chimeric constant domains, recombinant polypeptides, antibodies and fusion proteins are encoded by a single nucleic acid. In other embodiments where the antibody or fusion protein is composed of multiple polypeptide chains, the antibody or fusion protein may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0175] A single nucleic acid can encode an antibody or fusion protein comprising a single recombinant polypeptide chain, an antibody or fusion protein comprising two or more polypeptide chains, or a portion of an antibody or fusion protein comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an antibody or fusion protein comprising three, four or more polypeptide chains, or three polypeptide chains of an antibody or fusion protein comprising four or more polypeptide chains). For separate controls of expression, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by internal ribosome entry site (IRES) sequences, thereby allowing translation into separate polypeptides.

[0176] In some embodiments, a chimeric constant domain, recombinant polypeptide, antigen or fusion protein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding a chimeric constant domain, recombinant polypeptide, antigen or fusion protein may be equal to or less than the number of polypeptide chains in the chimeric constant domain, recombinant polypeptide, antigen or fusion protein (e.g., when one nucleic acid encodes more than one polypeptide chain).

[0177] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA).

[0178] In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids can be present in a single vector or in separate vectors in the same host cell or in separate host cells, as described in more detail below.

[0179] 6.3.1. Carrier

[0180] The present disclosure provides a vector comprising a nucleotide sequence encoding a chimeric constant domain, a recombinant polypeptide, an antigen or a fusion protein as described herein, such as one or two polypeptide chains of an antibody. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC).

[0181] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus and flavivirus.

[0182] In addition, can select the cell that DNA is stably integrated into its chromosome by introducing one or more markers that allow to select transfected host cells.Mark can provide for example the original plasm of auxotrophic host, biocide resistance (for example, antibiotic) or resistance to heavy metal (for example, copper) etc.Selectable marker gene can be directly linked to DNA sequence to be expressed, or is introduced in the same cell by cotransformation.The best synthesis of mRNA may also need additional elements.These elements can include splicing signal and transcription promoter, enhancer and termination signal.

[0183] Once the expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and for recovering and / or purifying the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the present specification, depending on the specific expression vector and mammalian host cell used.

[0184] 6.3.2. Cells

[0185] The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

[0186] In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids described herein.

[0187] In one embodiment, host cells are genetically engineered by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such sequence. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or helpful to affect expression may also be used, such as, for example, an inducible promoter.

[0188] The present disclosure also provides host cells comprising the vectors described herein.

[0189] Cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0190] 6.4. Pharmaceutical Compositions

[0191] The antibodies and fusion proteins of the present disclosure may be in the form of a composition comprising the antibody or fusion protein and one or more carriers, excipients and / or diluents. The composition may be formulated for specific uses, such as for veterinary use or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid preparation, etc.) and the excipients, diluents and / or carriers used will depend on the intended use of the antibody or fusion protein, and for therapeutic use, will depend on the mode of administration.

[0192] For therapeutic use, the composition can be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of applying it to the patient). The pharmaceutical composition can be applied to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, external application or topical. The most suitable route of administration in any given case will depend on the nature and severity of the specific antibody, the subject and the disease, and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0193] The pharmaceutical composition can conveniently exist in a unit dosage form containing a predetermined amount of the antibody or fusion protein of the present disclosure per dose. The amount of the antibody or fusion protein contained in the unit dosage will depend on the disease being treated and other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing the antibody or fusion protein of an amount suitable for a single administration, or in the form of a liquid. The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent and / or other components for administration. The unit dosage in liquid form can conveniently be provided in the form of a syringe, which is prefilled with an amount of the antibody or fusion protein suitable for a single administration.

[0194] Pharmaceutical compositions may also be supplied in bulk containing an amount of the antibody or fusion protein suitable for multiple administrations.

[0195] The pharmaceutical composition can be prepared as a lyophilized preparation or an aqueous solution for storage by mixing an antibody or fusion protein having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (all of which are referred to herein as "carriers") commonly used in the art, i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other miscellaneous additives. See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare, ed. 2020). Such additives should be nontoxic to the recipient at the dose and concentration used.

[0196] Buffers help maintain the pH in a range close to physiological conditions. They can be present in a variety of concentrations, but are generally present in a concentration ranging from about 2 mM to about 50 mM. Suitable buffers for use with the present invention include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-succinic acid monosodium salt mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-succinic acid disodium mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-fumaric acid monosodium salt mixtures, In some embodiments, the present invention provides a buffer solution containing at least one of the following buffers: a mixture of ...

[0197] Preservatives can be added to slow down microbial growth, and can be added in an amount within the range of about 0.2% to 1% (w / v). Suitable preservatives used with the present invention include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzylammonium halides (e.g., chloride, bromide and iodide), hexamethylammonium chloride and alkylparabens, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents, sometimes referred to as "stabilizers", can be added to ensure the isotonicity of the liquid composition of the present invention, and isotonic agents include polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a large class of excipients, and their functions range from fillers to dissolving therapeutic agents or additives that help prevent denaturation or adhesion to the container wall. Typical stabilizers may be polyols (listed above); amino acids (such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc.), including cyclic alcohols (such as inositol); polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea. , glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount of 0.5 to 10% by weight per weight of the antibody or fusion protein.

[0198] Nonionic surfactants or detergents (also referred to as "wetting agents") can be added to help dissolve glycoproteins and protect glycoproteins from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxysamers (184, 188, etc.) and pluronic polyols. Nonionic surfactants can exist in the range of about 0.05 mg / mL to about 1.0 mg / mL, for example, about 0.07 mg / mL to about 0.2 mg / mL.

[0199] Additional miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and co-solvents.

[0200] 6.4.1. Pharmaceutical compositions for delivery

[0201] The antibodies or fusion proteins of the present disclosure can be delivered by any method useful for gene therapy, such as as mRNA or by a viral vector encoding the antibody or fusion protein under the control of a suitable promoter.

[0202] Exemplary gene therapy vectors include adenovirus- or AAV-based therapeutics. Non-limiting examples of adenovirus- or AAV-based therapeutics for use in the methods, uses, or compositions herein include, but are not limited to: rAd-p53, which is a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53, for example, for the treatment of cancer (also known as Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5-d11520, an adenovirus lacking the E1B gene for inactivating host p53 (also known as H101 or ONYX-015; see, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670); AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF, e.g., for the treatment of cancer (Cerullo et al., 2010, Cancer Res. 70:4297); rAd-HSVtk, a replication-deficient adenovirus with the HSV thymidine kinase gene, e.g., for the treatment of cancer (developed for Ark Therapeutics, see, e.g., U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak TM ; International PCT Application No. WO2005 / 049094); rAd-TNFα, a replication-defective adenoviral vector expressing human tumor necrosis factor α (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter, for example, for the treatment of cancer (TNFerade TM , GenVec; Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7; Ad-IFNβ, an adenovirus serotype 5 vector in which the E1 and E3 genes have been deleted, expresses the human interferon β gene under the guidance of the cytomegalovirus (CMV) immediate early promoter, for example, for the treatment of cancer (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther. 18:852-860).

[0203] Nucleic acid molecules (e.g., mRNA) or viruses can be formulated into the only active pharmaceutical ingredient in a pharmaceutical composition, or can be combined with other active agents for a specific disease to be treated. Alternatively, other medicaments, pharmaceutical agents, carriers, adjuvants, diluents may be included in the compositions provided herein. For example, wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate) and colorants, release agents, coating agents, sweeteners, flavoring agents and flavoring agents, preservatives, antioxidants, chelating agents and inert gases may also be present in the composition. Other exemplary agents and excipients that may be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. 7. Specific embodiments

[0205] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. The present disclosure is illustrated by the numbered embodiments set forth below as Group A, Group B, Group C, and Group D. Unless otherwise specified, the features of any numbered embodiment of a particular group are applicable to the numbered embodiments of the other groups mutatis mutandis.

[0206] Embodiments numbered as Group A

[0207] 1. A recombinant polypeptide comprising a chimeric constant domain, comprising from N-terminus to C-terminus:

[0208] (a) a chimeric immunoglobulin hinge comprising:

[0209] (i) The amino acid sequence of the hinge of human IgG1 from position 216 to 225 (EU numbering)

[0210] EPKSCDKTHT (SEQ ID NO: 24)

[0211] (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) from position 226 to 229 (EU numbering);

[0212] (iii) Human IgG2 lower hinge amino acid sequence from position 230 to 238 (EU numbering)

[0213] PCPAPPVAGP (SEQ ID NO: 42);

[0214] (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur simultaneously; and

[0215] (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitution is selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing.

[0216] 2. The recombinant polypeptide of embodiment 1, wherein the chimeric constant domain comprises or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0217] 3. The recombinant polypeptide of embodiment 1 or embodiment 2, wherein the chimeric constant domain comprises or consists of an amino acid sequence that is at least 96% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0218] 4. The recombinant polypeptide of any one of embodiments 1 to 3, wherein the chimeric constant domain comprises or consists of an amino acid sequence that is at least 97% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0219] 5. The recombinant polypeptide of any one of embodiments 1 to 4, wherein the chimeric constant domain comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0220] 6. The recombinant polypeptide of any one of embodiments 1 to 5, wherein the chimeric constant domain comprises or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0221] 7. The recombinant polypeptide of any one of embodiments 1 to 6, wherein the chimeric constant domain comprises or consists of the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0222] 8. The recombinant polypeptide of any one of embodiments 1 to 7, wherein the optional one or more CH2 domain amino acid substitutions reduce binding of the chimeric constant domain to an Fc receptor and / or reduce effector function.

[0223] 9. The recombinant polypeptide of any one of embodiments 1 to 8, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position G237 (EU numbering).

[0224] 10. The recombinant polypeptide of any one of embodiments 1 to 9, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position D265 (EU numbering).

[0225] 11. The recombinant polypeptide of embodiment 10, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution D265A (EU numbering).

[0226] 12. The recombinant polypeptide of any one of embodiments 1 to 11, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position N297 (EU numbering).

[0227] 13. The recombinant polypeptide of embodiment 12, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution N297A (EU numbering).

[0228] 14. The recombinant polypeptide of embodiment 12, wherein the optional one or more CH2 domain amino acid substitutions comprise amino acid substitution N297D (EU numbering).

[0229] 15. The recombinant polypeptide of any one of embodiments 1 to 14, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position P329 (EU numbering).

[0230] 16. The recombinant polypeptide of embodiment 15, wherein the optional one or more CH2 domain amino acid substitutions comprise amino acid substitution P329A (EU numbering).

[0231] 17. The recombinant polypeptide of embodiment 15, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution P329G (EU numbering).

[0232] 18. The recombinant polypeptide of any one of embodiments 1 to 17, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position A330 (EU numbering).

[0233] 19. The recombinant polypeptide of any one of embodiments 1 to 18, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position P331 (EU numbering).

[0234] 20. The recombinant polypeptide of embodiment 19, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution P331S (EU numbering).

[0235] 21. The recombinant polypeptide of any one of embodiments 1 to 20, wherein the optional one or more CH2 domain amino acid substitutions comprise or consist of amino acid substitutions at positions D265 and N297 (EU numbering).

[0236] 22. The recombinant polypeptide of embodiment 21, wherein the optional one or more CH2 domain amino acid substitutions comprise or consist of D265A and N297A (EU numbering).

[0237] 23. The recombinant polypeptide of any one of embodiments 1 to 22, wherein the optional one or more CH3 domain amino acid substitutions provide heterodimeric association of the chimeric constant domain with another constant domain.

[0238] 24. The recombinant polypeptide of any one of embodiments 1 to 23, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position S354 or E357 (EU numbering).

[0239] 25. The recombinant polypeptide of embodiment 24, wherein the optional one or more CH3 domain amino acid substitutions comprise amino acid substitutions S354C or E357C (EU numbering).

[0240] 26. The recombinant polypeptide of any one of embodiments 1 to 25, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position T366 (EU numbering).

[0241] 27. The recombinant polypeptide of embodiment 26, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution T366W (EU numbering).

[0242] 28. The recombinant polypeptide of any one of embodiments 1 to 27, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of amino acid substitutions at positions S354 and T366 (EU numbering).

[0243] 29. The recombinant polypeptide of embodiment 28, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of S354C and T366W (EU numbering).

[0244] 30. The recombinant polypeptide of any one of embodiments 1 to 23, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y349 (EU numbering).

[0245] 31. A recombinant polypeptide according to embodiment 30, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y349C (EU numbering).

[0246] 32. The recombinant polypeptide of any one of embodiments 1 to 23, 30 or 31, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y407 (EU numbering).

[0247] 33. A recombinant polypeptide according to embodiment 32, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y407V (EU numbering).

[0248] 34. The recombinant polypeptide of embodiment 32 or embodiment 33, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position T366 (EU numbering).

[0249] 35. The recombinant polypeptide of embodiment 34, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution T366S (EU numbering).

[0250] 36. The recombinant polypeptide of any one of embodiments 32 to 35, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position L368 (EU numbering).

[0251] 37. A recombinant polypeptide according to embodiment 36, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution L368A (EU numbering).

[0252] 38. The recombinant polypeptide of any one of embodiments 1 to 23 or 30 to 37, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of amino acid substitutions at positions Y349, T366, L368 and Y407 (EU numbering).

[0253] 39. The recombinant polypeptide of embodiment 38, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of Y349C, T366S, L368A and Y407V (EU numbering).

[0254] 40. The recombinant polypeptide of any one of embodiments 1 to 39, wherein the optional one or more CH3 domain amino acid substitutions provide for selective purification of the recombinant polypeptide.

[0255] 41. A recombinant polypeptide according to embodiment 40, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position H435 (EU numbering).

[0256] 42. A recombinant polypeptide according to embodiment 41, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution H435R (EU numbering).

[0257] 43. A recombinant polypeptide according to embodiment 40, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y436 (EU numbering).

[0258] 44. A recombinant polypeptide according to embodiment 41, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y436F (EU numbering).

[0259] 45. The recombinant polypeptide of any one of embodiments 1 to 44, wherein the recombinant polypeptide is an antigen binding protein.

[0260] 46. ​​The recombinant polypeptide of any one of embodiments 1 to 45, further comprising an IgG1 CH1 domain or a fragment or variant thereof.

[0261] 47. The recombinant polypeptide of embodiment 46, wherein the fragment of IgG1 CH1 comprises or consists of DKKV (SEQ ID NO: 31).

[0262] 48. The recombinant polypeptide of embodiment 46, wherein the fragment of IgG1 CH1 comprises or consists of DKRV (SEQ ID NO: 32).

[0263] 49. The recombinant polypeptide of any one of embodiments 1 to 48, comprising at least one target binding domain.

[0264] 50. The recombinant polypeptide of any one of embodiments 1 to 49, comprising at least two target binding domains.

[0265] 51. The recombinant polypeptide of any one of embodiments 1 to 50, comprising at least three target binding domains.

[0266] 52. The recombinant polypeptide of any one of embodiments 49 to 51, wherein at least one target binding domain comprises a Fab.

[0267] 53. The recombinant polypeptide of any one of embodiments 49 to 52, wherein at least one target binding domain comprises a scFv.

[0268] 54. The recombinant polypeptide of any one of embodiments 49 to 53, comprising a Fab as a first target binding domain and a scFv as a target binding domain.

[0269] 55. The recombinant polypeptide of any one of embodiments 49 to 54, comprising at least one target binding domain at the N-terminus of the chimeric constant domain.

[0270] 56. The recombinant polypeptide of any one of embodiments 49 to 55, comprising at least one target binding domain at the C-terminus of the chimeric constant domain.

[0271] 57. The recombinant polypeptide of any one of embodiments 49 to 56, comprising at least a first target binding domain at the N-terminus of the chimeric constant domain and at least a second target binding domain at the C-terminus of the chimeric constant domain.

[0272] 58. The recombinant polypeptide of any one of embodiments 49 to 57, comprising, from N-terminus to C-terminus, scFv, Fab and chimeric constant domains.

[0273] 59. The recombinant polypeptide according to any one of embodiments 1 to 58, wherein the recombinant polypeptide is or forms part of an antibody, optionally a multispecific antibody.

[0274] 60. The recombinant polypeptide according to any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a monovalent antibody.

[0275] 61. The recombinant polypeptide of any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a bivalent antibody.

[0276] 62. The recombinant polypeptide of embodiment 61, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0277] 63. The recombinant polypeptide of any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a trivalent antibody.

[0278] 64. The recombinant polypeptide of embodiment 63, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0279] 65. The recombinant polypeptide of embodiment 63, wherein the recombinant polypeptide is a trispecific antibody or forms part of a trispecific antibody.

[0280] 66. The recombinant polypeptide of any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a tetravalent antibody.

[0281] 67. The recombinant polypeptide of embodiment 66, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0282] 68. The recombinant polypeptide of embodiment 66, wherein the recombinant polypeptide is a trispecific antibody or forms part of a trispecific antibody.

[0283] 69. The recombinant polypeptide of any one of embodiments 1 to 49, wherein the recombinant polypeptide is a fusion protein or forms part of a fusion protein.

[0284] 70. The recombinant polypeptide of any one of embodiments 59 to 69, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 20% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0285] 71. The recombinant polypeptide of any one of embodiments 59 to 69, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 10% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0286] 72. The recombinant polypeptide of any one of embodiments 59 to 71, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 5% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0287] 73. The recombinant polypeptide of any one of embodiments 59 to 72, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 4% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0288] 74. The recombinant polypeptide of any one of embodiments 59 to 73, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 3% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0289] 75. The recombinant polypeptide of any one of embodiments 59 to 74, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 2% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0290] 76. The recombinant polypeptide of any one of embodiments 59 to 75, wherein the antibody or fusion protein exhibits 0% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0291] 77. The recombinant polypeptide of any one of embodiments 59 to 69, wherein the antibody or fusion protein exhibits undetectable cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0292] 78. The recombinant polypeptide of any one of embodiments 59 to 77, wherein the antibody or fusion protein exhibits increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0293] 79. The recombinant polypeptide of embodiment 78, wherein the expression system is a Chinese hamster ovary (CHO) stable expression system.

[0294] 80. The recombinant polypeptide of embodiment 78 or embodiment 79, wherein the increase is at least 5% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0295] 81. The recombinant polypeptide of any one of embodiments 78 to 80, wherein the increase is at least 10% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0296] 82. The recombinant polypeptide of any one of embodiments 78 to 81, wherein the increase is at least 15% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0297] 83. The recombinant polypeptide of any one of embodiments 78 to 82, wherein the increase is at least 20% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0298] 84. The recombinant polypeptide of any one of embodiments 78 to 83, wherein the increase is at least 25% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0299] 85. The recombinant polypeptide of any one of embodiments 78 to 84, wherein the increase is at least 30% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0300] 86. A recombinant polypeptide according to any one of embodiments 78 to 85, wherein the amount of IgG1 constant domain is increased by at least 40% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0301] 87. A recombinant polypeptide according to any one of embodiments 78 to 86, wherein the protein content is increased by at least 50% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0302] 88. The recombinant polypeptide according to any one of embodiments 78 to 87, wherein the protein content is increased by at least 60% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0303] 89. The recombinant polypeptide according to any one of embodiments 78 to 88, wherein the protein content is increased by at least 70% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0304] 90. The recombinant polypeptide according to any one of embodiments 78 to 89, wherein the protein content is increased by at least 80% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0305] 91. A recombinant polypeptide according to any one of embodiments 78 to 90, wherein the protein content is increased by at least 90% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0306] 92. The recombinant polypeptide of any one of embodiments 78 to 91, wherein the increase is at least 100% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0307] 93. The recombinant polypeptide of any one of embodiments 1 to 92, wherein the recombinant polypeptide exhibits at least 5-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0308] 94. The recombinant polypeptide of any one of embodiments 1 to 93, wherein the recombinant polypeptide exhibits at least 10-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0309] 95. The recombinant polypeptide of any one of embodiments 1 to 94, wherein the recombinant polypeptide exhibits at least 50-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0310] 96. The recombinant polypeptide of any one of embodiments 1 to 95, wherein the recombinant polypeptide exhibits at least 100-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0311] 97. The recombinant polypeptide of any one of embodiments 1 to 96, wherein the recombinant polypeptide exhibits at least 500-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0312] 98. The recombinant polypeptide of any one of embodiments 1 to 97, wherein the recombinant polypeptide exhibits at least 1,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0313] 99. The recombinant polypeptide of any one of embodiments 1 to 98, wherein the recombinant polypeptide exhibits at least 5,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0314] 100. The recombinant polypeptide of any one of embodiments 1 to 99, wherein the recombinant polypeptide exhibits at least 10,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0315] 101. The recombinant polypeptide of any one of embodiments 93 to 100, wherein the FcR is FcRγ1.

[0316] 102. The recombinant polypeptide of any one of embodiments 93 to 100, wherein the FcR is FcRγ2A.

[0317] 103. The recombinant polypeptide of any one of embodiments 93 to 100, wherein the FcR is FcRγ2B.

[0318] 104. The recombinant polypeptide of any one of embodiments 93 to 100, wherein the FcR is FcRγ3A.

[0319] 105. The recombinant polypeptide of any one of embodiments 93 to 100, wherein the FcR is FcRγ3B.

[0320] 106. A composition comprising the recombinant polypeptide of any one of embodiments 1 to 105.

[0321] 107. The composition of embodiment 106, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharmaceutically acceptable carriers.

[0322] 108. A nucleic acid molecule or a plurality of nucleic acid molecules encoding a recombinant polypeptide according to any one of embodiments 1 to 105.

[0323] 109. A host cell engineered to express the recombinant polypeptide of any one of embodiments 1 to 105 or the nucleic acid molecule of embodiment 108.

[0324] 110. A recombinant polypeptide comprising a heavy chain constant (CH) region comprising or consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S and P331S (EU numbering), they do not occur simultaneously.

[0325] 111. A recombinant polypeptide according to embodiment 110, wherein the CH region comprises or consists of an amino acid sequence that is at least 96% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0326] 112. A recombinant polypeptide according to embodiment 110 or embodiment 111, wherein the CH region comprises or consists of an amino acid sequence that is at least 97% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0327] 113. A recombinant polypeptide according to any one of embodiments 110 to 112, wherein the CH region comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0328] 114. A recombinant polypeptide according to any one of embodiments 110 to 113, wherein the CH region comprises or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0329] 115. A recombinant polypeptide according to any one of embodiments 110 to 114, wherein the CH region comprises or consists of the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0330] 116. A recombinant polypeptide according to any one of embodiments 110 to 115, wherein the optional one or more CH2 domain amino acid substitutions reduce the binding of the chimeric constant domain to an Fc receptor and / or reduce effector function.

[0331] 117. The recombinant polypeptide of any one of embodiments 110 to 116, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position G237 (EU numbering).

[0332] 118. The recombinant polypeptide of any one of embodiments 110 to 117, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position D265 (EU numbering).

[0333] 119. A recombinant polypeptide according to embodiment 118, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution D265A (EU numbering).

[0334] 120. The recombinant polypeptide of any one of embodiments 110 to 119, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position N297 (EU numbering).

[0335] 121. A recombinant polypeptide according to embodiment 120, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution N297A (EU numbering).

[0336] 122. A recombinant polypeptide according to embodiment 120, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution N297D (EU numbering).

[0337] 123. A recombinant polypeptide according to any one of embodiments 110 to 122, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position P329 (EU numbering).

[0338] 124. A recombinant polypeptide according to embodiment 123, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution P329A (EU numbering).

[0339] 125. A recombinant polypeptide according to embodiment 123, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution P329G (EU numbering).

[0340] 126. A recombinant polypeptide according to any one of embodiments 110 to 125, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position A330 (EU numbering).

[0341] 127. A recombinant polypeptide according to any one of embodiments 110 to 126, wherein the optional one or more CH2 domain amino acid substitutions comprise an amino acid substitution at position P331 (EU numbering).

[0342] 128. A recombinant polypeptide according to embodiment 127, wherein the optional one or more CH2 domain amino acid substitutions comprise the amino acid substitution P331S (EU numbering).

[0343] 129. The recombinant polypeptide of any one of embodiments 110 to 128, wherein the optional one or more CH2 domain amino acid substitutions comprise or consist of amino acid substitutions at positions D265 and N297 (EU numbering).

[0344] 130. A recombinant polypeptide according to embodiment 129, wherein the optional one or more CH2 domain amino acid substitutions comprise or consist of D265A and N297A (EU numbering).

[0345] 131. A recombinant polypeptide according to any one of embodiments 110 to 130, wherein optional one or more CH3 domain amino acid substitutions provide heterodimeric association of the chimeric constant domain with another constant domain.

[0346] 132. The recombinant polypeptide of any one of embodiments 110 to 131, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position S354 or E357 (EU numbering).

[0347] 133. A recombinant polypeptide according to embodiment 132, wherein the optional one or more CH3 domain amino acid substitutions comprise amino acid substitutions S354C or E357C (EU numbering).

[0348] 134. A recombinant polypeptide according to any one of embodiments 110 to 133, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position T366 (EU numbering).

[0349] 135. A recombinant polypeptide according to embodiment 134, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution T366W (EU numbering).

[0350] 136. The recombinant polypeptide of any one of embodiments 110 to 135, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of amino acid substitutions at positions S354 and T366 (EU numbering).

[0351] 137. A recombinant polypeptide according to embodiment 136, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of S354C and T366W (EU numbering).

[0352] 138. A recombinant polypeptide according to any one of embodiments 110 to 137, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y349 (EU numbering).

[0353] 139. A recombinant polypeptide according to embodiment 138, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y349C (EU numbering).

[0354] 140. The recombinant polypeptide of any one of embodiments 110 to 131, 138 or 139, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y407 (EU numbering).

[0355] 141. A recombinant polypeptide according to embodiment 140, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y407V (EU numbering).

[0356] 142. A recombinant polypeptide according to embodiment 140 or embodiment 141, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position T366 (EU numbering).

[0357] 143. A recombinant polypeptide according to embodiment 142, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution T366S (EU numbering).

[0358] 144. A recombinant polypeptide according to any one of embodiments 140 to 143, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position L368 (EU numbering).

[0359] 145. A recombinant polypeptide according to embodiment 144, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution L368A (EU numbering).

[0360] 146. A recombinant polypeptide according to any one of embodiments 110 to 131 or 140 to 145, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of amino acid substitutions at positions Y349, T366, L368 and Y407 (EU numbering).

[0361] 147. A recombinant polypeptide according to embodiment 146, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of Y349C, T366S, L368A and Y407V (EU numbering).

[0362] 148. The recombinant polypeptide of any one of embodiments 110 to 147, wherein the optional one or more CH3 domain amino acid substitutions provide for selective purification of the recombinant polypeptide.

[0363] 149. A recombinant polypeptide according to embodiment 148, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position H435 (EU numbering).

[0364] 150. A recombinant polypeptide according to embodiment 149, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution H435R (EU numbering).

[0365] 151. A recombinant polypeptide according to embodiment 148, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y436 (EU numbering).

[0366] 152. A recombinant polypeptide according to embodiment 151, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y436F (EU numbering).

[0367] 153. The recombinant polypeptide of any one of embodiments 110 to 152, wherein the recombinant polypeptide is an antigen binding protein.

[0368] 154. The recombinant polypeptide of any one of embodiments 110 to 153, further comprising an IgG1 CH1 domain or a fragment thereof.

[0369] 155. The recombinant polypeptide of embodiment 154, wherein the fragment of IgG1 CH1 comprises or consists of DKKV (SEQ ID NO: 31).

[0370] 156. The recombinant polypeptide of embodiment 154, wherein the fragment of IgG1 CH1 comprises or consists of DKRV (SEQ ID NO: 32).

[0371] 157. The recombinant polypeptide of any one of embodiments 110 to 156, comprising at least one target binding domain.

[0372] 158. The recombinant polypeptide of any one of embodiments 110 to 157, comprising at least two target binding domains.

[0373] 159. The recombinant polypeptide of any one of embodiments 110 to 158, comprising at least three target binding domains.

[0374] 160. The recombinant polypeptide of any one of embodiments 157 to 159, wherein at least one target binding domain comprises a Fab.

[0375] 161. The recombinant polypeptide of any one of embodiments 157 to 160, wherein at least one target binding domain comprises a scFv.

[0376] 162. The recombinant polypeptide of any one of embodiments 157 to 161, comprising a Fab as a first target binding domain and a scFv as a second target binding domain.

[0377] 163. A recombinant polypeptide according to any one of embodiments 157 to 160, comprising at least one target binding domain at the N-terminus of the chimeric constant domain.

[0378] 164. A recombinant polypeptide according to any one of embodiments 157 to 163, comprising at least one target binding domain at the C-terminus of the chimeric constant domain.

[0379] 165. A recombinant polypeptide according to any one of embodiments 157 to 164, comprising at least a first target binding domain at the N-terminus of the chimeric constant domain and at least a second target binding domain at the C-terminus of the chimeric constant domain.

[0380] 166. The recombinant polypeptide of any one of embodiments 157 to 165, comprising, from N-terminus to C-terminus, scFv, Fab and chimeric constant domains.

[0381] 167. A recombinant polypeptide according to any one of embodiments 110 to 166, wherein the recombinant polypeptide is an antibody or forms part of an antibody, optionally a multispecific antibody.

[0382] 168. The recombinant polypeptide of any one of embodiments 110 to 167, wherein the recombinant polypeptide is or forms part of a monovalent antibody.

[0383] 169. The recombinant polypeptide of any one of embodiments 110 to 167, wherein the recombinant polypeptide is a bivalent antibody or forms part of a bivalent antibody.

[0384] 170. The recombinant polypeptide of embodiment 169, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0385] 171. The recombinant polypeptide of any one of embodiments 110 to 167, wherein the recombinant polypeptide is a trivalent antibody or forms part of a trivalent antibody.

[0386] 172. A recombinant polypeptide according to embodiment 171, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0387] 173. A recombinant polypeptide according to embodiment 171, wherein the recombinant polypeptide is a trispecific antibody or forms part of a trispecific antibody.

[0388] 174. A recombinant polypeptide according to any one of embodiments 110 to 167, wherein the recombinant polypeptide is a tetravalent antibody or forms part of a tetravalent antibody.

[0389] 175. The recombinant polypeptide of embodiment 174, wherein the recombinant polypeptide is a bispecific antibody or forms part of a bispecific antibody.

[0390] 176. A recombinant polypeptide according to embodiment 174, wherein the recombinant polypeptide is a trispecific antibody or forms part of a trispecific antibody.

[0391] 177. The recombinant polypeptide of any one of embodiments 110 to 157, wherein the recombinant polypeptide is a fusion protein or forms part of a fusion protein.

[0392] 178. A recombinant polypeptide according to any one of embodiments 167 to 171, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 20% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0393] 179. The recombinant polypeptide of any one of embodiments 167 to 178, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 10% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0394] 180. The recombinant polypeptide of any one of embodiments 167 to 179, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 5% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0395] 181. The recombinant polypeptide of any one of embodiments 167 to 180, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 4% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0396] 182. A recombinant polypeptide according to any one of embodiments 167 to 181, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 3% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0397] 183. A recombinant polypeptide according to any one of embodiments 167 to 182, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 2% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0398] 184. The recombinant polypeptide of any one of embodiments 167 to 183, wherein the antibody or fusion protein exhibits 0% cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0399] 185. The recombinant polypeptide of any one of embodiments 167 to 171, wherein the antibody or fusion protein exhibits undetectable cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0400] 186. The recombinant polypeptide of any one of embodiments 167 to 185, wherein the antibody or fusion protein exhibits increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0401] 187. A recombinant polypeptide according to embodiment 186, wherein the expression system is a Chinese hamster ovary (CHO) stable expression system.

[0402] 188. The recombinant polypeptide of embodiment 186 or embodiment 187, wherein the increase is at least 5% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0403] 189. The recombinant polypeptide of any one of embodiments 186 to 188, wherein the increase is at least 10% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0404] 190. The recombinant polypeptide of any one of embodiments 186 to 189, wherein the increase is at least 15% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0405] 191. The recombinant polypeptide of any one of embodiments 186 to 190, wherein the increase is at least 20% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0406] 192. The recombinant polypeptide of any one of embodiments 186 to 191, wherein the increase is at least 25% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0407] 193. The recombinant polypeptide of any one of embodiments 186 to 192, wherein the increase is at least 30% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0408] 194. A recombinant polypeptide according to any one of embodiments 186 to 193, wherein the amount of the recombinant polypeptide is increased by at least 40% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0409] 195. A recombinant polypeptide according to any one of embodiments 186 to 194, wherein the amount of the recombinant polypeptide is increased by at least 50% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0410] 196. A recombinant polypeptide according to any one of embodiments 186 to 195, wherein the amount of the recombinant polypeptide is increased by at least 60% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0411] 197. A recombinant polypeptide according to any one of embodiments 186 to 196, wherein the amount of the recombinant polypeptide is increased by at least 70% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0412] 198. A recombinant polypeptide according to any one of embodiments 186 to 197, wherein the amount of the recombinant polypeptide is increased by at least 80% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0413] 199. A recombinant polypeptide according to any one of embodiments 186 to 198, wherein the amount of the recombinant polypeptide is increased by at least 90% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0414] 200. The recombinant polypeptide of any one of embodiments 186 to 199, wherein the increase is at least 100% compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain.

[0415] 201. The recombinant polypeptide of any one of embodiments 110 to 200, wherein the recombinant polypeptide exhibits at least 5-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0416] 202. The recombinant polypeptide of any one of embodiments 110 to 201, wherein the recombinant polypeptide exhibits at least 10-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0417] 203. The recombinant polypeptide of any one of embodiments 110 to 202, wherein the recombinant polypeptide exhibits at least 50-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0418] 204. The recombinant polypeptide of any one of embodiments 110 to 203, wherein the recombinant polypeptide exhibits at least 100-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0419] 205. The recombinant polypeptide of any one of embodiments 110 to 204, wherein the recombinant polypeptide exhibits at least 500-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0420] 206. The recombinant polypeptide of any one of embodiments 110 to 205, wherein the recombinant polypeptide exhibits at least 1,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0421] 207. The recombinant polypeptide of any one of embodiments 110 to 206, wherein the recombinant polypeptide exhibits at least 5,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0422] 208. The recombinant polypeptide of any one of embodiments 110 to 207, wherein the recombinant polypeptide exhibits at least 10,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain, wherein the wild-type IgG1 constant domain comprises an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

[0423] 209. The recombinant polypeptide of any one of embodiments 201 to 208, wherein the FcR is FcRγ1.

[0424] 210. The recombinant polypeptide of any one of embodiments 201 to 208, wherein the FcR is FcRγ2A.

[0425] 211. The recombinant polypeptide of any one of embodiments 201 to 208, wherein the FcR is FcRγ2B.

[0426] 212. The recombinant polypeptide of any one of embodiments 201 to 208, wherein the FcR is FcRγ3A.

[0427] 213. The recombinant polypeptide of any one of embodiments 201 to 208, wherein the FcR is FcRγ3B.

[0428] 214. A composition comprising the recombinant polypeptide of any one of embodiments 110 to 213.

[0429] 215. The composition of embodiment 214, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharmaceutically acceptable carriers.

[0430] 216. A nucleic acid molecule or multiple nucleic acid molecules encoding a recombinant polypeptide according to any one of embodiments 110 to 215.

[0431] 217. A host cell engineered to express the recombinant polypeptide of any one of embodiments 110 to 213 or the nucleic acid molecule of embodiment 216.

[0432] 218. A method for producing a recombinant polypeptide comprising a chimeric constant domain, the method comprising culturing the host cell of claim 109 or claim 217 under conditions that express the recombinant polypeptide.

[0433] 219. The method of claim 218, further comprising recovering and optionally purifying the recombinant polypeptide.

[0434] Embodiments numbered as Group B

[0435] 1. A method for increasing the production of a fusion protein comprising a constant domain, the method comprising expressing a polypeptide having a chimeric constant domain comprising, from N-terminus to C-terminus:

[0436] (a) a chimeric immunoglobulin hinge comprising:

[0437] (i) The amino acid sequence of the hinge of human IgG1 from position 216 to 225 (EU numbering)

[0438] EPKSCDKTHT (SEQ ID NO: 24)

[0439] (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) from position 226 to 229 (EU numbering);

[0440] (iii) Human IgG2 lower hinge amino acid sequence from position 230 to 238 (EU numbering)

[0441] PCPAPPVAGP (SEQ ID NO: 42);

[0442] (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur simultaneously; and

[0443] (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitution is selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing.

[0444] 2. The method of embodiment 1, wherein the method increases expression relative to expression of a variant fusion protein comprising a control constant domain.

[0445] 3. The method according to embodiment 2, wherein the control constant domain is the constant domain of SEQ ID NO: 1, with or without mutations allowing heterodimerization or purification, such as knob-to-hole mutations and / or star mutations.

[0446] 4. The method according to embodiment 2, wherein the control constant domain is the constant domain of SEQ ID NO: 9, with or without mutations that allow heterodimerization or purification, such as knob-to-hole mutations and / or star mutations.

[0447] 5. The method of embodiment 2, wherein the control constant domain is the constant domain of SEQ ID NO: 10, with or without mutations that allow heterodimerization or purification, such as knob-to-hole mutations and / or star mutations.

[0448] 6. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 10% relative to the variant fusion protein.

[0449] 7. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 15% relative to the variant fusion protein.

[0450] 8. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 20% relative to the variant fusion protein.

[0451] 9. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 30% relative to the variant fusion protein.

[0452] 10. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 40% relative to the variant fusion protein.

[0453] 11. The method of any one of embodiments 2 to 5, wherein expression is increased by at least 50% relative to the variant fusion protein.

[0454] 12. A method for increasing the activity of a fusion protein having a constant domain, the method comprising expressing a polypeptide having a chimeric constant domain, the chimeric constant domain comprising, from N-terminus to C-terminus:

[0455] (a) a chimeric immunoglobulin hinge comprising:

[0456] (i) human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) from position 216 to 225 (EU numbering)

[0457] (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) from position 226 to 229 (EU numbering);

[0458] (iii) Human IgG2 lower hinge amino acid sequence from position 230 to 238 (EU numbering)

[0459] PCPAPPVAGP (SEQ ID NO: 42);

[0460] (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur simultaneously; and

[0461] (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitution is selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing.

[0462] 13. The method of embodiment 6, wherein the method increases expression relative to expression of a variant fusion protein comprising a control constant domain.

[0463] 14. The method of embodiment 13, wherein the control constant domain is the constant domain of SEQ ID NO: 1, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0464] 15. The method of embodiment 13, wherein the control constant domain is the constant domain of SEQ ID NO: 9, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0465] 16. The method of embodiment 13, wherein the control constant domain is the constant domain of SEQ ID NO: 10, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0466] 17. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 10% relative to the variant fusion protein.

[0467] 18. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 15% relative to the variant fusion protein.

[0468] 19. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 20% relative to the variant fusion protein.

[0469] 20. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 30% relative to the variant fusion protein.

[0470] 21. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 40% relative to the variant fusion protein.

[0471] 22. The method of any one of embodiments 13 to 16, wherein the activity is increased by at least 50% relative to the variant fusion protein.

[0472] 23. A method for increasing the expression and activity of a fusion protein having a constant domain, the method comprising expressing a polypeptide having a chimeric constant domain comprising, from N-terminus to C-terminus:

[0473] (a) a chimeric immunoglobulin hinge comprising:

[0474] (i) human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) from position 216 to 225 (EU numbering)

[0475] (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) from position 226 to 229 (EU numbering);

[0476] (iii) Human IgG2 lower hinge amino acid sequence from position 230 to 238 (EU numbering)

[0477] PCPAPPVAGP (SEQ ID NO: 42);

[0478] (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur simultaneously; and

[0479] (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitution is selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing.

[0480] 24. The method of embodiment 23, wherein the method increases expression and activity relative to expression of a variant fusion protein comprising a control constant domain.

[0481] 25. The method of embodiment 24, wherein the control constant domain is the constant domain of SEQ ID NO: 1, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0482] 26. The method of embodiment 24, wherein the control constant domain is the constant domain of SEQ ID NO: 9, with or without mutations that allow heterodimerization or purification, e.g., knob-to-hole mutations and / or star mutations.

[0483] 27. The method of embodiment 24, wherein the control constant domain is the constant domain of SEQ ID NO: 10, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0484] 28. The method of any one of embodiments 24 to 27, wherein:

[0485] (a) expression is increased by at least 10% relative to a variant fusion protein; and

[0486] (b) The activity is increased by at least 10% relative to the variant fusion protein.

[0487] 29. The method of embodiment 28, wherein expression is increased by at least 15% relative to the variant fusion polypeptide.

[0488] 30. The method of embodiment 28, wherein expression is increased by at least 20% relative to the variant fusion polypeptide.

[0489] 31. The method of embodiment 28, wherein expression is increased by at least 30% relative to the variant fusion polypeptide.

[0490] 32. The method of embodiment 28, wherein expression is increased by at least 40% relative to the variant fusion polypeptide.

[0491] 33. The method of embodiment 28, wherein expression is increased by at least 50% relative to the variant fusion polypeptide.

[0492] 34. The method of any one of embodiments 28 to 33, wherein the activity is increased by at least 15% relative to the variant fusion polypeptide.

[0493] 35. The method of any one of embodiments 28 to 33, wherein the activity is increased by at least 20% relative to the variant fusion polypeptide.

[0494] 36. The method of any one of embodiments 28 to 33, wherein the activity is increased by at least 30% relative to the variant fusion polypeptide.

[0495] 37. The method of any one of embodiments 28 to 33, wherein the activity is increased by at least 40% relative to the variant fusion polypeptide.

[0496] 38. The method of any one of embodiments 28 to 33, wherein the activity is increased by at least 50% relative to the variant fusion polypeptide.

[0497] 39. The method of any one of embodiments 1 to 38, wherein the optional one or more CH2 domain amino acid substitutions reduce binding of the chimeric constant domain to an Fc receptor and / or reduce effector function.

[0498] 40. The method of any one of embodiments 1 to 39, wherein the optional one or more CH3 domain amino acid substitutions provide for heterodimeric association of the chimeric constant domain with another constant domain.

[0499] 41. The method according to any one of embodiments 1 to 40, wherein the optional one or more CH3 domain amino acid substitutions provide for selective purification of the fusion protein.

[0500] 42. The method of any one of embodiments 1 to 41, wherein the fusion protein is an antigen binding protein.

[0501] 43. The method of any one of embodiments 1 to 42, wherein the chimeric constant domain comprises an IgG1 CH1 domain or a fragment thereof, optionally wherein the fragment of IgG1 CH1 comprises DKKV (SEQ ID NO: 31) or

[0502] DKRV (SEQ ID NO: 32) or consisting thereof.

[0503] 44. The method of any one of embodiments 1 to 43, wherein the fusion protein comprises at least one target binding domain.

[0504] 45. The method of any one of embodiments 1 to 44, wherein the fusion protein comprises at least two target binding domains.

[0505] 46. ​​The method of any one of embodiments 1 to 45, wherein the fusion protein comprises at least three target binding domains.

[0506] 47. The method of any one of embodiments 1 to 46, wherein the fusion protein comprises at least one Fab.

[0507] 48. The method of any one of embodiments 1 to 47, wherein the fusion protein comprises at least one scFv.

[0508] 49. The method of any one of embodiments 1 to 48, wherein the fusion protein comprises at least one Fab and at least one scFv.

[0509] 50. The method of any one of embodiments 1 to 49, wherein the fusion protein comprises two Fabs and one scFv.

[0510] 51. The method of any one of embodiments 1 to 50, wherein the fusion protein comprises at least one target binding domain at the N-terminus of the chimeric constant domain.

[0511] 52. The method of any one of embodiments 1 to 51, wherein the fusion protein comprises at least one target binding domain at the C-terminus of the chimeric constant domain.

[0512] 53. The method of any one of embodiments 1 to 52, wherein the fusion protein comprises a first target binding domain at the N-terminus of the chimeric constant domain and at least a second target binding domain at the C-terminus of the chimeric constant domain.

[0513] 54. The method of any one of embodiments 1 to 53, wherein the fusion protein comprises, from N-terminus to C-terminus, a scFv, a Fab, and a chimeric constant domain.

[0514] 55. A method according to any one of embodiments 1 to 54, wherein the fusion protein is or forms part of an antibody, optionally a multispecific antibody.

[0515] 56. A method according to any one of embodiments 1 to 55, wherein the fusion protein is a dimer comprising two chimeric constant domains as defined in any one of embodiments 1 and 39 to 41, optionally wherein each chimeric constant domain is independently selected from a constant domain having an amino acid sequence of any one of SEQ ID NO: 3, 15, 16, 17, 18, 19, 20, 21, 22.

[0516] 57. The method of embodiment 56, wherein the fusion protein is a homodimer.

[0517] 58. A method according to embodiment 56, wherein the fusion protein is a heterodimer.

[0518] 59. The method of embodiment 58, wherein one chimeric constant domain comprises a knob substitution and the other chimeric constant domain comprises a hole substitution.

[0519] 60. A method according to embodiment 58 or embodiment 59, wherein the chimeric constant domain comprises a star mutation.

[0520] 61. The method of any one of embodiments 1 to 60, wherein the fusion protein comprises two polypeptide chains.

[0521] 62. The method of any one of embodiments 1 to 60, wherein the fusion protein comprises three polypeptide chains.

[0522] 63. The method of any one of embodiments 1 to 60, wherein the fusion protein comprises four polypeptide chains.

[0523] 64. The method of any one of embodiments 1 to 63, wherein expressing the fusion protein comprises culturing a host cell engineered to express the fusion protein under conditions where the fusion protein is expressed.

[0524] 65. The method of embodiment 64, wherein the host cell is a mammalian host cell.

[0525] 66. The method of embodiment 64 or embodiment 65, further comprising recovering and optionally purifying the expressed fusion protein.

[0526] 67. The method of any one of embodiments 64 to 66, wherein the host cell is a Chinese Hamster Ovary (CHO) cell or a HEK293 or derivative (e.g., Expi293F) cell.

[0527] 68. The method of any one of embodiments 1 to 67, further comprising, prior to the expressing step, introducing a nucleic acid molecule or multiple nucleic acid molecules encoding the fusion protein into a host cell.

[0528] 69. The method of embodiment 64, wherein the nucleic acid molecule or multiple nucleic acid molecules are contained in one or more host cell expression vectors.

[0529] 70. A population of fusion proteins produced by the method of any one of embodiments 1 to 69.

[0530] 71. The population of embodiment 70, comprising at least 10,000 fusion proteins.

[0531] 72. A population according to embodiment 70 or embodiment 71, characterized in that it has higher activity compared to a population of fusion proteins comprising a control constant domain, wherein the control constant domain comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:9 or SEQ ID NO:10, with or without mutations allowing heterodimerization or purification, such as knob-to-hole mutations and / or star mutations).

[0532] Embodiments numbered as Group C

[0533] 1. A fusion protein comprising a first polypeptide chain associated with a second polypeptide chain, wherein:

[0534] (a) the first polypeptide chain comprises a first chimeric constant domain, the first chimeric constant domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, provided that the chimeric constant domain has:

[0535] (i) a PVA-deletion of the sequence at amino acids 233 to 236 as defined by EU numbering;

[0536] (ii) one or more amino acid substitutions that promote heterodimerization with a second polypeptide chain; and

[0537] (b) the second polypeptide chain comprises a second chimeric constant domain comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, provided that the chimeric constant domain has:

[0538] (i) a PVA-deletion of the sequence at amino acids 233 to 236 as defined by EU numbering; and

[0539] (ii) one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain;

[0540] 2. A fusion protein comprising a first polypeptide chain associated with a second polypeptide chain, wherein:

[0541] (a) the first polypeptide chain comprises a first chimeric constant domain, the first chimeric constant domain comprising, from N-terminus to C-terminus:

[0542] (i) a chimeric immunoglobulin hinge comprising:

[0543] (1) Human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) from position 216 to 225 (EU numbering)

[0544] (2) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) from position 226 to 229 (EU numbering);

[0545] (3) the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) from position 230 to 238 (EU numbering);

[0546] (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 237 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S and P331S (EU numbering), they do not occur simultaneously;

[0547] (iii) a CH3 domain comprising the human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1;

[0548] (iv) one or more amino acid substitutions that promote heterodimerization with a second polypeptide chain; and

[0549] (v) optionally, one or more amino acid substitutions to reduce effector function and / or facilitate purification; and

[0550] (b) the second polypeptide chain comprises a second chimeric constant domain, the second chimeric constant domain comprising, from N-terminus to C-terminus:

[0551] (i) a chimeric immunoglobulin hinge comprising the human IgG1 upper hinge amino acid sequence EPKSCDKTHTCP (SEQ ID NO: 43) from position 216 to 227 (EU numbering) of human IgG1 and the human IgG2 lower hinge amino acid sequence PCPAPPVA (SEQ ID NO: 44) from position 228 to 236 (EU numbering) of human IgG2;

[0552] (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 237 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S and P331S (EU numbering), they do not occur simultaneously;

[0553] (iii) a CH3 domain comprising the human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1;

[0554] (iv) one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain; and

[0555] (c) Optionally, one or more amino acid substitutions are made to reduce effector function and / or to facilitate purification.

[0556] 3. The fusion protein of embodiment 2, wherein the first chimeric constant domain and the second chimeric constant domain each comprise or consist of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0557] 4. The fusion protein of embodiment 1 or embodiment 2, wherein the first chimeric constant domain and the second chimeric constant domain each comprise or consist of an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0558] 5. The fusion protein of embodiment 1 or embodiment 2, wherein the first chimeric constant domain and the second chimeric constant domain each comprise or consist of an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0559] 6. The fusion protein of embodiment 1 or embodiment 2, wherein the first chimeric constant domain and the second chimeric constant domain each comprise or consist of an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0560] 7. The fusion protein of embodiment 1 or embodiment 2, wherein the first chimeric constant domain and the second chimeric constant domain each comprise or consist of an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0561] 8. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 15, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 16.

[0562] 9. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 17, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 16.

[0563] 10. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 15, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 18.

[0564] 11. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 17, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 18.

[0565] 12. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 19, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 20.

[0566] 13. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 19, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 22.

[0567] 14. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 21, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 20.

[0568] 15. The fusion protein of any one of embodiments 1 to 7, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 21, and wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 22.

[0569] 16. The fusion protein of any one of embodiments 1 to 15, wherein the fusion protein is an antigen binding protein comprising at least one target binding domain.

[0570] 17. A fusion protein according to any one of embodiments 1 to 16, wherein the fusion protein is an antibody or forms part of an antibody, optionally a multispecific antibody.

[0571] 18. The fusion protein of embodiment 16 or embodiment 17, comprising at least two target binding domains.

[0572] 19. The fusion protein of embodiment 18, wherein the fusion protein is a bivalent antibody or forms part of a bivalent antibody.

[0573] 20. The fusion protein of any one of embodiments 16 to 18, comprising at least three target binding domains.

[0574] 21. The fusion protein of embodiment 20, wherein the fusion protein is a trivalent antibody or forms part of a trivalent antibody.

[0575] 22. The fusion protein of any one of embodiments 16 to 21, wherein at least one target binding domain comprises a Fab.

[0576] 23. The fusion protein of any one of embodiments 16 to 21, wherein at least one target binding domain comprises a scFv.

[0577] 24. The fusion protein of any one of embodiments 16 to 21, comprising a Fab as a first target binding domain and a scFv as a second target binding domain.

[0578] 25. The fusion protein of embodiment 20 or embodiment 21, comprising Fab as the first target binding domain, scFv as the second target binding domain, and Fab as the third target binding domain.

[0579] 26. The fusion protein of any one of embodiments 16 to 25, comprising a third polypeptide chain and a fourth polypeptide chain, wherein:

[0580] (a) the first polypeptide chain comprises, from N-terminus to C-terminus, a first heavy chain variable region (VH1), a CH1 domain and a first constant domain; and

[0581] (b) the second polypeptide chain comprises, from N-terminus to C-terminus, a second heavy chain variable region (VH2), a CH1 domain, and a second constant domain;

[0582] (c) the third polypeptide chain comprises a first light chain comprising a first light chain variable region (VL1) associated with VH1 to form a first Fab domain; and

[0583] (d) the fourth polypeptide chain comprises a first light chain comprising a second light chain variable region (VL2) associated with VH2 to form a second Fab domain.

[0584] 27. The fusion protein of embodiment 26, wherein VH1 and VH2 are identical.

[0585] 28. The fusion protein of embodiment 26, wherein VH1 and VH2 are different.

[0586] 29. The fusion protein of embodiment 28, wherein the first Fab domain and the second Fab domain bind to different epitopes on the same target molecule.

[0587] 30. The fusion protein of embodiment 28, wherein the first Fab domain and the second Fab domain bind to different target molecules.

[0588] 31. The fusion protein of embodiment 20 or embodiment 21, comprising a third polypeptide chain and a fourth polypeptide chain, wherein:

[0589] (a) the first polypeptide chain comprises, from N-terminus to C-terminus, an scFv, an optional linker, a first heavy chain variable region (VH1), a CH1 domain and a first constant domain; and

[0590] (b) the second polypeptide chain comprises, from N-terminus to C-terminus, a second heavy chain variable region (VH2), a CH1 domain, and a second constant domain, optionally, wherein VH2 is identical to VH1;

[0591] (c) the third polypeptide chain comprises a first light chain comprising a first light chain variable region (VL1) associated with VH1 to form a first Fab domain; and

[0592] (d) the fourth polypeptide chain comprises a first light chain comprising a second light chain variable region (VL2) associated with VH2 to form a second Fab domain.

[0593] 32. The fusion protein of embodiment 20 or embodiment 21, comprising a third polypeptide chain and a fourth polypeptide chain, wherein:

[0594] (a) the first polypeptide chain comprises, from N-terminus to C-terminus, a first heavy chain variable region (VH1), a CH1 domain and a first constant domain; and

[0595] (b) a second polypeptide chain comprising, from N-terminus to C-terminus, a scFv, an optional linker, a second heavy chain variable region (VH2), a CH1 domain, and a second constant domain, optionally, wherein VH2 is identical to VH1;

[0596] (c) the third polypeptide chain comprises a first light chain comprising a first light chain variable region (VL1) associated with VH1 to form a first Fab domain; and

[0597] (d) the fourth polypeptide chain comprises a first light chain comprising a second light chain variable region (VL2) associated with VH2 to form a second Fab domain.

[0598] 33. A fusion protein according to embodiment 31 or embodiment 32, wherein the scFv comprises a third heavy chain variable region (VH2) and a third light chain variable region (VL3) arranged from N-terminus to C-terminus, VH3-optional linker-VL3.

[0599] 34. A fusion protein according to embodiment 31 or embodiment 32, wherein the scFv comprises a third heavy chain variable region (VH2) and a third light chain variable region (VL3) arranged from N-terminus to C-terminus, VL3-optional linker-VH3.

[0600] 35. The fusion protein of any one of embodiments 31 to 34, wherein the scFv binds to a target molecule different from the first Fab domain and / or the second Fab domain.

[0601] 36. The fusion protein of any one of embodiments 1 to 35, which exhibits at least 10% increased expression in an expression system compared to a variant protein comprising:

[0602] (a) the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering in its first chimeric constant domain and its second chimeric constant domain; and / or

[0603] (b) a first chimeric constant domain and a second chimeric constant domain having the amino acid sequence of SEQ ID NO: 10, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0604] 37. The fusion protein of embodiment 36, wherein the expression system is a Chinese hamster ovary (CHO) expression system or a HEK293 or derivative (e.g., Expi293F) expression system.

[0605] 38. The fusion protein of embodiment 36 or embodiment 37, wherein the increase in expression is at least 20% or at least 30%.

[0606] 39. The fusion protein of any one of embodiments 1 to 38, which exhibits at least 5-fold reduced binding to a human Fc receptor (FcR) compared to a variant protein comprising:

[0607] (a) the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering in its first chimeric constant domain and its second chimeric constant domain; and / or

[0608] (b) a first chimeric constant domain and a second chimeric constant domain having the amino acid sequence of SEQ ID NO: 9, with or without mutations that allow heterodimerization or purification, such as, for example, knob-to-hole mutations and / or star mutations.

[0609] 40. The fusion protein of embodiment 39, wherein the reduction in binding is at least 10-fold or at least 20-fold.

[0610] 41. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ1.

[0611] 42. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ2A.

[0612] 43. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ2B.

[0613] 44. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ3A.

[0614] 45. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ3B.

[0615] 46. ​​A composition comprising the fusion protein of any one of embodiments 1 to 45.

[0616] 47. The composition of embodiment 46, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharmaceutically acceptable carriers.

[0617] 48. A nucleic acid molecule or a plurality of nucleic acid molecules encoding the fusion protein of any one of embodiments 1 to 45.

[0618] 49. A host cell engineered to express the fusion protein of any one of embodiments 1 to 45.

[0619] 50. A host cell comprising one or more expression vectors encoding the fusion protein of any one of embodiments 1 to 45.

[0620] 51. The host cell according to embodiment 49 or embodiment 50, which is a CHO cell or a HEK293 or derivative (e.g., Expi293F) cell.

[0621] 52. A method of producing the fusion protein of any one of embodiments 1 to 45, the method comprising culturing the host cell of any one of embodiments 49 to 51 and optionally recovering and / or purifying the expressed protein.

[0622] 53. A population of fusion proteins according to any one of embodiments 1 to 45.

[0623] 54. A population of fusion proteins according to embodiment 53, produced by the method according to any one of embodiments 52.

[0624] 55. The population of embodiment 53 or embodiment 54, comprising at least 10,000 fusion proteins.

[0625] Embodiment No. D

[0626] 1. A recombinant protein comprising a constant domain, the constant domain having:

[0627] (a) the amino acid sequence of SEQ ID NO: 3;

[0628] (b) the amino acid sequence of SEQ ID NO: 15;

[0629] (c) the amino acid sequence of SEQ ID NO: 16;

[0630] (d) the amino acid sequence of SEQ ID NO: 17;

[0631] (e) the amino acid sequence of SEQ ID NO: 18;

[0632] (f) the amino acid sequence of SEQ ID NO: 19;

[0633] (g) Amino acid sequence of SEQ ID NO: 20

[0634] (h) the amino acid sequence of SEQ ID NO: 21;

[0635] (i) the amino acid sequence of SEQ ID NO: 22;

[0636] (j) an amino acid sequence having at least 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, with the proviso that the Fc domain (i) has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering and (ii) optionally has (1) a star mutation and / or (2) a knob or hole mutation and / or (3) an S354C or E357C mutation (as defined by EU numbering).

[0637] 2. The recombinant protein according to embodiment 1, which is a multimer.

[0638] 3. The recombinant protein according to embodiment 2, comprising two polypeptide chains.

[0639] 4. The recombinant protein according to embodiment 2, comprising three polypeptide chains.

[0640] 5. The recombinant protein according to embodiment 2, comprising four polypeptide chains.

[0641] 6. The recombinant protein of any one of embodiments 1 to 5, comprising an Fc homodimer.

[0642] 7. The recombinant protein according to embodiment 6, comprising two constant domains, each of which has the amino acid sequence of SEQ ID NO: 3.

[0643] 8. A recombinant protein according to embodiment 6, comprising two identical constant domains, each constant domain having an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, provided that the Fc region has the sequence PVA-deletion at amino acids 233 to 236.

[0644] 9. A recombinant protein according to embodiment 6, comprising two identical constant domains, each constant domain having an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, provided that the Fc region has a sequence PVA-deletion at amino acids 233 to 236.

[0645] 10. A recombinant protein according to embodiment 6, comprising two identical constant domains, each constant domain having an amino acid sequence that is at least 99% identical to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, provided that the Fc region has a sequence PVA-deletion at amino acids 233 to 236.

[0646] 11. The recombinant protein according to embodiment 6, comprising two identical constant domains, each constant domain having an amino acid sequence having 100% sequence identity with SEQ ID NO: 3.

[0647] 12. The recombinant protein of any one of embodiments 1 to 5, comprising an Fc heterodimer.

[0648] 13. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 15 and a second constant domain having the amino acid sequence of SEQ ID NO: 16.

[0649] 14. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 15 and a second constant domain having the amino acid sequence of SEQ ID NO: 18.

[0650] 15. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 17 and a second constant domain having the amino acid sequence of SEQ ID NO: 16.

[0651] 16. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 17 and a second constant domain having the amino acid sequence of SEQ ID NO: 18.

[0652] 17. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 19 and a second constant domain having the amino acid sequence of SEQ ID NO: 20.

[0653] 18. The recombinant protein according to embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 19 and a second constant domain having the amino acid sequence of SEQ ID NO: 22.

[0654] 19. The recombinant protein of embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 21 and a second constant domain having the amino acid sequence of SEQ ID NO: 20.

[0655] 20. The recombinant protein of embodiment 12, comprising a first constant domain having the amino acid sequence of SEQ ID NO: 21 and a second constant domain having the amino acid sequence of SEQ ID NO: 22.

[0656] 21. A recombinant protein according to embodiment 12, comprising two different constant domains, each having an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, provided that the Fc domain (i) has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering and (ii) optionally has (1) a star mutation and / or (2) a knob or hole mutation and / or (3) an S354C or E357C mutation (as defined by EU numbering).

[0657] 22. A recombinant protein according to embodiment 12, comprising two different constant domains, each having an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, provided that the Fc domain (i) has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering and (ii) optionally has (1) a star mutation and / or (2) a knob or hole mutation and / or (3) an S354C or E357C mutation (as defined by EU numbering).

[0658] 23. A recombinant protein according to embodiment 12, comprising two different constant domains, each having an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, provided that the Fc domain (i) has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering and (ii) optionally has (1) a star mutation and / or (2) a knob or hole mutation and / or (3) an S354C or E357C mutation (as defined by EU numbering).

[0659] 24. A recombinant protein according to embodiment 12, comprising two different constant domains, each having an amino acid sequence having 100% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, with the proviso that the Fc domain (i) has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering and (ii) optionally has (1) a star mutation and / or (2) a knob or hole mutation and / or (3) an S354C or E357C mutation (as defined by EU numbering).

[0660] 25. A nucleic acid or multiple nucleic acids encoding the recombinant protein according to any one of embodiments 1 to 24.

[0661] 26. A host cell:

[0662] (a) which is engineered to express a recombinant protein according to any one of embodiments 1 to 24; and / or

[0663] (b) comprising one or more expression vectors encoding the recombinant protein according to any one of embodiments 1 to 24.

[0664] 27. The host cell according to embodiment 26, which is a CHO cell or a HEK293 or derivative (eg, Expi293F) cell.

[0665] 28. A method of producing a recombinant protein according to any one of embodiments 1 to 24, the method comprising culturing the host cell according to embodiment 26 or embodiment 27, so that the recombinant protein is expressed and optionally recovering and / or purifying the expressed protein.

[0666] 29. A method for increasing the production of a polypeptide, the method comprising expressing the polypeptide as a protein as defined in any one of embodiments 1 to 24.

[0667] 30. The method of embodiment 29, comprising culturing the host cell of any one of embodiments 25 to 27, such that the protein is expressed and optionally recovering and / or purifying the expressed protein.

[0668] 31. The method of embodiment 29 or embodiment 30, wherein the method increases expression relative to expression of a variant protein comprising a control constant domain or constant domain pair.

[0669] 32. The method of embodiment 31, wherein the control constant domain or constant domain pair has the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering.

[0670] 33. A method according to embodiment 31, wherein the control constant domain or constant domain pair has an amino acid sequence of SEQ IDNO:1, with or without one or more mutations that allow heterodimerization or purification, for example, a knob-to-hole mutation and / or a star mutation and / or a mutation introducing a disulfide bridge (e.g., S354C or E357C).

[0671] 34. A method according to embodiment 31, wherein the control constant domain or constant domain pair has an amino acid sequence of SEQ IDNO:9, with or without one or more mutations that allow heterodimerization or purification, for example, a knob-to-hole mutation and / or a star mutation and / or a mutation introducing a disulfide bridge (e.g., S354C or E357C).

[0672] 35. A method according to embodiment 31, wherein the control constant domain or constant domain pair has an amino acid sequence of SEQ IDNO: 10, with or without one or more mutations that allow heterodimerization or purification, for example, a knob-to-hole mutation and / or a star mutation and / or a mutation that introduces a disulfide bridge (e.g., S354C or E357C).

[0673] 36. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 10%.

[0674] 37. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 15%.

[0675] 38. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 20%.

[0676] 39. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 30%.

[0677] 40. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 40%.

[0678] 41. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity relative to the variant protein is at least 50%.

[0679] 42. A population of proteins according to any one of embodiments 1 to 24.

[0680] 43. A population of proteins according to embodiment 42, produced by the method of any one of embodiments 28 to 41.

[0681] 44. The population of embodiment 42 comprising at least 10,000 proteins.

[0682] 45. A population according to any one of embodiments 42 to 44, characterized in that it has greater activity compared to a population of proteins comprising a control constant domain or constant domain pair as defined in any one of embodiments 32 to 35.

[0683] 46. ​​The population according to any one of embodiments 42 to 45, which is in the form of a pharmaceutical composition comprising the population of proteins and one or more excipients and / or pharmaceutically acceptable carriers. 8. Examples

[0685] Materials and methods

[0686] 8.1.1. Experimental constructs

[0687] Antibody constructs were generated comprising an IgG1 PVA domain and a control constant domain as shown below. The components of the test and control constructs are listed in Table 2 below.

[0688]

[0689]

[0690]

[0691]

[0692]

[0693] Test and control constructs include various bispecific and trispecific binding molecules, as shown in Table 3 below, which provides a description of various controls and test constructs used in the studies described herein. Certain bispecific antibodies include three antigen binding sites, the first antigen binding site binds FGFR1c, the second antigen binding site binds the GH1 domain of KLB, and the third antigen binding site binds the GH2 domain of KLB; IgG Fc domain (e.g., IgG1 PVA, IgG1N180G (N297G according to EU numbering), IgG4 or IgG4s) and linkers of varying lengths. In some cases, the antigen binding site that binds to the GH2 domain of KLB is a Fab or scFv, and is connected to the N-terminus of the Fab that binds to the FGFR1c on the first arm of the bispecific antibody, while the Fab that binds to the GH1 domain of KLB is located on the second arm of the bispecific antibody.

[0694]

[0695]

[0696]

[0697] 8.1.2. Vector construction

[0698] DNA fragments (encoding anti-KLB GH1 Fab, anti-KLB GH2 Fab, anti-KLB GH2 scFv, and anti-FGFR1 cFab domains; various amino acid linkers; and various IgG hinge and Fc domains) were synthesized by Integrated DNA Technologies, Inc. (San Diego, CA) or Geneart / Thermo Fisher Scientific (Regensburg, Germany).

[0699] Mammalian expression vectors for single polypeptide chains were created by one of the following methods: using the NEBuilder HiFi DNA assembly kit (New England BioLabs Inc.); by restriction digestion followed by ligation according to the standard molecular cloning protocol provided by New England BioLabs Inc; or by DNA synthesis and cloning into the pcDNA3.4 Topo expression system (Life Technologies) in a ready-to-use construct. DNA was transfected as a single plasmid or as a heavy and light chain pair according to the manufacturer's protocol. 50 ml of cell culture supernatant was collected and processed for purification via HiTrap™ protein G HP, HiTrap protein A FF or MabSelect SuRe pcc columns (Cytiva).

[0700] Some constructs were expressed by transient transfection (Thermo Fisher Scientific) in Expi293F TM The protein in the Expi293F supernatant was purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, Maryland) and HiTrap™ Protein GHP or MabSelect SuRe pcc columns (Cytiva). After a single-step elution, the antibody was neutralized, dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted and stored at -80°C. For some constructs, an additional step of size exclusion chromatography using a HiPrep 26 / 60 Sephacryl S-200 column was used.

[0701] Other expression vectors were stably expressed in the Chinese Hamster Ovary (CHO) expression system. Purification of these antibodies from CHO stable expression followed similar procedures as described above.

[0702] 8.1.3. Kinetic measurements of Fc receptor binding by Biacore

[0703] In brief, surface plasmon resonance (SPR) experiments were performed at 25°C on a Biacore T200 instrument using a carboxymethyl dextran coated (CM-5) chip. Mouse monoclonal anti-pentahistidine antibodies (GE Healthcare) were immobilized on the surface of a CM-5 sensor chip using standard amine coupling chemistry. 140RU-376RU of His-tagged human, monkey or mouse FcγR proteins were captured on an anti-pentahistidine amine-coupled CM-5 chip, and a stock solution of the antibody was injected over the captured protein at 50μl / min within 2 minutes and serially diluted (6uM-24.7nM). The mAb binding reaction was monitored, and for low affinity receptors, the steady-state binding equilibrium was calculated. Kinetic association (ka) and dissociation (kd) rate constants were determined by processing the data and fitting it to a 1:1 binding model using Scrubber 2.0 curve fitting software. The binding dissociation equilibrium constant (KD) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as: KD(M)=kd / ka; and t1 / 2(min)=(In2 / (60*kd). Some KDs were derived using steady-state equilibrium dissociation constants; NB=no binding observed; IC=indeterminate affinity determination due to low specific RU signal.

[0704] 8.1.4. Enzyme-linked immunosorbent assay (ELISA)

[0705] Wells of microtiter plates were coated (18 h, 4 °C) with 6x-His tag (SEQ ID NO: 47) monoclonal antibody (4E3D10H2 / E3) (Thermo scientific) at 4 μg / ml in 100 μl of PBS and then blocked with blocking buffer (2% BSA in PBS) for 1 h at room temperature. Different Fc receptors (2 μg / ml, 100 μl / well) were loaded in duplicate and incubated for 1 h at room temperature. At the same time, antibodies were added in blocking buffer at a ratio of 1:5 from 6.0x10 -06 The diluted antibody (100ul) was then added to the wells and incubated for 1h at room temperature. Peroxidase-conjugated goat anti-human IgG, F(ab') 2 Detection antibody 100ul / well (1:5000, in blocking buffer) 1h, and by adding 100μl peroxidase substrate (KPL-TMB) 30min to make the reaction visual. The reaction was stopped with 100μl TMB stop buffer, and the absorbance was measured at 450nm using an ELISA plate reader (Envision, PerkinElmer). After each step, the plate was washed three times with washing buffer (PBS, pH 7.4, containing 0.05% (v / v) Tween 20).

[0706] 8.1.5. Alternative ADCC assays

[0707] 8.1.5.1. Target cells

[0708] HEK293 / hFGFR1c / hKLB / hCD20: HEK293 cells with endogenous FGFR1 excised by CRISPR-Cas9 were engineered to constitutively express full-length human CD20 (hCD20, amino acids M1-P297 of accession number NP_690605.1), FGFR1c (hFGFR1c, amino acids M1-R731 of accession number NP_075594), and KLB (hKLB, amino acids M1-S1044 of accession number NP_783864.1). Cells were sorted for high expression of all receptors.

[0709] 8.1.5.2. Reporter cells

[0710] Jurkat / NFAT-Luc / FcγR3a 176Val: Jurkat T cells were engineered to stably express the nuclear factor of activated T cells (NFAT) luciferase reporter construct and the high affinity human FcγR3a 176Val alloreceptor (amino acids M1-K254 of accession number P08637 VAR_003960).

[0711] 8.1.5.3. Measurement settings

[0712] Three days before the experiment, Jurkat reporter cells were split into 1.25 x 10 cells in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / mL puromycin + 500 μg / mL G418 growth medium. 5 On the day of the experiment, target cells and reporter cells were transferred to assay medium (RPMI + 10% FBS + P / S / G) and cultured at a 1:1 ratio (3 x 10 cells / ml for each cell type). 4 / well) was added to a 96-well white microtiter plate. Multispecific anti-FGFR1c / KLB antibody and hIgG4 S108P isotype control antibody were titrated at 7 points, 1:4 serial dilutions ranging from 73.2pM to 300nM final concentrations, and the last 8th point contained no antibody and was added to the cells in duplicate. The plate was incubated at 37°C / 5% CO2 for 4.6h, followed by the addition of an equal amount of ONE-Glo TM(Promega) reagent was used to lyse cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer). The EC50 value of the antibody was determined from a 4-parameter logistic equation on an 8-point dose-response curve (including background signal) using GraphPad Prism software. The maximum multiple induction was calculated using the following formula:

[0713] Fold induction = maximum mean RLU / mean RLU (background signal = no antibody) within the tested dose range for each antibody

[0714] 8.1.6. Stable expression and antibody titer

[0715] Recombinant proteins encoding different antibodies with various IgG subclasses were cloned into expression plasmids, transfected into CHO cells, and stable transfected pools were isolated after selection with 400 mg / L hygromycin for 12 to 14 days. Stable CHO cell pools grown in suspension in chemically defined protein-free medium were used to produce the proteins for testing.

[0716] Protein was produced by inducing cell cultures with 0.5 mg / L doxycycline for five days and harvesting the conditioned media. Protein titers were determined using an Octet instrument (ForteBio) using a protein A sensor against known standards at varying concentrations.

[0717] 8.1.7. Luciferase reporter assay

[0718] HEK293.SREluc.hFGFR1c / hKLB cells were used to detect the agonist activity of antibodies containing different IgG hinges and Fc domains, which stably expressed human FGFR1c and KLB and a luciferase reporter gene controlled by a promoter containing a serum response element (SRE). Recombinant human FGF21 (SEQ ID NO:47) with a 6xHis tag was used as a positive control, where the maximum reported activity obtained from FGF21 was defined as 100% activity. Cells were treated with each antibody or 6xHis-FGF21 ("HHHHHH" is disclosed as SEQ ID NO:47) for 6 hours, and then luciferase assays were performed. The percentage of activity induced by each antibody was standardized relative to the maximum activity of FGF21. Dose response assays were performed to determine EC50. Anti-FelD1 isotype (hIgG4-S108P) control antibody was used as a negative control.

[0719] 8.1.8. Human primary adipocyte signaling assay

[0720] Human primary adipocytes differentiated from subcutaneous preadipocytes were obtained from Zen-Bio Inc (Durham, NC). Cells were cultured in serum-free medium for 4 hours and then treated with serially diluted antibodies for 15 minutes. TM SureFire TM Cells were lysed with lysis buffer from the ERK Assay Kit, which measures phospho-ERK in the treated cell lysate (PerkinElmer, Shelton, CT). SureFire TM The ERK assay was performed according to the manufacturer's protocol. His-tagged human FGF21 and isotype control human IgG4 antibody were tested as positive and negative controls, respectively. FGFR1c / KLB bispecific antibody was also included in the experiment.

[0721] 8.1.9. Flow Binding Assay

[0722] BaF3 cells overexpressing target protein X were cultured at 1×10 6 Resuspend in FACS wash buffer (PBS containing 1% FBS). 5 The antibody was added at a ratio of 1:5 from 1.3 x 10 -07 The diluted antibodies were then added to the wells containing the cells. The cells were stained for 30 min at 2 to 8°C and washed twice with FACS wash buffer. AF647-conjugated goat anti-hFc Fab (Jackson Nutrition Research, 109-607-008, 1:400) was added to the wells and the cells were incubated for 30 min at 2 to 8°C. Next, the cells were washed to remove excess antibody and fixed in 2% paraformaldehyde at 2 to 8°C for 30 min. After two washes, the cells were fixed using BD LSR Fortessa TM The stained cells were analyzed by FACS instrument. The results were analyzed by FlowJo. The FSC / SSC gate was used to select monocytes.

[0723] 8.1.10. Negative stain electron microscopy (EM)

[0724] Complexes of C-terminally myc-myc-6xHis-tagged CD40 ectodomain (res.20-193) and anti-CD40 antibodies containing different hinge sequences (IgG1, IgG1-PVA, and IgG2) were separated by size exclusion chromatography using a Superdex 200 Increase 10 / 300 column. Purified samples with a protein concentration of approximately 0.02 mg / mL were applied to 400-mesh carbon-film Cu grids (Electron Microscopy Sciences) and negatively stained with NanoW (Nanoprobes) or Vitroease methylamine tungstate (Thermo Fisher).

[0725] Negatively stained EM grids were inserted into a Glacios TEM (Thermo Fisher) and imaged with a Ceta camera (ThermoFisher). Automated data collection was performed using EPU at a nominal magnification of 73,000 times. EM data were processed using RELION4.0. Particles were first picked using a Gaussian Laplace algorithm to generate a 2D template for subsequent template-based particle picking. Particle images were subjected to multiple rounds of 2D classification, and particles belonging to the class average with clear IgG features were selected after each round. The diffusion density of binding CD40 allowed the assignment of two Fab arms, which otherwise lacked distinguishing features from the Fc region. The class average with discernible features of the two Fabs each bound to CD40 was selected for Fab-Fab angle determination. The Fab-Fab angle, measured manually using the angle tool in ImageJ, is the angle between lines drawn through the long axes of the two CD40-bound Fab arms.

[0726] 8.2. Example 1: Design, cloning and expression of IgG1 PVA

[0727] 8.2.1. Overview

[0728] IgG1 Fc and IgG4 Fc have different Fcγ receptor binding abilities and charge distributions, which provide options for optimal Fc functional engagement and different compatibilities with antibody building blocks (such as Fab, scFv and alternative forms of antibody fusion proteins). The hinge regions of IgG1 and IgG4 also have different lengths and flexibility. IgG4 (S108P or S228P, EU numbering) has been used in a variety of approved antibody products, such as pembrolizumab, nivolumab and ixekizumab, which require reduction of Fc effector functions. Due to the preference of antibody building blocks (e.g., Fab, scFv) for specific immunoglobulin subclasses, alternative and natural sequence variants based on human IgG1 Fc that are different from IgG4 (S108P) are sought, which show that Fcγ receptor binding is reduced, and Fc receptor effector functions are reduced.

[0729] Figure 4 Alignments of sequences between various IgG hinge / Fc variants and various wild-type and modified human IgG1 and IgG4 hinge regions are presented, along with a description of the CH2 and CH3 Fc regions used, from amino acids 226 to 447 (EU numbering). The hIgG1 PVA was designed to include PVA mutations in the lower hinge region in an otherwise complete IgG1 background (e.g., IgG1 upper hinge region, CH2 and CH3 regions).

[0730] To test the properties of hIgG1 PVA, it was incorporated into alternative antibody formats having either a 2+1 N-scFv or a 2+1 N-Fab format (see, e.g., Figure 5 Illustration of the 2+1 N-scFv format; in the 2+1 N-Fab format, the N-terminal scFv domain is replaced by the Fab domain).

[0731] Results

[0732] Control and bispecific antibodies containing various IgG hinge and Fc domains were successfully expressed and purified.

[0733] When expressed in CHO cells, AF1 constructs in an IgG1 PVA backbone with different linker lengths between scFv and Fab had higher antibody titers (as measured by total antibody species) than constructs containing IgG4 S108P ( Figure 5 ).

[0734] 8.3. Example 2: Binding kinetics of Fcγ receptors

[0735] 8.3.1. Overview

[0736] The binding affinity and signal of various antibodies with different hinge-Fc regions to Fcγ receptors were measured by Biacore as described in Section 0.

[0737] 8.3.1.1. Results

[0738] The results are shown in Tables 4 and 5 below.

[0739]

[0740]

[0741]

[0742] In Table 5, NB means no binding; WB means weak binding

[0743] IgG1 PVA had no binding signals to FcγR1, FcγR2b, FcγR3a (F176), FcγR3b. It had low binding signals to FcγR2a (R131 and H131), but the levels were significantly reduced compared to IgG1 and IgG4 S108P (91 and 21 RU, respectively). IgG1 PVA had weak to moderate binding signals (144 RU) to FcγR3a (V176), with a KD = 7.2 x 10 -05 M, which is much weaker than IgG1 and IgG4 S108P (Tables 4 and 5).

[0744] 8.4. Example 3: ELISA binding to Fcγ receptors

[0745] 8.4.1. Overview

[0746] Binding of FGFR1c / KLB bispecific antibodies (comprising various IgG hinge and Fc regions) was assessed by ELISA as described in Section 5.1.4.

[0747] 8.4.2. Results

[0748] Binding curves indicating the ability of control and test antibodies to bind to various Fcγ receptors are depicted in FIG. 6A to FIG. 6G Antibodies containing the wild-type IgG1 hinge and Fc domain showed the highest binding to hFCRγ1. Binding of hFCRγ1 was significantly reduced with IgG1 PVA, showing similar binding to IgG4s ( Fig. 6A ). Binding with IgG1 N180G was similarly reduced. Binding of IgG4 S108P to hFCRγ1 was only slightly reduced relative to wild-type IgG1. Similar trends were observed for binding of hFCRγ3A(V158) and hFCRγ3A(F158) ( Fig. 6E , 6F Very small differences in binding were observed with hFCRγ2A(H131) and hFCRγ2A(R131) ( Figure 6B and 6C ). However, IgG1 PVA binds weaker to hFCRγ2B than IgG4 S108P and slightly weaker than IgG1 ( Fig.6D ) In hFCRγ3B, IgG1PVA had less binding than IgG1 ( Figure 6G ).

[0749] 8.5. Example 4: Antibody-dependent cellular cytotoxicity

[0750] 8.5.1. Overview

[0751] The cytotoxic activity of IgG1 PVA was determined using the alternative antibody-dependent cellular cytotoxicity (ADCC) assay described in Section 0 and compared to that of other IgG variants (e.g., IgG1 N180G and IgG4 S108P).

[0752] The ability of multispecific antibodies targeting hFGFR1c and hKLB to interact with FcγR3a, an Fc receptor prominently expressed on NK cells that can induce antibody-dependent cell-mediated cytotoxicity (ADCC), was measured in an alternative bioassay using reporter cells and target cells conjugated to the antibodies. In this assay, Jurkat T cells engineered to express the transcription factor NFAT (NFAT-Luc) as well as the high-affinity human FcγR3a 176Val allotype receptor (Jurkat / NFAT-Luc / hFcγR3a 176 Val) under the control of the reporter luciferase. The target cell is a HEK293 cell, which is engineered to express a combination of human CD20 and full-length human FGFR1c and human KLB. The reporter cells are incubated with the target cells, and FcγR3a is engaged via the Fc domain of the human IgG1 antibody bound to the target cells, resulting in the activation of the transcription factor NFAT in the reporter cells, and driving the expression of luciferase, which is then measured via luminescent readings.

[0753] 8.5.2. Results

[0754] Representative data from ADCC assays are depicted in Figure 7 and Figure 8 Only the alternative format antibodies with wild-type IgG1, AF1dIgG1, and AF2d IgG1 showed induction of luciferase signal, 1.9-fold (EC50=307pM) and 3.4-fold (EC50=1.04nM), respectively. None of the alternative format antibodies with 2+1N-scFv or 2+1N-Fab format of IgG1 PVA, IgG1 N180G, or IgG4 S108P showed activity in the alternative ADCC assay.

[0755] 8.6. Example 5: Molecular activity

[0756] 8.6.1. Overview

[0757] The activity of FGFR1c / KLB bispecific antibodies (including IgG1 PVA and control) was tested using the luciferase reporter assay and human primary adipocyte signaling assay described in Sections 6.1.7 and 0.

[0758] 8.6.2. Results

[0759] Activity of the alternative forms of the antibody in HEK.293SREluc.hFGFR1c / hKLB was shown Fig. 9 (2+1N-scFv, AF1a, 1b) and Fig.10 (2+1N-Fab, AF2a, 2b, 3a, 3b). Activity in human adipocytes was shown Fig.11 (AF1a, 1b, 3a, 3b). The antibody in the form of 2+1N-scFv incorporated with IgG1 PVA (AF1a) was expressed in HEK FGFR1c / KLB cells ( Fig. 9 ) and human adipocytes ( Fig.11 ) showed superior agonist activity to IgG4 S108P (AF1b). In the reporter cell assay, antibody AF2a (2+1 N-Fab format in IgG1 PVA) elicited greater maximal activation than AF2b (the same antibody as IgG4 S108P) ( Fig.10 ).

[0760] 8.7. Example 6: Target Cell Binding

[0761] 8.7.1. Overview

[0762] AF5-a and AF5-b (both multispecific tetravalent antibodies with four antigen binding domains that bind to protein X) were designed and produced as described in Sections 8.1.1 and 8.1.2, wherein AF5-a and AF5-b were designed to contain the same set of antigen binding arms linked to the human IgG1 PVA or IgG4 S108P backbone, respectively. The binding properties of AF5-a, AF5-b, and a non-binding isotype control antibody were evaluated using the flow binding assay described in Section 0.

[0763] 8.7.2. Results

[0764] As expected, the isotype control antibody showed no binding affinity. In contrast, both AF5-a and AF5-b showed binding to protein X overexpressing cells. More specifically, AF5-a had a higher maximum MFI signal than AF5-b ( Fig.12 ), indicating that the IgG1 PVA backbone has a higher efficacy relevance compared to the IgG4 S108P backbone.

[0765] 8.8. Example 7: Fab-Fab angle associated with three different hinge sequences

[0766] 8.8.1. Overview

[0767] The Fab-fab angles of CD40 binding anti-CD40 IgG antibodies linked to different IgG hinge sequences were calculated as described in Section 0.

[0768] 8.8.2. Results

[0769] Three IgG-CD40 complexes were evaluated: IgG1-CD40 complex, IgG1-PVA-CD40 complex, and IgG2-CD40 complex. In the 2D classification runs for IgG1-CD40, IgG1-PVA-CD40, and IgG2-CD40, respectively, approximately 35%, 35%, and 11% of the particles had IgG-like features but were not assigned a Fab-Fab corner due to ambiguity in Fab arm recognition. FIG. 13C to FIG. 13E For the 2D classification run shown, only a small fraction of particles (ie, less than 2%) were classified as a "junk" 2D class lacking IgG-like features.

[0770] Table 6 lists the Fab-Fab angles determined based on the 2D class averages and particle population percentages.

[0771]

[0772]

[0773] Without being bound by theory, it is believed that the IgG1-PVA-Fc domain causes increased target binding of an antibody preparation by increasing the percentage of molecules in the antibody preparation that have a Fab-Fab angle suitable for target binding. 9. References

[0775] All publications, patents, patent applications, or other documents cited in this application are hereby incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes. If there is an inconsistency between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present specification are intended to control.

Claims

1. A multispecific antibody comprising a first polypeptide chain, wherein the first polypeptide chain comprises a chimeric constant domain, wherein the chimeric constant domain comprises, from N-terminus to C-terminus: (a) a chimeric immunoglobulin hinge comprising: (i) Human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) from position 216 to 225 (EU numbering) (ii) the human IgG1 core hinge amino acid sequence CPPC from position 226 to 229 (EU numbering) (SEQ ID NO: 25); (iii) the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) from position 230 to 238 (EU numbering); (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, wherein if CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur simultaneously; and (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions selected from knob substitutions (e.g., T366W), hole substitutions (e.g., T366S, L368A, Y407V), star mutations (e.g., H435R, Y436F), mutations introducing disulfide bridges (e.g., S354C or E357C), or a combination of two or more of the foregoing.

2. A multispecific antibody comprising a first polypeptide chain associated with a second polypeptide chain, wherein: (a) the first polypeptide chain comprises: a first chimeric constant domain, the first chimeric constant domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1, with the proviso that the chimeric constant domain has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering; and (b) the second polypeptide chain comprises a second chimeric constant domain, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1, with the proviso that the chimeric constant domain has a sequence PVA-deletion at amino acids 233 to 236 as defined by EU numbering.

3. A multispecific antibody comprising a first polypeptide chain associated with a second polypeptide chain, wherein: (a) the first polypeptide chain comprises a first chimeric constant domain, wherein the first chimeric constant domain comprises, from N-terminus to C-terminus: (i) a chimeric immunoglobulin hinge comprising: (1) Human IgG1 upper hinge amino acid sequence EPKSCDKTHT from position 216 to 225 (EU numbering) (2) the human IgG1 core hinge amino acid sequence CPPC from positions 226 to 229 (EU numbering); (3) the human IgG2 lower hinge amino acid sequence PCPAPPVAGP from positions 230 to 238 (EU numbering); (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur together; and (iii) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing; and (b) the second polypeptide chain comprises a second chimeric constant domain, the second chimeric constant domain comprising, from N-terminus to C-terminus: (i) a chimeric immunoglobulin hinge comprising: (1) Human IgG1 upper hinge amino acid sequence EPKSCDKTHT from position 216 to 225 (EU numbering) (2) the human IgG1 core hinge amino acid sequence CPPC from positions 226 to 229 (EU numbering); (3) the human IgG2 lower hinge amino acid sequence PCPAPPVAGP from positions 230 to 238 (EU numbering); (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from position 239 to 340 (EU numbering) of human IgG1, optionally comprising one or more CH2 domain amino acid substitutions, and optionally, wherein if CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not occur together; and (iii) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from position 341 to 447 (EU numbering) of human IgG1, optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitution is selected from a knob substitution (e.g., T366W), a hole substitution (e.g., T366S, L368A, Y407V), a star mutation (e.g., H435R, Y436F), a mutation introducing a disulfide bridge (e.g., S354C or E357C), or a combination of two or more of the foregoing.

4. The multispecific antibody according to any one of claims 1 to 3, wherein the first chimeric constant domain comprises one or more amino acid substitutions that promote heterodimerization with the second polypeptide chain. 5 . The multispecific antibody of claim 4 , wherein the one or more amino acid substitutions that promote heterodimerization with the second polypeptide chain are one or more of S354C and T366W (EU numbering). 6 . The multispecific antibody of claim 4 , wherein the one or more amino acid substitutions that promote heterodimerization with the second polypeptide chain are one or more of Y349C, T366S, L368A, and Y407V (EU numbering).

7. The multispecific antibody of any one of claims 1 to 6, wherein the first chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

8. The multispecific antibody of any one of claims 1 to 7, wherein the first chimeric constant domain comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

9. The multispecific antibody of any one of claims 1 to 8, wherein the first chimeric constant domain comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

10. The multispecific antibody of any one of claims 1 to 9, wherein the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:

22.

11. The multispecific antibody according to any one of claims 1 to 10, wherein the second chimeric constant domain comprises one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain. 12 . The multispecific antibody of claim 11 , wherein the one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain are one or more of S354C and T366W (EU numbering).

13. The multispecific antibody of claim 11, wherein the one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain are one or more of Y349C, T366S, L368A, and Y407V (EU numbering).

14. The multispecific antibody of any one of claims 1 to 13, wherein the second chimeric constant domain comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

15. The multispecific antibody of any one of claims 1 to 14, wherein the second chimeric constant domain comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

16. The multispecific antibody of any one of claims 1 to 15, wherein the second chimeric constant domain comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

17. The multispecific antibody of any one of claims 1 to 16, wherein the second chimeric constant domain comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:

22.

18. The multispecific antibody of any one of claims 1 to 16, wherein the first chimeric constant domain and the second chimeric constant domain are identical. The multispecific antibody of claim 18 , wherein the multispecific antibody is a homodimer.

20. The multispecific antibody of any one of claims 1 to 16, wherein the first chimeric constant domain is different from the second chimeric constant domain.

21. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 15, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

16.

22. The multispecific antibody of claim 20, the multispecific antibody of any one of claims 1 to 17, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 17, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

16.

23. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 15, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

18.

24. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 17, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

18.

25. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 19, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

20.

26. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO: 19, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

22.

27. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO:21, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

20.

28. The multispecific antibody of claim 20, wherein the first constant domain comprises the amino acid sequence of SEQ ID NO:21, and the second constant domain comprises the amino acid sequence of SEQ ID NO:

22.

29. The multispecific antibody of any one of claims 20 to 28, wherein the multispecific antibody is a heterodimer.

30. The multispecific antibody of any one of claims 1 to 29, further comprising a first target binding domain and a second target binding domain.

31. The multispecific antibody of claim 30, wherein the first target binding domain binds to a first target molecule and the second target binding domain binds to a second target molecule.

32. The multispecific antibody of claim 30, wherein the first target binding domain and the second target binding domain bind to the same target molecule.

33. The multispecific antibody of any one of claims 30 to 32, further comprising a third target binding domain that binds to a different target molecule than the first target binding domain and / or the second target binding domain.

34. The multispecific antibody of any one of claims 1 to 33, wherein the multispecific antibody is a bispecific antibody.

35. The multispecific antibody of any one of claims 1 to 33, wherein the multispecific antibody is a trispecific antibody.

36. The multispecific antibody of any one of claims 1 to 35, wherein the multispecific antibody is a trivalent antibody.

37. The multispecific antibody of any one of claims 1 to 35, wherein the multispecific antibody is a tetravalent antibody.

38. The multispecific antibody of any one of claims 1 to 37, wherein the multispecific antibody exhibits at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

39. The multispecific antibody of any one of claims 1 to 38, wherein the multispecific antibody exhibits at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold reduced binding to a human Fc receptor (FcR) compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, IgG1 CH2, and IgG1 CH3.

40. The multispecific antibody of claim 39, wherein the FcR is FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B.

41. A composition comprising the multispecific antibody according to any one of claims 1 to 40.

42. The composition of claim 41, wherein the composition is a pharmaceutical composition and further comprises one or more excipients and / or pharmaceutically acceptable carriers.

43. A nucleic acid molecule or a plurality of nucleic acid molecules encoding the multispecific antibody according to any one of claims 1 to 40.

44. A host cell engineered to express the multispecific antibody of any one of claims 1 to 40.

45. A method of producing a multispecific antibody according to any one of claims 1 to 40, the method comprising culturing the host cell according to claim 44 and optionally recovering and / or purifying the expressed protein.

46. ​​A method for increasing the production of a multispecific antibody, the method comprising expressing the multispecific antibody as a multispecific antibody as defined in any one of claims 1 to 40.

47. The method of claim 46, comprising culturing the host cell of claim 44 so that the protein is expressed, and recovering the expressed protein.

48. The method of claim 46 or 47, wherein the method increases expression by at least 10%, at least 15%, at least 20%, at least 30%, at least 40% or at least 50% relative to expression of a variant protein comprising a control pair of constant domains, each of which comprises the sequence ELLG at amino acids 233 to 236 as defined by EU numbering.

49. A population of multispecific antibodies according to any one of claims 1 to 40, optionally comprising at least 10,000 multispecific antibodies.

50. The population of multispecific antibodies according to claim 49, characterized in that The protein has higher activity compared to a population of proteins comprising a control pair of constant domains each comprising the sequence ELLG at amino acids 233 to 236 as defined by EU numbering.

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