Chimeric heavy chain constant domains with reduced binding to Fc gamma receptors and uses thereof - Patents.com

Abstract

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

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 333,293, filed April 21, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically, which is incorporated herein 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. [Background technology]

[0003] 3.Background technology Antibodies of the IgG class are attractive therapeutic agents. IgG exists in four subclasses in humans: IgG1, IgG2, IgG3, and IgG4. The heavy chain constant (CH) region of IgG contains three domains, CH1, CH2, CH3, and a hinge linking CH1 and CH2. Although the role of each subclass appears to vary from species to species, the heavy chain constant domains are involved in various biological effector functions. Human IgG subclasses mediate several cellular immune responses through interactions with Fcγ (FcγR), such as cell death, phagocytosis, and opsonization. Such interactions involve binding of at least functional CH2 and CH3 domains of the heavy chain constant region to FcγR on the surface of effector cells, such as natural killer cells and activated macrophages. Complement-mediated lysis can also be triggered by the interaction of the Fc region (e.g., CH2 and CH3 domains) with various complement components.

[0004] Effector functions are useful in some antibody therapies, such as the treatment of some cancers or pathogens, where effector functions are primarily or at least partially responsible for killing cancer cells or pathogens. However, other antibody therapies are entirely or primarily mediated by effector-independent mechanisms, such as inhibition of receptor-ligand interactions or agonization of receptors. In such therapies, antibody effector functions serve little or no useful purpose, but may result in undesirable effects, including inflammation. In such situations, it may be advantageous to engineer the Fc receptor binding properties of antibodies to inhibit some or all of the available effector mechanisms without substantially affecting the pharmacokinetic properties, immunogenicity, and specificity and affinity of the variable region of the antibody. Several groups have performed such antibody engineering.

[0005] To test the effect of amino acids on IgG / FcγR interaction, IgG heavy chain constant regions have been mutated at various positions (see, for example, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). Several amino acid residues in the hinge region and CH2 domain of the heavy chain constant region have been proposed to mediate binding to Fcγ receptors (see Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, and Non-Patent Document 7). Glycosylation of a site (N297) in the CH2 domain and variation in its carbohydrate composition also strongly affect IgG / FcγR interaction (Non-Patent Document 7, Non-Patent Document 8). Patent Document 1 describes a chimeric IgG comprising an IgG1 upper hinge, an IgG1 lower hinge, an IgG4 CH2, and an IgG1 CH3, in which amino acids in the lower hinge region are replaced with corresponding amino acids from the human IgG2 isotype to reduce FcγR binding without unacceptable conformational changes and resulting immunogenicity. Patent document 2 describes an engineered IgG heavy chain constant domain with a modified hinge region in which amino acids 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, the positions being numbered according to EU numbering (shown in Figure 1 of Patent document 2).

[0006] It would be valuable to have alternatives to those chimeric heavy chain constant domains known in the art. Having alternative chimeric heavy chain constant regions based on different IgG subclasses would, for example, allow for optimization of antibody activity and improved expression and production. Thus, there is a need for heavy chain constant regions with reduced binding to FcγR, and therapeutic polypeptides (e.g., recombinant antibodies) comprising them, which can be used to treat diseases or conditions in which effector functions should be minimized. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2014 / 121087 [Patent Document 2] WO2016 / 161010A2 [Non-patent literature]

[0008] [Non-Patent Document 1] Canfield and Morrison, 1991, J Exp Med 73:1483-1491 [Non-Patent Document 2] Chappel et al., 1993, JSC 268(33):25124-31 [Non-Patent Document 3] Armour et al., Eur J Immunol 29:2613-24 [Non-Patent Document 4] Sarmay et al., 1992, Mol Immunol 29:633-9 [Non-Patent Document 5] Greenwood et al., Eur J Immunol 23(5):1098 [Non-Patent Document 6] Morgan et al., 1995, Immunology 86:319 [Non-Patent Document 7] Stevenson, 1997, Chem Immunol 65:57-72 [Non-Patent Document 8] Siberil et al., 2006, Immunol Ltrs 106:111-118 Summary of the Invention

[0009] 4. Summary of the Invention 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 an IgG1 heavy chain constant domain with an engineered hinge region. Unexpectedly, incorporation of the chimeric constant domains of the present disclosure into exemplary antibodies improved their expression and activity, in addition to reducing Fc receptor and effector function (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] Thus, the present disclosure provides a recombinant protein, e.g., a fusion protein, comprising the chimeric heavy chain constant domain of the present disclosure. The chimeric heavy chain constant domain can be advantageously used as a dimerization moiety in various 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 can include one or more target binding domains (e.g., one or more Fab moieties, one or more scFv moieties, or a combination thereof) and / or one or more linker moieties that separate one or more moieties in the recombinant protein. In some embodiments, the recombinant protein is a recombinant antibody, e.g., 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, Group A: numbered embodiments 1-105 and 110-214, Group C: numbered embodiments 1-45, and Group D: numbered embodiments 1-24. When the recombinant protein is an antibody, exemplary target binding domains for incorporation into the antibody are disclosed in Section 6.2.2, and exemplary linkers useful for connecting the constant domain to the target binding domain or to different components of the target binding domain are described in Section 6.2.5.

[0013] The present disclosure further provides nucleic acids encoding the recombinant proteins of the disclosure. The nucleic acids can 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 present disclosure further provides host cells and cell lines engineered to express the nucleic acids and recombinant proteins of the disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 6.3, Group A: numbered embodiments 108, 109, 216, and 217, Group C: numbered embodiments 48-51, and Group D: numbered embodiments 25-27.

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

[0015] The present disclosure further provides compositions, e.g., populations of proteins, and pharmaceutical compositions comprising the recombinant proteins of the present disclosure. Exemplary compositions are described in Section 6.4, Group A: numbered embodiments 106, 107, 214, and 215, Group B: numbered embodiments 70-72, Group C: numbered embodiments 46, 47, and 53-55, and Group D: numbered embodiments 42-46.

[0016] 5. Brief description of the drawings [Brief description of the drawings]

[0017] [Figure 1]The wild type sequence of the heavy chain constant region of human IgG1 (human IGHG1 heavy chain constant region; UniProt accession number P01857) is depicted. CH1 = amino acids 1-98; upper hinge = amino acids 99-108; core hinge = 109-112; lower hinge = 113-121; CH2 = 120-223; CH3 = 224-330. The amino acid numbering shown is relative to the depicted sequence. The upper, core, and lower hinge regions are boxed. 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 discloses SEQ ID NO: 48. [Diagram 2] The wild type sequence of the heavy chain constant region of human IgG2 (human IGHG2 heavy chain constant region; UniProt accession number P01859) is depicted. CH1 = amino acids 1-98; upper hinge = amino acids 99-105; core hinge = 106-109; lower hinge = 110-117; CH2 = 116-219; CH3 = 220-326. The numbering of the amino acids shown is with respect 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 discloses SEQ ID NO: 49. [Diagram 3] The wild type sequence of the heavy chain constant region of human IgG4 (human IGHG4 heavy chain constant region; UniProt accession number P01861) is depicted. CH1 = amino acids 1-98; upper hinge = amino acids 99-105; core hinge = 106-109; lower hinge = 110-118; CH2 = 117-220; CH3 = 221-227. The numbering of the amino acids shown is 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 discloses SEQ ID NO: 50. [Figure 4]4 depicts the amino acid sequence alignment of the upper hinge, core hinge, lower hinge, CH2, and CH3 of the accepted chimeric IgG heavy chain constant domain constructs. The amino acid numbering shown is EU numbering. The shaded cells in the lower hinge indicate the amino acids that also correspond to the first two amino acids of the CH2 domain. FIG. 4 discloses SEQ ID NO:51 (columns 3 and 4), SEQ ID NO:52 (column 5), SEQ ID NO:53 (column 6), SEQ ID NO:54 (column 7), and SEQ ID NO:55 (column 8). [Diagram 5] Representative data are presented demonstrating the antibody titers of alternative format antibodies drawn containing either IgG4 S108P / IgG4 S108P star (H315R, Y316F) or IgG1 PVA / IgG1 PVA star (H315R, Y316F) heterodimers and having a 2+1 N-scFv format drawn following stable expression in Chinese Hamster Ovary (CHO) cells. Various lengths of linkers between Fab and scFv were tested. [Figure 6A] Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6B] Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6C]Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6D] Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6E] Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6F] Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 6G]Representative enzyme-linked immunosorbent assay (ELISA) data are plotted demonstrating binding of observed controls and antibodies to hFCRγ1 (FIG. 6A), hFCRγ2A(H131) (FIG. 6B), hFCRγ2A(R131) (FIG. 6C), hFCRγ2B (FIG. 6D), hFCRγ3A(V158) (FIG. 6E), hFCRγ3A(F158) (FIG. 6F), and hFCRγ3B (FIG. 7G). Descriptions of control and test antibodies are provided in Table 3. [Figure 7] Representative results are depicted from a surrogate antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which the indicated 2+1 N-scFv alternative format antibody (AF1) with different Fc regions was tested along with a control. [Figure 8] Representative results from a surrogate ADCC assay are depicted in which the indicated 2+1 N-Fab alternative format antibodies (AF2) with different Fc regions were tested along with controls. [Figure 9] Representative results from a luciferase reporter assay demonstrating a 2+1 N-scFv format antibody (AF1) with a different Fc region, along with a control that caused activation of HEK293.SREluc.hFGFR1c.hKLB cells are depicted. [Figure 10] Representative results from a luciferase reporter assay demonstrating 2+1 N-Fab format antibodies with different Fc regions (AF2 and AF3) along with a control that caused activation of HEK293.SREluc.hFGFR1c.hKLB cells are depicted. [Figure 11] Representative results from a phospho-ERK activation assay are depicted demonstrating that 2+1 N-scFv (AF1) or 2+1 N-Fab (AF3) format antibodies, together with either IgG4 S108P or IgG1 PVA Fc regions or His.hFGF21, caused activation in primary human adipocytes. [Figure 12] FIG. 1 depicts representative flow binding assay results demonstrating that a bispecific antibody with an IgG1 PVA Fc region binds to cell surface targets with a higher maximum MFI signal than a bispecific antibody with an IgG4 S108P Fc region. [Figure 13A] Negative stain EM 2D class averages of IgG-CD40 complexes are plotted. Figure 13A is an exemplary negative stain EM 2D image displaying features corresponding to Fc, Fab, and CD40, as well as Fab-Fab angles. Figure 13B is a diagram of Figure 13A including its components. Figure 13C shows the 2D class averages of IgG1-CD40 complexes, Figure 13D shows the 2D class averages of IgG1-PVA-CD40 complexes, and Figure 13E shows the 2D class averages of IgG2-CD40 complexes. [Figure 13B] Negative stain EM 2D class averages of IgG-CD40 complexes are plotted. Figure 13A is an exemplary negative stain EM 2D image displaying features corresponding to Fc, Fab, and CD40, as well as Fab-Fab angles. Figure 13B is a diagram of Figure 13A including its components. Figure 13C shows the 2D class averages of IgG1-CD40 complexes, Figure 13D shows the 2D class averages of IgG1-PVA-CD40 complexes, and Figure 13E shows the 2D class averages of IgG2-CD40 complexes. [Figure 13C] Negative stain EM 2D class averages of IgG-CD40 complexes are plotted. Figure 13A is an exemplary negative stain EM 2D image displaying features corresponding to Fc, Fab, and CD40, as well as Fab-Fab angles. Figure 13B is a diagram of Figure 13A including its components. Figure 13C shows the 2D class averages of IgG1-CD40 complexes, Figure 13D shows the 2D class averages of IgG1-PVA-CD40 complexes, and Figure 13E shows the 2D class averages of IgG2-CD40 complexes. [Figure 13D] Negative stain EM 2D class averages of IgG-CD40 complexes are plotted. Figure 13A is an exemplary negative stain EM 2D image displaying features corresponding to Fc, Fab, and CD40, as well as Fab-Fab angles. Figure 13B is a diagram of Figure 13A including its components. Figure 13C shows the 2D class averages of IgG1-CD40 complexes, Figure 13D shows the 2D class averages of IgG1-PVA-CD40 complexes, and Figure 13E shows the 2D class averages of IgG2-CD40 complexes. [Figure 13E]Negative stain EM 2D class averages of IgG-CD40 complexes are plotted. Figure 13A is an exemplary negative stain EM 2D image displaying features corresponding to Fc, Fab, and CD40, as well as Fab-Fab angles. Figure 13B is a diagram of Figure 13A including its components. Figure 13C shows the 2D class averages of IgG1-CD40 complexes, Figure 13D shows the 2D class averages of IgG1-PVA-CD40 complexes, and Figure 13E shows the 2D class averages of IgG2-CD40 complexes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 6. MODE FOR CARRYING OUT THEINVENTION 6.1.Definition About, Approximately: The terms "about," "approximately," and the like are used throughout the specification preceding a numerical value to indicate that the numerical value is not necessarily precise (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, is understood to also be a disclosure of "X." Thus, for example, a disclosure of an embodiment in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of an embodiment in which the sequence has "X% sequence identity" to the other sequence.

[0019] And and Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where the selection of A is mutually exclusive of B) and the selection of conjoint features (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used with the same purpose and should not be interpreted to mean that "or" is used in reference to mutually exclusive alternatives.

[0020] 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 these. Antibodies can be monospecific (in which case all binding regions have the same specificity) or multispecific, in which the binding sites have at least two specificities (e.g., bispecific). The term "antibody" encompasses monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, and the like.

[0021] Associated: The term "associated" in relation to 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 portions of polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, e.g., non-covalently through molecular interactions or covalently through one or more disulfide or chemical bridges, to generate 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) an association between homodimeric or heterodimeric chimeric heavy chain constant domains of the present disclosure, an association between a VH region and a VL region in a Fab or scFv, an association between a CH1 and a CL in a Fab, and an association between a CH3 and a CH3 in a domain-substituted Fab.

[0022] Bivalent: As used herein with respect to an antibody comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure, the term "bivalent" means that the antibody has two antigen-binding moieties (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 chimeric constant domain of the present disclosure may be bivalent for one type of moiety (e.g., two antigen-binding fragments of a first antibody) or monovalent for another type of moiety (e.g., a single antigen-binding fragment of a second antibody).

[0023] Chimeric heavy chain constant domain and chimeric constant domain: The terms "chimeric heavy chain constant domain" and "chimeric constant domain", when used in reference to 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, an IgG1 lower hinge domain with a substitution / deletion mutation ELLG (SEQ ID NO: 23)→PVA- (or "PVA-absent") at amino acid positions 233-236 (EU numbering), an IgG1 CH2 domain, and an IgG1 CH3 domain. In some embodiments, the chimeric constant domain also comprises an IgG1 CH1 domain. In some embodiments, the chimeric constant domain can be further modified, for example, to further alter effector function and / or to provide heterodimerization. Chimeric constant domains of the present disclosure are further described in Section 6.2.

[0024] 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 identical or different. Thus, the resulting dimer can be a homodimer or a heterodimer.

[0025] A chimeric constant domain comprising an IgG1 lower hinge domain with the substitution / deletion mutation ELLG (SEQ ID NO: 23)→PVA- (or "PVA-absent") may be referred to as having the "IgG1 PVA" isotype or similar terminology.

[0026] Dimerization moiety: The term "dimerization moiety" refers to a polypeptide chain or an amino acid sequence that can promote association between two polypeptide chains to form a dimer. A first dimerization moiety can associate with an identical 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, and the association of the two recombinant constant domains forms an Fc region. Thus, the Fc region can be a homodimer or a heterodimer.

[0027] 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, that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an antibody that gives half-maximal activation in a luciferase reporter assay.

[0028] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain comprises an antibody variable heavy (VH) domain N-terminal to a first constant domain (herein referred to as C1), and the second polypeptide chain comprises an antibody variable light (VL) domain N-terminal to a second constant domain (herein referred to as C2) that can pair with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain, and the VL is N-terminal to the constant domain (CL) of the light chain. The Fabs of the present disclosure can be arranged according to the native orientation, or can include domain substitutions or swaps that facilitate correct VH and VL pairing. For example, the CH1 and CL domain pair in the Fab can be replaced with a CH3 domain pair to facilitate correct altered Fab-chain pairing in a heterodimeric molecule. The CH1 and CL can also be reversed, such that the CH1 is attached to the VL and the CL is attached to the VH, a configuration commonly known as a Crossmab.

[0029] Fc domain and Fc region: The term "Fc domain" refers to the portion of an immunoglobulin heavy chain that pairs 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 an Fc region may be identical to each other or different. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, e.g., via knob-in-hole interactions, and / or to allow purification, e.g., via a star mutation. The chimeric constant domain of the present disclosure comprises an Fc domain.

[0030] Hinge Region or Hinge Domain: As used herein, the terms "hinge region" and "hinge domain" refer to the consecutive amino acid residues that connect the C-terminus of CH1 to the N-terminus of the CH2 domain of an immunoglobulin. In human IgG1, IgG2, and IgG4, the hinge region extends from residues 216 to 238 according to EU numbering, with residues 237 and 238 overlapping with the CH2 domain. Residues 216-225 (EU numbering) form the upper hinge, residues 226-229 (EU numbering) form the middle (or core) hinge, and residues 230-238 (EU numbering) form the lower hinge. The upper and middle hinges of IgG1, IgG2, and IgG4 are 12-15 consecutive amino acids that are encoded by different hinge exons. The lower hinge contains 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., FIG. 4.

[0031] Host cell or recombinant host cell: 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 acid. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such cells. Since certain modifications may occur in successive generations, either by mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Host cells can harbor heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing 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 for higher density growth), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., HepG2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, for example, glycan profile modifications and / or site-specific integration site derivatives.

[0032] Monovalent: As used herein with respect to antibodies comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure, the term "monovalent" means that the antibody has one antigen-binding moiety specific for a first target molecule. Antibodies comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure may be monovalent for one type of moiety (e.g., a single antigen-binding fragment of a first antibody) or bivalent for another type of moiety (e.g., two antigen-binding fragments of a second antibody).

[0033] Multivalent: As used herein with respect to an antibody comprising a recombinant polypeptide and / or chimeric constant domain of the present disclosure, the term "multivalent" means that the antibody has two or more antigen-binding moieties (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 chimeric constant domain of the present disclosure may be multivalent with respect to one type of moiety (e.g., two or more antigen-binding fragments of a first antibody) or monovalent with respect to another type of moiety (e.g., a single antigen-binding fragment of a second antibody).

[0034] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, where the two or more regions are joined to produce a functional polypeptide or two or more nucleic acid sequences, e.g., to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0035] Recombinant Polypeptide: 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.

[0036] In the context of the present disclosure, the term "recombinant polypeptide" may refer to the core component(s) of the molecule, i.e., the chimeric constant domain, and may also refer to the antigen-binding portion. It should be understood that the term "recombinant polypeptide" also extends to polypeptides that include additional features, such as one or more stabilizing moieties, one or more linker moieties, and any combination of the foregoing, unless the context indicates otherwise.

[0037] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0038] Specifically (or selectively) binds: As used herein, the term "specifically (or selectively) binds" means that the antigen-binding portion thereof, e.g., an antibody or antigen-binding domain ("ABD"), forms a complex with a target molecule that is relatively stable under physiological conditions. Specific binding is on the order of about 5×10 -2 M or less (e.g., 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M or 10 -10 K (less than M) D Methods for determining the binding affinity of an antibody or antibody fragment to a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assay), fluorescence activated cell sorting (FACS) binding assays, etc. An antigen-binding moiety that specifically binds to a target molecule from one species may, however, have cross-reactivity to target molecules from one or more other species.

[0039] Target binding domain: As used herein, the term "target binding domain" refers to a polypeptide sequence or a group of related polypeptide sequences that can specifically, non-covalently, and reversibly bind to a target molecule. This term includes "antigen binding domain" and "antigen binding fragment," which refer to a portion of an antibody that can specifically, non-covalently, and reversibly bind to a target molecule. "Target binding domain" also encompasses the target binding portion of a receptor (e.g., a TNF receptor) that can specifically, non-covalently, and reversibly bind to a target molecule (e.g., TNF). Target binding domains can be incorporated into recombinant polypeptides of the present disclosure. In some embodiments, recombinant polypeptides that include one or more target binding domains can be included in an antibody or a fusion protein. Such antibodies are described below in Section 6.2.2. Fusion proteins are described below in Section 6.2.3.

[0040] Target molecule: As used herein, the term "target molecule" refers to any biological molecule (e.g., protein, carbohydrate, lipid, or combination thereof) expressed on a cell surface or in the extracellular matrix that can be specifically bound by an antigen-binding portion in an antibody comprising a recombinant polypeptide of the present disclosure.

[0041] Universal light chain: The term "universal light chain" as used herein in the context of an antigen-binding moiety refers to a light chain polypeptide that is capable of pairing with the heavy chain region of the antigen-binding moiety and is also capable of pairing with other heavy chain regions. The universal light chain is also known as the "common light chain."

[0042] VH: The term "VH" refers to the variable region of an antibody immunoglobulin heavy chain, including the heavy chain of an scFv or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or Fab.

[0043] 6.2. Recombinant Polypeptides The present disclosure provides recombinant polypeptides comprising a chimeric constant domain based on an IgG1 heavy chain constant domain in which the hinge region is modified to reduce Fc receptor and / or effector function. The recombinant polypeptides of the present disclosure can function as dimerization moieties that can promote association between two polypeptide chains to form dimers. In some embodiments, the recombinant polypeptides of the present disclosure can dimerize to form antibodies. Antibodies comprising recombinant polypeptides of the present disclosure are described below in Section 6.2.2. In other embodiments, the recombinant polypeptides of the present disclosure can dimerize to form fusion proteins. Fusion proteins comprising recombinant polypeptides of the present disclosure are described below in Section 6.2.3. The recombinant polypeptides of the present disclosure comprised in an antibody or fusion protein of the present disclosure can comprise, in addition to the chimeric constant domains of the present disclosure, one or more target binding domains (e.g., one or more Fab moieties, one or more scFv moieties, one or more target receptors or binding fragments thereof) and / or one or more linker moieties separating one or more moieties in the recombinant polypeptide. Target binding domains are described in Section 6.2.4. Linkers useful in the recombinant polypeptides of the disclosure are described in Section 6.2.5.

[0044] 6.2.1. Chimeric Constant Domains The present disclosure provides chimeric constant domains 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 with a substitution / deletion mutation ELLG (SEQ ID NO: 23)→PVA- at amino acid positions 233-236 (EU numbering), an IgG1 CH2 domain, and an IgG1 CH3 domain. In some embodiments, the chimeric constant domain also comprises an IgG1 CH1 domain or a fragment thereof.

[0045] 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 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 variation in its carbohydrate composition also strongly influences IgG / FcγR interactions (Stevenson, 1997, Chem Immunol 65:57-72; Siberil et al., 2006, Immunol Ltrs 106:111-118).

[0046] Alanine residues have typically been the preferred substitution for replacing natural amino acids with non-natural amino acids to reduce function, since alanine has a side chain that does not bear any functional group. For example, the well-known technique of alanine scanning mutagenesis systematically replaces all natural residues in a protein or protein domain with alanine to identify which natural residues primarily contribute to function. Replacing an amino acid with an alanine-bearing functional group eliminates the functional group and its contribution to binding to any receptor, while the presence of the alanine side chain substantially preserves the conformation, reducing the possibility of immunogenicity or other complications due to conformational changes. An alternative strategy replaces amino acids in the lower hinge region of a chimeric IgG, including IgG1 upper hinge, IgG1 lower hinge, IgG4 CH2, and IgG1 CH3, with the corresponding amino acids from the human IgG2 isotype to reduce FcγR binding without unacceptable conformational changes and resulting immunogenicity (see WO14 / 121087). In yet another alternative strategy, amino acids 233-236 (EU numbering) of the modified hinge region can be G, G, G and empty, G, G, empty and empty, G, empty, empty and empty, or all empty, with positions numbered according to EU numbering (as shown in Figure 1 of WO2016 / 161010A2).

[0047] An IgG1 heavy chain constant region is provided having a modified hinge region to reduce Fc receptor and / or effector function. The modification occurs at amino acid positions 233-236 (EU numbering) by substitution / deletion with PVA- of ELLG (SEQ ID NO: 23), with amino acid 236 deleted. In a particular 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 activating Fc receptor. In a specific embodiment, the Fc receptor is an activating 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 of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0048] As presented in this example, it was found that this substitution / deletion mutation (ELLG (SEQ ID NO: 23) → PVA- at amino acids 233-236 (EU numbering)) in an IgG1 background reduces FcR engagement and maximizes hinge flexibility while minimizing immunogenicity, thereby providing an alternative chimeric heavy chain constant region for inclusion in, for example, therapeutic antibodies and Fc fusion proteins. Having alternatives to those chimeric heavy chain constant regions known in the art is valuable. Having alternative chimeric heavy chain constant regions based on different IgG subclasses allows, for example, optimization of antibody activity. This modified immunoglobulin constant region can be incorporated into virtually any format of antibody or Fc fusion protein. Such antibodies or fusion proteins can be used in therapeutic methods, particularly those where the mechanism of action of the antibody or Fc fusion protein is primarily or entirely independent of effector function, such as when the antibody inhibits receptor-ligand interaction or agonizes the receptor.

[0049] With the exception of the →ELLG (SEQ ID NO: 23) →PVA-substitution / deletion at amino acid positions 233-236 (EU numbering), a 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 exception that the CH1 domain, as well as the upper hinge region, can optionally be omitted entirely. The CH1, CH2, and CH3 domains are considered to be of the IgG1 isotype if they differ from the CH1, CH2, and CH3 regions of the IgG1 wild-type sequence, respectively, by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, deletions, or insertions. The substitutions, deletions, and / or insertions (except for the ELLG (SEQ ID NO: 23)→PVA-substitution / deletion at amino acid positions 233-236 (EU numbering)) can be any substitution, deletion, and / or insertion. In some embodiments, the substitutions, deletions, and / or insertions do not result in a sequence identical to, for example, the CH1, CH2, or CH3 of another IgG isotype (e.g., IgG2, IgG3, or IgG4). For example, in some embodiments, if a chimeric constant domain of the present disclosure includes one or more of H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), they do not all occur simultaneously. The wild-type sequences of the heavy chain constant regions of IgG1, IgG2, and IgG4 are depicted in Figures 1, 2, and 3, respectively, with the CH1, hinge, CH2, and CH3 regions illustrated. Exemplary mutations that may be included in the chimeric constant domains of the present disclosure are provided below.

[0050] The sequence of the wild type IgG1 heavy chain constant region (amino acids 216-447, EU numbering) comprises: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1), wherein the upper hinge region comprises EPKSCDKTHT (SEQ ID NO:24) (amino acids 216-225 (EU numbering); amino acids 1-10 of the wild type IgG1 constant domain sequence), the core hinge comprises CPPC (SEQ ID NO:25) (amino acids 226-229 (EU numbering); amino acids 11-14 of the wild type IgG1 constant domain sequence), the lower hinge region comprises PAPELLG (SEQ ID NO:26) (amino acids 230-236 (EU numbering); amino acids 15-21 of the wild type IgG1 constant domain sequence), and CH2 comprises GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:27) (amino acids 237-340 (EU numbering); amino acids 22-125 of the wild type IgG1 constant domain sequence) and CH3 comprises GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:28) (amino acids 341-447 (EU numbering); amino acids 126-232 of the wild type IgG1 constant sequence). It will be appreciated by those skilled in the art that amino acids 237-238 (EU numbering) represent both the C-terminus of the lower hinge and the N-terminus of the CH2 region. However, for purposes of this disclosure, amino acids 237-238 (EU numbering) are designated as part of the lower hinge.

[0051] In some embodiments, the sequence of the chimeric constant region of the disclosure, also referred to herein as IgG1 PVA (amino acids 216-447; EU numbering), comprises: EPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2), wherein the upper hinge region comprises EPKSCDKTHT (SEQ ID NO:56) (amino acids 216-225 (EU numbering); amino acids 1-10 of the IgG1 PVA constant domain sequence), the core hinge comprises CPPC (SEQ ID NO:57) (amino acids 226-229 (EU numbering); amino acids 11-14 of the IgG1 PVA constant domain sequence), the lower hinge region comprises PAPPVA (SEQ ID NO:29) (amino acids 230-236 (EU numbering); amino acids 15-20 of the IgG1 PVA constant domain sequence), and CH2 comprises GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:58) (amino acids 237-340 (EU numbering); IgG1 CH3 contains GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:59) (amino acids 341-447) (EU numbering); IgG1 PVA sequence).

[0052] 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 the following sequence: EPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2).

[0053] The chimeric constant domains can also contain knob mutations, hole mutations, star mutations, disulfide bridge forming mutations, etc. to facilitate heterodimerization and / or purification. 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).

[0054] 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)).

[0055] 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)).

[0056] 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)).

[0057] 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)).

[0058] 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)).

[0059] 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 all mutations T366S, L368A, Y407V and Cys mutation S354C (EU numbering)).

[0060] 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 mutations H435R, Y436F, and Cys mutation S354C (EU numbering)).

[0061] 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)).

[0062] 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 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 30) or a fragment or variant thereof, such as 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).

[0063] 6.2.1.1. Engineered Chimeric Constant Domains In some embodiments, the chimeric constant domain is modified to further alter effector function and / or provide heterodimerization. Exemplary modifications are described in Sections 6.2.1.1.1 and 6.2.1.1.2.

[0064] 6.2.1.1.1. Chimeric constant domains with further modification of effector function 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 acids 233-236 (EU numbering), which additional amino acid substitutions further reduce binding to Fc receptors and / or effector function.

[0065] In some embodiments, the Fc receptor is an Fcγ receptor. In certain embodiments, the Fc receptor is a human Fc receptor. In certain embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating 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 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.

[0066] 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).

[0067] In some embodiments, the chimeric constant domain comprises an amino acid substitution at position P329 (EU numbering). In more specific embodiments, the amino acid substitution is P329A or P329G, in particular P329G (EU numbering). In one embodiment, the chimeric constant domain comprises an amino acid substitution at position P329 and a further amino acid substitution at positions N297 and / or P331 (EU numbering). In more specific embodiments, the further amino acid substitution is N297A or N297D and / or P331S.

[0068] 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 (numbering according to the Kabat EU index).

[0069] In some embodiments, the chimeric constant domain contains the D265A and N297A mutations (EU numbering) to reduce effector function. 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. Thus, in certain embodiments, each chimeric constant domain contains the amino acid substitutions G237A, A330S, and P331S (EU numbering), i.e., in each of the first and second chimeric constant domains, the glycine residue at position 237 is replaced with an alanine residue (G237A), the alanine residue at position 330 is replaced with a serine residue (A330S), and the proline residue at position 331 is replaced with a serine residue (P331S) (EU numbering).

[0070] 6.2.1.1.2. Chimeric Constant Domain Heterodimerization Variants In certain embodiments, the recombinant polypeptides of the present disclosure are dimerized through a chimeric constant domain. In some embodiments, the resulting dimer forms or forms part of an antibody or an Fc fusion protein. In certain embodiments, a first recombinant polypeptide dimerizes with an identical second recombinant polypeptide to form a homodimer. In other embodiments, a 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 can include a first target binding domain specific for a first target molecule, and the second recombinant polypeptide can include a second target binding domain specific for a second target molecule. When the first and second recombinant polypeptides dimerize through their respective chimeric constant domains, the result is a heterodimer. Inappropriate heterodimerization of recombinant polypeptides (e.g., heterodimerization of the chimeric constant domains of each recombinant polypeptide to form an Fc region) can be an obstacle to increasing the yield of the desired heterodimeric molecule and poses purification challenges. Various approaches available in the art can be used to enhance dimerization of recombinant polypeptides of the present disclosure, for example, as disclosed in EP 1870459 A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006 / 204493 A1; and PCT Publication No. WO 2009 / 089004 A1.

[0071] 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. Heterodimerization of recombinant polypeptides via the CH3 domain of a chimeric constant domain can produce the desired heterodimer (e.g., antibody or fusion protein), but homodimerization of the same chimeric constant domain will reduce yield. Thus, in some embodiments, recombinant polypeptides that assemble to form an antibody or fusion protein of the present disclosure will comprise a chimeric constant domain that contains a CH3 domain with a modification that favors heterodimer association compared to a non-modified constant domain.

[0072] In a specific embodiment, the modification that promotes heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification that includes a "knob" modification in one of the chimeric constant domains and a "hole" modification in the other chimeric constant domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 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 a corresponding cavity ("hole") at the interface of a second polypeptide such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent 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 identical or similar size to the protrusion are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0073] Thus, in some embodiments, an amino acid residue in the CH3 domain of a first chimeric constant domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance in the CH3 domain of the first chimeric constant domain that can be positioned in a cavity in the CH3 domain of the second chimeric constant domain, and an amino acid residue in the CH3 domain of a second chimeric constant domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second chimeric constant domain into which the protuberance in the CH3 domain of the first chimeric constant domain can be positioned. Preferably, the amino acid residue having 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 having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0074] In a specific embodiment, the first chimeric constant domain has the threonine residue at position 366 replaced with a tryptophan residue (T366W) and the second chimeric constant domain has the tyrosine residue at position 407 replaced with a valine residue (Y407V), and optionally the threonine residue at position 366 replaced with a serine residue (T366S) and the leucine residue at position 368 replaced with an alanine residue (L368A) (EU numbering). In a further embodiment, the first chimeric constant domain additionally has the serine residue at position 354 replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue). In the second chimeric constant domain, additionally, the tyrosine residue at position 349 is replaced with 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).

[0075] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of the first and second chimeric constant domains.

[0076] As an alternative or in addition to the use of chimeric constant domains modified to promote heterodimerization, chimeric constant domains can be modified to allow for purification strategies that allow for the selection of chimeric constant domain heterodimers. In one such embodiment, one recombinant polypeptide comprises a modified chimeric constant domain that abrogates its binding to Protein A, thus allowing for a purification method that results in a heterodimeric protein. See, for example, U.S. Pat. No. 8,586,713. Such an antibody or fusion protein comprises a first CH3 domain and a second CH3 domain, the first and second CH3 domains differing from each other by at least one amino acid, the at least one amino acid difference reducing binding of the antibody or fusion protein to Protein A compared to a corresponding antibody or fusion protein lacking the amino acid difference. 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 a H435R modification (EU numbering). The second CH3 may further comprise a Y436F modification (EU numbering). This class of alterations is referred to herein as "star" mutations.

[0077] In some embodiments, the chimeric constant domain may contain one or more mutations to promote heterodimerization (e.g., knob and hole mutations) and star mutations to promote purification.

[0078] 6.2.2. Antibodies In some embodiments, recombinant polypeptides comprising chimeric constant domains dimerize to form antibodies. The recombinant polypeptides can be used in any type of engineered antibody, including chimeric, humanized, veneered, or human antibodies. The antibodies can be monoclonal or engineered polyclonal antibodies.

[0079] Antibodies of the disclosure typically comprise or consist of recombinant polypeptides of the disclosure, each of which comprises a chimeric constant domain, one or more antigen-binding domains, and, optionally, one or more linkers separating the one or more domains in the recombinant polypeptide.

[0080] An antibody may be composed of one or more recombinant polypeptides of the present disclosure. In some embodiments, an antibody is composed of two recombinant polypeptides, optionally associated with one or more additional polypeptide chains (e.g., a polypeptide chain comprising the light chain of the antigen-binding domain, such as in the case of a Fab). The two recombinant polypeptides may be identical, thereby forming a homodimer, or different, thereby forming a heterodimer. The two recombinant polypeptides are generally configured to dimerize together via the chimeric constant domain of each recombinant polypeptide. In some embodiments, one or more additional polypeptide chains are associated with the dimerizing recombinant polypeptide. Thus, an antibody may comprise two, three, four or more polypeptide chains.

[0081] An antibody is monospecific if the two recombinant polypeptides contain antigen binding domains that target the same epitope on the same target molecule. An antibody or fusion protein is multispecific (e.g., bispecific) if the two recombinant polypeptides contain antibody binding domains that, individually or together, target two or more different epitopes on the same target molecule, or two or more different target molecules. Most antibodies are multimeric by dimerization via the chimeric constant domains of the recombinant polypeptides. A multispecific antibody or fusion protein may be monovalent for the first antigen binding domain, monovalent for the second and subsequent antigen binding domains, or monovalent for the first antigen binding domain, multivalent for the second antigen binding domain, and monovalent or multivalent for any additional (e.g., third) antigen binding domain.

[0082] Particularly preferred formats of antibodies comprising the constant domains of the present disclosure are N-terminal scFv formats (e.g., as described in WO2021 / 091953A1, the contents of which are incorporated herein by reference in their entirety; an example of an N-terminal scFv format is shown in Figure 5 herein) or antibodies in which the N-terminal scFv is replaced with an N-terminal Fab. scFv and Fab domains are described in Sections 6.2.4.2 and 6.2.4.1, respectively. In some embodiments, the antibody is trispecific and is in a "2+1 N-scFv" or "2+1 N-Fab" format, while traditional bispecific antibodies have an scFv or Fab domain added to the N-terminus of one of their VH domains, preferably via a linker. Suitable linkers are disclosed in Section 6.2.5.

[0083] As disclosed in WO2021 / 091953A1, a "2+1 N-scFv" format typically comprises: (a) a first polypeptide chain comprising an scFv comprising (i) a first antigen binding site ("ABS1"), in an N-terminal to C-terminal orientation, operably linked to (ii) a first heavy chain region of a first Fab ("Fab1"), which is operably linked to (iii) an Fc domain; and (b) a second heavy chain region of a second Fab ("Fab2"), in an N-terminal to C-terminal orientation, operably linked to (ii) an Fc domain. (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form Fab1, where Fab1 comprises a second antigen binding site ("ABS2"); and (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form Fab2, where Fab2 comprises a third antigen binding site ("ABS3"). In some embodiments, ABS1, ABS2, and ABS3 each bind to a different epitope. In some embodiments, two of ABS1, ABS2, and ABS3 specifically bind to different epitopes of the same target molecule. In some embodiments, the 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 having a first tissue expression profile, and at least one of ABS1, ABS2, and ABS3 specifically binds to a target molecule having a second tissue expression profile that overlaps, but is not identical to, the first tissue expression profile. In some embodiments, the scFv is linked to the first heavy chain region via a linker. In some embodiments, the antigen binding molecule is trivalent. In some embodiments, the scFv is linked to the first heavy chain region via a linker. In some embodiments, the linker is 5, 6, 7, or more amino acids in length. In some embodiments, the linker is up to 30, up to 40, or up to 50 amino acids in length.

[0084] In some embodiments, effector functions dependent on Fcγ receptor binding, such as ADCC or ADCP, are reduced by at least 90%, at least 95%, at least 99%, or to background levels, for example, compared to an antibody comprising a wild-type IgG1 constant heavy domain. Such functions include cell killing or phagocytosis, B cell activation, and release of inflammatory mediators, such as cytokines. Some such effects may be quantified by measuring EC50. In certain embodiments, an antibody comprising a chimeric constant domain of the present disclosure exhibits a cytotoxic activity of less than 20% cell lysis (e.g., % cytotoxicity), less than 10% cell lysis, less than 5% cell lysis, less than 4% cell lysis, less than 3% cell lysis, less than 2% cell lysis, or 0% cell lysis or undetectable cell lysis, as measured in an in vitro or ex vivo cell killing assay (optionally measured at an antibody concentration of at least 10 nM), compared to a suitable isotype-matched control antibody having a wild-type constant region.

[0085] In some embodiments, an antibody comprising a recombinant polypeptide of the disclosure exhibits at least 5-fold reduced binding, 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 to a human Fc receptor (FcR) than an antibody comprising a wild-type IgG1 constant domain. The FcR can be, for example, FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B. FcR binding can be determined by ELISA, for example, as described in Section 8.1.4.

[0086] In some embodiments, the binding affinity of an antibody incorporating a chimeric constant domain of the present disclosure to a target is not substantially affected by the chimeric constant domain compared to an appropriate heavy chain constant domain (e.g., wild-type IgG1). That is, the binding affinity is typically the same within experimental error, or at least within a factor of 2 or 3, of a suitable control antibody having an isotype-matched wild-type constant domain. The same is true for functional properties that are not dependent on FcγR binding, such as the ability to inhibit receptor-ligand binding (e.g., EC50) or the ability to agonize a receptor.

[0087] The immunogenicity of antibodies incorporating recombinant polypeptides of the present disclosure (and thus chimeric constant domains of the present disclosure) compared to an isotype-matched control can be assessed in vitro from 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 antibody between populations. In some embodiments, the immunogenicity of antibodies incorporating recombinant polypeptides of the present disclosure (and thus chimeric constant domains of the present disclosure) is reduced. In other embodiments, the immunogenicity is not significantly different from an isotype-matched control, or is not more than 2-fold, 3-fold, or 5-fold lower than an isotype-matched control. Similarly, C max , C 平均 Pharmacokinetic parameters such as kinetics, area under the curve, and half-life are preferably not significantly different from, or at least not more than 2-, 3-, or 5-fold lower than, an isotype-matched control. Substantial retention or improvement of such PK parameters can indicate that the antibodies of the disclosure have not undergone substantial conformational changes that result in enhanced clearance mechanisms.

[0088] In certain embodiments, the antibodies of the present disclosure exhibit increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain (e.g., comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3). In some embodiments, the increase compared to an antibody comprising a wild-type IgG1 heavy chain constant domain is at least a 5% increase, at least a 10% increase, at least a 10% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, or at least a 100% increase. In certain aspects, the increased expression of the antibody is evidenced by increased production of secreted protein, e.g., antibody, and / or increased total protein yield obtained from an in vitro expression system, e.g., an antibody expression system. In some embodiments, the expression system is a Chinese Hamster Ovary (CHO) stable expression system. Increased expression in CHO stable expression systems can be suitably assessed as described in Section 8.1.6, where protein production is stimulated by inducing the cultures with doxycycline for multiple days. In other embodiments, the expression system is a HEK293-based expression system, such as the Expi293F™ expression system.

[0089] Fusion Proteins In some embodiments, recombinant polypeptides comprising chimeric constant domains dimerize to form fusion proteins. Fusion proteins of the present disclosure typically comprise or consist of recombinant polypeptides of the present disclosure, each of which comprises a chimeric constant domain, one or more heterologous polypeptides, and, optionally, one or more linkers separating one or more domains within the recombinant polypeptide.

[0090] A fusion protein may be composed of one or more recombinant polypeptides of the present disclosure. In some embodiments, a fusion protein is composed of two recombinant polypeptides. The two recombinant polypeptides may be identical, thereby forming a homodimer, or may be different, thereby forming a heterodimer. The two recombinant polypeptides are generally configured to dimerize together via the chimeric constant domain of each recombinant polypeptide. In some embodiments, one or more additional polypeptide chains are associated with the dimerized recombinant polypeptide. Thus, a fusion protein may include two, three, four or more polypeptide chains.

[0091] 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 not naturally linked to an immunoglobulin constant domain, including an engineered form of the 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-natural antigen binding domains (e.g., engineered antigen binding domains such as scFvs, domain-swapped Fabs, etc.). Thus, in some embodiments, the fusion protein can be an antibody. Exemplary receptor proteins that can combine chimeric constant and extracellular domains in a recombinant polypeptide 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 & Muller-Newen, 2012, Eur J Cell Biol 91(6-7):428-34).

[0092] In some embodiments, the fusion protein is a homodimer. In other embodiments, the fusion protein is a heterodimer. In certain embodiments, the fusion protein is monospecific (e.g., contains target binding domain(s) specific for a single target molecule or contains a single ligand domain). In other embodiments, the fusion protein is multispecific (e.g., contains two or more target binding domains each specific for a different target molecule or contains two or more different ligand domains). Most fusion proteins are multimeric by dimerization via chimeric constant domains of the recombinant polypeptides.

[0093] In some embodiments, effector functions dependent on Fcγ receptor binding, such as ADCC or ADCP, are reduced by at least 90%, at least 95%, at least 99%, or to background levels, for example, compared to a fusion protein comprising a wild-type IgG1 constant heavy domain. Such functions include cell killing or phagocytosis, B cell activation, and release of inflammatory mediators, such as cytokines. Some such effects may be quantified by measuring EC50. In certain embodiments, a fusion protein comprising a chimeric constant domain of the present disclosure exhibits a cytotoxic activity of less than 20% cell lysis (e.g., % cytotoxicity), less than 10% cell lysis, less than 5% cell lysis, less than 4% cell lysis, less than 3% cell lysis, less than 2% cell lysis, or 0% cell lysis or undetectable cell lysis, as measured in an in vitro or ex vivo cell killing assay (optionally measured at a fusion protein concentration of at least 10 nM), compared to a suitable isotype-matched control fusion protein having a wild-type constant region.

[0094] In some embodiments, a fusion protein comprising a recombinant polypeptide of the disclosure exhibits at least 5-fold reduced binding, 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 to a human Fc receptor (FcR) than a fusion protein comprising a wild-type IgG1 constant domain. The FcR can be, for example, FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B. FcR binding can be determined by ELISA, for example, as described in Section 8.1.4.

[0095] In some embodiments, the binding affinity of a fusion protein incorporating a chimeric constant domain of the present disclosure to a target is not substantially affected by the chimeric constant domain compared to an appropriate heavy chain constant domain (e.g., wild-type IgG1). That is, the binding affinity is typically the same within experimental error, or at least within a factor of 2 or 3, of a suitable control fusion protein having an isotype-matched wild-type constant domain. The same is true for functional properties that are not dependent on FcγR binding, such as the ability to inhibit receptor-ligand binding (e.g., EC50) or the ability to antagonize a receptor.

[0096] In some embodiments, the immunogenicity of a fusion protein incorporating a recombinant polypeptide of the present disclosure (and thus a chimeric constant domain of the present disclosure) is reduced. In other embodiments, the immunogenicity is not significantly different from an isotype-matched control, or is not more than 2-fold, 3-fold, or 5-fold lower than an isotype-matched control. Similarly, C max , C 平均Pharmacokinetic parameters such as PK, area under the curve, and half-life are preferably not significantly different from, or at least not more than 2-, 3-, or 5-fold lower than, an isotype-matched control. Substantial retention or improvement of such PK parameters can indicate that the fusion proteins of the present disclosure have not undergone substantial conformational changes that result in enhanced clearance mechanisms.

[0097] In certain embodiments, the fusion proteins of the present disclosure may exhibit increased expression in an expression system compared to a fusion protein comprising a wild-type IgG1 constant domain (e.g., comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3). In some embodiments, the increase compared to a fusion protein comprising a wild-type IgG1 heavy chain constant domain is at least a 5% increase, at least a 10% increase, at least a 10% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, or at least a 100% increase. In certain aspects, the increased expression of the fusion protein is evidenced by increased production of secreted protein, e.g., the fusion protein, and / or increased total protein yield obtained from an in vitro expression system, e.g., a fusion protein expression system. In some embodiments, the expression system is a Chinese Hamster Ovary (CHO) stable expression system. Increased expression in CHO stable expression systems can be conveniently assessed as described in Section 8.1.6, where protein production is stimulated by inducing the cultures with doxycycline for multiple days. In other embodiments, the expression system is a HEK293-based expression system, such as the Expi293F expression system.

[0098] 6.2.4. Target Binding Domains A recombinant polypeptide of the present disclosure can include one or more target binding domains (e.g., one, two, three, or more target binding domains), where each target binding domain specifically binds to a selected target molecule. In some embodiments, a recombinant polypeptide includes a single target binding domain. The target binding domain can be N-terminal to the chimeric constant domain or can be C-terminal to the chimeric constant domain. In some embodiments, the single target binding domain is attached to the chimeric constant domain via a linker.

[0099] In some embodiments, the recombinant polypeptide comprises two or more target binding domains. The two or more target binding domains may be identical or different. In some embodiments, the two or more target binding domains are identical and bind to the same epitope on the target molecule. In other embodiments, the 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 are identical and can bind to the same epitope on the first target molecule, and the remaining target binding domain(s) 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 can be N-terminal to the chimeric constant domain, each of the two or more target binding domains can be C-terminal to the chimeric constant domain, or one(s) target binding domain can be N-terminal to the chimeric constant domain and the other(s) target binding domain(s) C-terminal to the chimeric constant domain. The two or more target binding domains and the chimeric constant domains can be separated via one or more linkers.

[0100] The target binding domain may be specific for any epitope and / or target molecule of interest. The target molecule may be human, mammalian, or bacterial. Targets may be antigens such as proteins, glycoproteins, and carbohydrates from microbial pathogens, both viral and bacterial, as well as tumors.

[0101] The target binding domain can be an antigen binding domain or an antigen binding fragment of an antibody. In some embodiments, the antigen binding domain is a domain of a commercially available antibody. 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 VH (or V H ) fragment, VL (or V L) fragment, Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, minibody, diabody, triabody, and tetrabody. When the antigen-binding domain comprises two separate polypeptide chains (e.g., Fab), the first polypeptide chain (e.g., comprising a VH) can be comprised in the recombinant polypeptide, and the second polypeptide chain (e.g., comprising a VL) can be comprised in a polypeptide that can associate with the VH comprised in the recombinant polypeptide. Exemplary targets of the antibody molecules comprising the constant domain of the present disclosure are cell surface expressed antigens, such as proteins, carbohydrates, and lipids. In some embodiments, the target molecule is a protein molecule. Exemplary target molecules include human Klotho beta ("KLB"), human fibroblast growth factor receptor 1c isoform ("FGFR1c"), human fibroblast growth factor receptor 3 ("FGFR3"), human CD63, and human amyloid precursor-like protein 2 (APLP2). Exemplary KLB targeting domains are described in Tables 2A and 2B of WO2021 / 091953A1. Exemplary FGFR1c targeting domains are shown in Tables 3A and 3B of WO2021 / 091953A1. Exemplary FGFR3 binding domains are described in Table 4 of WO2021 / 091953A1. Exemplary APLP2 binding domains are described in Table 5 of WO2021 / 091953A1. Exemplary CD3 binding domains are described in Table 6 of WO2021 / 091953A1. The binding domains can be configured in a 2+1 N-terminal scFv format as described in WO2021 / 091953A1, or in a 2+1 N-Fab format in which the N-terminal scFv of WO2021 / 091953 is replaced with an N-terminal Fab. The contents of WO2021 / 091953A1 are incorporated herein by reference in their entirety.

[0102] In other aspects, the target binding domain contained in a recombinant polypeptide of the present disclosure can be any type of receptor or target binding portion thereof that specifically binds to a selected target molecule. 6.2.4.1.Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In antibodies comprising recombinant polypeptides of the present disclosure, the Fab domain is typically recombinantly expressed in at least a portion of the Fab contained in the recombinant polypeptide.

[0103] The Fab domain can comprise constant and variable region sequences from any suitable species, and thus may be murine, chimeric, human, or humanized. In some embodiments, the variable and / or constant domain region sequences are derived from a known antibody. Examples of known antibodies are provided above.

[0104] A Fab domain typically comprises a CH1 domain attached to a VH domain that is paired with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0105] For antibodies comprising the 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 correct association of Fab domains belonging to the same antigen-binding domain and to minimize aberrant pairing of Fab domains belonging to different antigen-binding domains. For example, the Fab heterodimerization strategy shown in Table 1 below can be used:

[0106] [Table 1]

[0107] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is promoted by swapping the VL and VH domains of the Fab with one another, or swapping the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0108] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. 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 other Fab components.

[0109] In one embodiment, the one or more amino acid modifications are restricted to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains 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, Chothia, and IMGT numbering schemes.

[0110] In one embodiment, the modifications introduced in the VH and CH1 domains and / or the VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of different interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of structural and chemical correspondence between the two interacting surfaces.

[0111] In one embodiment, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0112] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and a 114A and 137K substitution 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).

[0113] 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 hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0114] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, the 39K, 62E modifications are introduced in the VH domain, the H172A, F174G modifications are introduced in the CH1 domain, the 1R, 38D, (36F) modifications are introduced in the VL domain, and the L135Y, S176W modifications are introduced in the CL domain. In another embodiment, the 39Y modification is introduced in the VH domain and the 38R modification is introduced in the VL domain.

[0115] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0116] Fab domains can also be modified by replacing the CH1 and CL domains 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 a T cell receptor and the CL domain with the bb domain of a T cell receptor, pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing the 38D modification in the VL domain and the 39K modification in the VH domain.

[0117] Instead of or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, a 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, using a common light chain as described herein reduces the number of inappropriate species of the recombinant polypeptide or antibody compared to using the original cognate VL. In various embodiments, the VL domain of the recombinant polypeptide or antibody is identified from a monospecific antibody that includes a common light chain. In various embodiments, the VH region of the recombinant polypeptide or antibody includes a limited human light chain repertoire or human heavy chain variable gene segments that are rearranged in vivo in a mouse B cell that has been previously engineered to express a single human light chain, cognate to a human heavy chain, and in response to exposure to an antigen of interest, generate an antibody repertoire that includes one of two possible human VLs or multiple human VHs that are cognate to one of the human heavy chains, and this antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0118] 6.2.4.2.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as single-chain polypeptides (e.g., recombinant polypeptides of the present disclosure), and retain the specificity of the intact antibody from which they are derived. Generally, scFv polypeptides further comprise 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 an scFV are the linkers identified in Section 6.2.5.

[0119] As used herein, unless otherwise specified, an scFv may have the VL variable region and the VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0120] An scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. In some embodiments, an scFv can comprise VH and VL sequences from a known antibody. Examples of known antibodies are provided above.

[0121] To generate an scFv-encoding nucleic acid, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.2.5 (typically repeats of the amino acids glycine and serine, e.g., a sequence containing the amino acid sequence (Gly4 to Ser)3 (SEQ ID NO: 13)), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, the VL and VH regions being 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).

[0122] Linker In certain aspects, the present disclosure provides recombinant polypeptides in which two or more components of the recombinant polypeptide are connected to each other by a peptide linker (which for convenience may be referred to herein as a "linker"). By way of 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., a VH domain and a VL domain within an scFv).

[0123] The peptide linker can be in the range of 2 to 60 or more amino acids, and in certain embodiments, the peptide linker can be in the range of 3 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 in length.

[0124] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0125] In some of the aforementioned embodiments, the linker is in the range of 5 amino acids to 50 amino acids in length, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, or 5-20 amino acids in length. In other of the aforementioned embodiments, the linker is in the range of 6 amino acids to 50 amino acids in length, e.g., 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, or 6-20 amino acids in length. In yet other of the aforementioned embodiments, the linker is in the range of 7 amino acids to 50 amino acids in length, e.g., 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, or 7-20 amino acids in length.

[0126] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used in the recombinant polypeptides of the present disclosure include those disclosed in 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 include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 33) or SG n(SEQ ID NO: 34), where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is G4S (SEQ ID NO: 11), e.g., (GGGGS) n (SEQ ID NO: 35)

[0127] Polyglycine linkers can be suitably used in the recombinant polypeptides of the present disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO:36), five consecutive glycines (5Gly) (SEQ ID NO:37), six consecutive glycines (6Gly) (SEQ ID NO:38), seven consecutive glycines (7Gly) (SEQ ID NO:39), eight consecutive glycines (8Gly) (SEQ ID NO:40), or nine consecutive glycines (9Gly) (SEQ ID NO:41).

[0128] 6.3. Nucleic Acids and Host Cells In another aspect, the disclosure provides nucleic acids encoding the chimeric constant domains, recombinant polypeptides, antibodies, and fusion proteins of the 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 an antibody or fusion protein is composed of more than one polypeptide chain, the antibody or fusion protein can be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0129] A single nucleic acid can encode a portion of an antibody or fusion protein comprising a single polypeptide chain, an antibody or fusion protein comprising two or more polypeptide chains, or an antibody or fusion protein comprising three or more 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). In order to separately control expression, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding the two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0130] 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 can 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 two or more polypeptide chains are encoded by a single nucleic acid).

[0131] The nucleic acids of the disclosure can be DNA or RNA (eg, mRNA). In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0132] Vectors The present disclosure provides a vector comprising a nucleotide sequence encoding one or two of the polypeptide chains of a chimeric constant domain, recombinant polypeptide, antigen, or fusion protein, such as an antibody, described herein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0133] A number of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as 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 flaviviruses.

[0134] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0135] Once the DNA sequence containing expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions for culturing the resulting transfected cells and 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.

[0136] 6.3.2.Cells The disclosure also provides a host cell comprising a nucleic acid of the disclosure. In one embodiment, the host cell is genetically engineered to contain one or more of the nucleic acids described herein.

[0137] In one embodiment, the host cell is genetically engineered by using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without an intron, and a termination signal. Additional factors necessary or helpful in effecting expression, such as an inducible promoter, can also be used.

[0138] The present disclosure also provides a host cell comprising the vector described herein. The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. 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.

[0139] Pharmaceutical Compositions The antibodies and fusion proteins of the present disclosure may be in the form of a composition comprising the antibodies and fusion proteins and one or more carriers, excipients, and / or diluents. The compositions may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the antibodies or fusion proteins and, in the case of therapeutic use, the mode of administration.

[0140] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition that includes a pharma- ceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0141] The pharmaceutical composition can be conveniently presented in a unit dosage form containing a predetermined amount of the antibody or fusion protein of the present disclosure per dose. The amount of antibody or fusion protein contained in the unit dose will depend on the disease being treated, as well as other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of antibody or fusion protein suitable for single administration, or in the form of a liquid. The dry powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. The unit dosage in liquid form can be conveniently supplied in the form of a syringe pre-filled with an amount of antibody or fusion protein suitable for single administration.

[0142] The pharmaceutical compositions may also be provided in bulk, containing an amount of antibody or fusion protein suitable for multiple administrations. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody or fusion protein having the desired purity with any pharma- ceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, isotonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare, ed. 2020). Such additives should be non-toxic to the recipient at the dosages and concentrations used.

[0143] Buffering agents serve to maintain the pH in a range close to physiological conditions. They may be present in a wide variety of concentrations, but will typically be present in a range of about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, 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-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate 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-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconic acid-sodium hydroxide mixtures, potassium gluconate glyconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffer (e.g., lactate-sodium lactate mixture, lactate-sodium hydroxide mixture, lactate-potassium lactate mixture, etc.), and acetate buffer (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffer, histidine buffer, and trimethylamine salt (e.g., Tris) may be used.

[0144] Preservatives may be added to retard microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity agents, sometimes known as "stabilizers," may be added to ensure isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives that help solubilize the therapeutic agent or prevent denaturation or adhesion to container walls.Exemplary stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and the like, including cyclitols such as inositol), polyethylene glycols, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, , thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate), low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone, etc.), monosaccharides (e.g., xylose, mannose, fructose, glucose, etc.), disaccharides (e.g., lactose, maltose, sucrose, and trehalose, etc.), trisaccharides (e.g., raffinose, etc.), and polysaccharides (e.g., dextran, etc.). Stabilizers may be present in an amount ranging from 0.5 to 10% by weight per weight of the antibody or fusion protein.

[0145] Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the glycoprotein and to protect the glycoprotein from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pluronic® polyols. Non-ionic surfactants can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0146] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0147] 6.4.1. Pharmaceutical Compositions for Delivery The antibodies or fusion proteins of the disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or via a viral vector encoding the antibody or fusion protein under the control of a suitable promoter.

[0148] 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, for example, rAd-p53, a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53 (Gendicine®, also known as Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297), for use in the treatment of cancer; Ad5 d11520 (also called H101 or ONYX-015, see, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670), an adenovirus lacking the E1B gene to inactivate host p53; AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF (Cerullo et al., 2010, Cancer Res. 70:4297; rAd-HSVtk, a replication-deficient adenovirus carrying the HSV thymidine kinase gene, for use in, for example, the treatment of cancer (Cerepro®, developed by Ark Therapeutics, see, for example, U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak™; International PCT Application No. WO 2005 / 049094), a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter, for example, the treatment of cancer (TNFerade™, GenVec, Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7, for example, Ad-IFNβ (BG 00001 and H5.110CMVhIFN-β, Biogen), an adenovirus serotype 5 vector with deleted E1 and E3 genes that expresses the human interferon beta gene under the direction of the cytomegalovirus (CMV) immediate early promoter for the treatment of cancer, but are not limited to these.

[0149] The nucleic acid molecule (e.g., mRNA) or virus may be formulated as the only pharmacoactive ingredient in the pharmaceutical composition, or may be combined with other active agents for the particular disease to be treated. Optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents may be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Exemplary other agents and excipients that may be included in the compositions include, for example, water soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid, and the like.

[0150] 7. Specific embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the disclosure(s). The disclosure is exemplified by numbered groups A, B, C, and D described below. Unless otherwise specified, any feature of a numbered embodiment of a particular group applies mutatis mutandis to the numbered embodiments of other groups.

[0151] Group A: Numbered Embodiments 1. A recombinant polypeptide comprising a chimeric constant domain, comprising, from N-terminus to C-terminus: (a) a chimeric immunoglobulin hinge, (i) the human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) at positions 216 to 225 (EU numbering); (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) at positions 226 to 229 (EU numbering); (iii) a chimeric immunoglobulin hinge comprising the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) at positions 230 to 238 (EU numbering); (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 239 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (c) a recombinant polypeptide comprising a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from positions 341 to 447 (EU numbering) of human IgG1, and optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitutions are 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.

[0152] 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 any one of the amino acid sequences 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.

[0153] 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 any one of the amino acid sequences 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.

[0154] 4. A recombinant polypeptide according to 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 any one of the amino acid sequences 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.

[0155] 5. A recombinant polypeptide according to 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 any one of the amino acid sequences 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.

[0156] 6. A recombinant polypeptide according to 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 any one of the amino acid sequences 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.

[0157] 7. A recombinant polypeptide according to 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.

[0158] 8. A recombinant polypeptide according to any one of embodiments 1 to 7, wherein optionally one or more CH2 domain amino acid substitutions reduce binding to Fc receptors and / or reduce effector function of the chimeric constant domain.

[0159] 9. A recombinant polypeptide according to any one of embodiments 1 to 8, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position G237 (EU numbering).

[0160] 10. A recombinant polypeptide according to any one of embodiments 1 to 9, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position D265 (EU numbering).

[0161] 11. The recombinant polypeptide of embodiment 10, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution D265A (EU numbering). 12. A recombinant polypeptide according to any one of embodiments 1 to 11, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position N297 (EU numbering).

[0162] 13. The recombinant polypeptide of embodiment 12, wherein the optional one or more CH2 domain amino acid substitutions comprises the amino acid substitution N297A (EU numbering). 14. The recombinant polypeptide of embodiment 12, wherein the optional one or more CH2 domain amino acid substitutions comprises the amino acid substitution N297D (EU numbering).

[0163] 15. A recombinant polypeptide according to any one of embodiments 1 to 14, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position P329 (EU numbering).

[0164] 16. The recombinant polypeptide of embodiment 15, wherein the optional one or more CH2 domain amino acid substitutions comprises the amino acid substitution P329A (EU numbering). 17. The recombinant polypeptide of embodiment 15, wherein the optional one or more CH2 domain amino acid substitutions comprises the amino acid substitution P329G (EU numbering).

[0165] 18. A recombinant polypeptide according to any one of embodiments 1 to 17, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position A330 (EU numbering).

[0166] 19. A recombinant polypeptide according to any one of embodiments 1 to 18, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position P331 (EU numbering).

[0167] 20. The recombinant polypeptide of embodiment 19, wherein the optional one or more CH2 domain amino acid substitutions comprises the amino acid substitution P331S (EU numbering). 21. A recombinant polypeptide according to 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).

[0168] 22. The recombinant polypeptide of embodiment 21, wherein the optional one or more CH2 domain amino acid substitutions include or consist of D265A and N297A (EU numbering).

[0169] 23. A recombinant polypeptide according to any one of embodiments 1 to 22, wherein the optional one or more CH3 domain amino acid substitutions provide for heterodimeric association of the chimeric constant domain with another constant domain.

[0170] 24. A recombinant polypeptide according to 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).

[0171] 25. The recombinant polypeptide of embodiment 24, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitutions S354C or E357C (EU numbering). 26. A recombinant polypeptide according to any one of embodiments 1 to 25, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position T366 (EU numbering).

[0172] 27. The recombinant polypeptide of embodiment 26, wherein the optional one or more CH3 domain amino acid substitutions comprises the amino acid substitution T366W (EU numbering). 28. A recombinant polypeptide according to 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).

[0173] 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).

[0174] 30. A recombinant polypeptide according to any one of embodiments 1 to 23, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position Y349 (EU numbering).

[0175] 31. The recombinant polypeptide of embodiment 30, wherein the optional one or more CH3 domain amino acid substitutions comprises the amino acid substitution Y349C (EU numbering). 32. A recombinant polypeptide according to any one of embodiments 1 to 23, 30 or 31, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position Y407 (EU numbering).

[0176] 33. The recombinant polypeptide of embodiment 32, wherein the optional one or more CH3 domain amino acid substitutions comprises the amino acid substitution Y407V (EU numbering). 34. A recombinant polypeptide according to 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).

[0177] 35. The recombinant polypeptide of embodiment 34, wherein the optional one or more CH3 domain amino acid substitutions comprises the amino acid substitution T366S (EU numbering). 36. A recombinant polypeptide according to any one of embodiments 32 to 35, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position L368 (EU numbering).

[0178] 37. The recombinant polypeptide of embodiment 36, wherein the optional one or more CH3 domain amino acid substitutions include the amino acid substitution L368A (EU numbering). 38. A recombinant polypeptide according to 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).

[0179] 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).

[0180] 40. A recombinant polypeptide according to any one of embodiments 1 to 39, wherein optionally one or more CH3 domain amino acid substitutions provide for selective purification of the recombinant polypeptide. 41. The recombinant polypeptide of embodiment 40, wherein the optional one or more CH3 domain amino acid substitutions include an amino acid substitution at position H435 (EU numbering).

[0181] 42. The recombinant polypeptide of embodiment 41, wherein the optional one or more CH3 domain amino acid substitutions include the amino acid substitution H435R (EU numbering). 43. The recombinant polypeptide of embodiment 40, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y436 (EU numbering).

[0182] 44. The recombinant polypeptide of embodiment 41, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y436F (EU numbering). 45. A recombinant polypeptide according to any one of embodiments 1 to 44, wherein the recombinant polypeptide is an antigen-binding protein.

[0183] 46. ​​A recombinant polypeptide according to any one of embodiments 1 to 45, further comprising an IgG1 CH1 domain or a fragment or variant thereof. 47. The recombinant polypeptide according to embodiment 46, wherein the fragment of IgG1 CH1 comprises or consists of DKKV (SEQ ID NO: 31).

[0184] 48. The recombinant polypeptide of embodiment 46, wherein the fragment of IgG1 CH1 comprises or consists of DKRV (SEQ ID NO: 32). 49. A recombinant polypeptide according to any one of embodiments 1 to 48, comprising at least one target binding domain.

[0185] 50. A recombinant polypeptide according to any one of embodiments 1 to 49, comprising at least two target binding domains. 51. A recombinant polypeptide according to any one of embodiments 1 to 50, comprising at least three target binding domains.

[0186] 52. A recombinant polypeptide according to any one of embodiments 49 to 51, wherein at least one target binding domain comprises a Fab. 53. A recombinant polypeptide according to any one of embodiments 49 to 52, wherein at least one target binding domain comprises an scFv.

[0187] 54. A recombinant polypeptide according to any one of embodiments 49 to 53, comprising a Fab as the first target binding domain and an scFv as the target binding domain. 55. A recombinant polypeptide according to any one of embodiments 49 to 54, comprising at least one target binding domain at the N-terminus of the chimeric constant domain.

[0188] 56. A recombinant polypeptide according to any one of embodiments 49 to 55, comprising at least one target binding domain at the C-terminus of the chimeric constant domain. 57. A recombinant polypeptide according to 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.

[0189] 58. A recombinant polypeptide according to any one of embodiments 49 to 57, comprising, from N-terminus to C-terminus, an scFv, a Fab, and a chimeric constant domain. 59. A recombinant polypeptide according to any one of embodiments 1 to 58, wherein the recombinant polypeptide is an antibody (optionally a multispecific antibody) or forms part of an antibody (optionally a multispecific antibody).

[0190] 60. A recombinant polypeptide according to any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a monovalent antibody. 61. A recombinant polypeptide according to any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a bivalent antibody.

[0191] 62. A recombinant polypeptide according to embodiment 61, wherein the recombinant polypeptide is or forms part of a bispecific antibody. 63. A recombinant polypeptide according to any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a trivalent antibody.

[0192] 64. A recombinant polypeptide according to embodiment 63, wherein the recombinant polypeptide is or forms part of a bispecific antibody. 65. The recombinant polypeptide according to embodiment 63, wherein the recombinant polypeptide is or forms part of a trispecific antibody.

[0193] 66. A recombinant polypeptide according to any one of embodiments 1 to 59, wherein the recombinant polypeptide is or forms part of a tetravalent antibody. 67. The recombinant polypeptide according to embodiment 66, wherein the recombinant polypeptide is or forms part of a bispecific antibody.

[0194] 68. The recombinant polypeptide according to embodiment 66, wherein the recombinant polypeptide is or forms part of a trispecific antibody. 69. A recombinant polypeptide according to any one of embodiments 1 to 49, wherein the recombinant polypeptide is or forms part of a fusion protein.

[0195] 70. A recombinant polypeptide according to any one of embodiments 59 to 69, wherein the antibody or fusion protein exhibits less than 20% cytotoxic activity at an antibody or fusion protein concentration of at least 10 nM.

[0196] 71. A recombinant polypeptide according to any one of embodiments 59 to 69, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 10% at an antibody or fusion protein concentration of at least 10 nM.

[0197] 72. A recombinant polypeptide according to any one of embodiments 59 to 71, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 5% at an antibody or fusion protein concentration of at least 10 nM.

[0198] 73. A recombinant polypeptide according to any one of embodiments 59 to 72, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 4% at an antibody or fusion protein concentration of at least 10 nM.

[0199] 74. A recombinant polypeptide according to any one of embodiments 59 to 73, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 3% at an antibody or fusion protein concentration of at least 10 nM.

[0200] 75. A recombinant polypeptide according to any one of embodiments 59 to 74, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 2% at an antibody or fusion protein concentration of at least 10 nM.

[0201] 76. A recombinant polypeptide according to 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.

[0202] 77. A recombinant polypeptide according to any one of embodiments 59-69, wherein the antibody or fusion protein exhibits undetectable cell lysis at an antibody or fusion protein concentration of at least 10 nM.

[0203] 78. A recombinant polypeptide according to any one of embodiments 59 to 77, wherein the antibody or fusion protein shows increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0204] 79. The recombinant polypeptide according to embodiment 78, wherein the expression system is a Chinese Hamster Ovary (CHO) stable expression system. 80. A recombinant polypeptide according to embodiment 78 or embodiment 79, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 5%.

[0205] 81. A recombinant polypeptide according to any one of embodiments 78 to 80, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 10%.

[0206] 82. A recombinant polypeptide according to any one of embodiments 78 to 81, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 15%.

[0207] 83. A recombinant polypeptide according to any one of embodiments 78 to 82, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 20%.

[0208] 84. A recombinant polypeptide according to any one of embodiments 78 to 83, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 25%.

[0209] 85. A recombinant polypeptide according to any one of embodiments 78 to 84, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 30%.

[0210] 86. A recombinant polypeptide according to any one of embodiments 78 to 85, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 40%.

[0211] 87. A recombinant polypeptide according to any one of embodiments 78 to 86, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 50%.

[0212] 88. A recombinant polypeptide according to any one of embodiments 78 to 87, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 60%.

[0213] 89. A recombinant polypeptide according to any one of embodiments 78 to 88, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 70%.

[0214] 90. A recombinant polypeptide according to any one of embodiments 78 to 89, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 80%.

[0215] 91. A recombinant polypeptide according to any one of embodiments 78 to 90, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 90%.

[0216] 92. A recombinant polypeptide according to any one of embodiments 78 to 91, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 100%.

[0217] 93. The recombinant polypeptide of any one of embodiments 1-92, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 5-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0218] 94. The recombinant polypeptide of any one of embodiments 1 to 93, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 10-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0219] 95. The recombinant polypeptide of any one of embodiments 1-94, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 50-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0220] 96. The recombinant polypeptide of any one of embodiments 1 to 95, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 100-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0221] 97. The recombinant polypeptide of any one of embodiments 1-96, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 500-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0222] 98. The recombinant polypeptide of any one of embodiments 1 to 97, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 1,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0223] 99. The recombinant polypeptide of any one of embodiments 1-98, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 5,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0224] 100. The recombinant polypeptide of any one of embodiments 1-99, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 10,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0225] 101. A recombinant polypeptide according to any one of embodiments 93 to 100, wherein the FcR is FcRγ1. 102. A recombinant polypeptide according to any one of embodiments 93 to 100, wherein the FcR is FcRγ2A.

[0226] 103. A recombinant polypeptide according to any one of embodiments 93 to 100, wherein the FcR is FcRγ2B. 104. A recombinant polypeptide according to any one of embodiments 93 to 100, wherein the FcR is FcRγ3A.

[0227] 105. A recombinant polypeptide according to any one of embodiments 93 to 100, wherein the FcR is FcRγ3B. 106. A composition comprising a recombinant polypeptide according to any one of embodiments 1 to 105.

[0228] 107. The composition according to embodiment 106, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharma- ceutically acceptable carriers. 108. A nucleic acid or a plurality of nucleic acids encoding a recombinant polypeptide according to any one of embodiments 1 to 105.

[0229] 109. A host cell engineered to express a recombinant polypeptide according to any one of embodiments 1 to 105 or a nucleic acid(s) according to embodiment 108. 110. A recombinant polypeptide comprising a heavy chain constant (CH) region comprising or consisting of an amino acid sequence 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, and optionally comprising one or more CH2 domain amino acid substitutions, optionally wherein the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which, if they do not occur simultaneously.

[0230] 111. The recombinant polypeptide of embodiment 110, wherein the CH region comprises or consists of an amino acid sequence that is at least 96% identical to any one of the amino acid sequences 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.

[0231] 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 any one of the amino acid sequences 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.

[0232] 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 any one of the amino acid sequences 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.

[0233] 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 any one of the amino acid sequences set forth in 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.

[0234] 115. A recombinant polypeptide according to any one of embodiments 110 to 114, wherein the CH region comprises or consists of any one of the amino acid sequences 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.

[0235] 116. A recombinant polypeptide according to any one of embodiments 110 to 115, wherein optionally one or more CH2 domain amino acid substitutions reduce binding of the chimeric constant domain to an Fc receptor and / or reduce effector function.

[0236] 117. A recombinant polypeptide according to any one of embodiments 110 to 116, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position G237 (EU numbering).

[0237] 118. A recombinant polypeptide according to any one of embodiments 110 to 117, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position D265 (EU numbering).

[0238] 119. The recombinant polypeptide of embodiment 118, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution D265A (EU numbering). 120. A recombinant polypeptide according to any one of embodiments 110 to 119, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position N297 (EU numbering).

[0239] 121. The recombinant polypeptide of embodiment 120, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution N297A (EU numbering). 122. The recombinant polypeptide of embodiment 120, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution N297D (EU numbering).

[0240] 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).

[0241] 124. The recombinant polypeptide of embodiment 123, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution P329A (EU numbering). 125. The recombinant polypeptide of embodiment 123, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution P329G (EU numbering).

[0242] 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).

[0243] 127. A recombinant polypeptide according to any one of embodiments 110 to 126, wherein the optional one or more CH2 domain amino acid substitutions comprises an amino acid substitution at position P331 (EU numbering).

[0244] 128. The recombinant polypeptide of embodiment 127, wherein the optional one or more CH2 domain amino acid substitutions include the amino acid substitution P331S (EU numbering). 129. A recombinant polypeptide according to 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).

[0245] 130. The recombinant polypeptide of embodiment 129, wherein the optional one or more CH2 domain amino acid substitutions comprise or consist of D265A and N297A (EU numbering).

[0246] 131. A recombinant polypeptide according to any one of embodiments 110 to 130, wherein optionally one or more CH3 domain amino acid substitutions provide for heterodimeric association of the chimeric constant domain with another constant domain.

[0247] 132. A recombinant polypeptide according to 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).

[0248] 133. The recombinant polypeptide of embodiment 132, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitutions S354C or E357C (EU numbering).

[0249] 134. A recombinant polypeptide according to any one of embodiments 110 to 133, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position T366 (EU numbering).

[0250] 135. The recombinant polypeptide of embodiment 134, wherein the optional one or more CH3 domain amino acid substitutions comprises the amino acid substitution T366W (EU numbering). 136. A recombinant polypeptide according to 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).

[0251] 137. The recombinant polypeptide of embodiment 136, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of S354C and T366W (EU numbering).

[0252] 138. A recombinant polypeptide according to any one of embodiments 110 to 137, wherein the optional one or more CH3 domain amino acid substitutions comprises an amino acid substitution at position Y349 (EU numbering).

[0253] 139. The recombinant polypeptide of embodiment 138, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y349C (EU numbering). 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 comprises an amino acid substitution at position Y407 (EU numbering).

[0254] 141. The recombinant polypeptide of embodiment 140, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y407V (EU numbering). 142. The 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).

[0255] 143. The recombinant polypeptide of embodiment 142, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution T366S (EU numbering). 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).

[0256] 145. The recombinant polypeptide of embodiment 144, wherein the optional one or more CH3 domain amino acid substitutions include the amino acid substitution L368A (EU numbering). 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).

[0257] 147. The recombinant polypeptide of embodiment 146, wherein the optional one or more CH3 domain amino acid substitutions comprise or consist of Y349C, T366S, L368A, and Y407V (EU numbering).

[0258] 148. A recombinant polypeptide according to any one of embodiments 110 to 147, wherein optionally one or more CH3 domain amino acid substitutions provide for selective purification of the recombinant polypeptide.

[0259] 149. The recombinant polypeptide of embodiment 148, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position H435 (EU numbering). 150. The recombinant polypeptide of embodiment 149, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution H435R (EU numbering).

[0260] 151. The recombinant polypeptide of embodiment 148, wherein the optional one or more CH3 domain amino acid substitutions comprise an amino acid substitution at position Y436 (EU numbering). 152. The recombinant polypeptide of embodiment 151, wherein the optional one or more CH3 domain amino acid substitutions comprise the amino acid substitution Y436F (EU numbering).

[0261] 153. A recombinant polypeptide according to any one of embodiments 110 to 152, wherein the recombinant polypeptide is an antigen-binding protein. 154. A recombinant polypeptide according to any one of embodiments 110 to 153, further comprising an IgG1 CH1 domain or a fragment thereof.

[0262] 155. The recombinant polypeptide according to embodiment 154, wherein the fragment of IgG1 CH1 comprises or consists of DKKV (SEQ ID NO: 31). 156. The recombinant polypeptide according to embodiment 154, wherein the fragment of IgG1 CH1 comprises or consists of DKRV (SEQ ID NO: 32).

[0263] 157. A recombinant polypeptide according to any one of embodiments 110 to 156, comprising at least one target binding domain. 158. A recombinant polypeptide according to any one of embodiments 110 to 157, comprising at least two target binding domains.

[0264] 159. A recombinant polypeptide according to any one of embodiments 110 to 158, comprising at least three target binding domains. 160. A recombinant polypeptide according to any one of embodiments 157 to 159, wherein at least one target binding domain comprises a Fab.

[0265] 161. A recombinant polypeptide according to any one of embodiments 157 to 160, wherein at least one target binding domain comprises an scFv. 162. A recombinant polypeptide according to any one of embodiments 157 to 161, comprising a Fab as the first target binding domain and an scFv as the second target binding domain.

[0266] 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. 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.

[0267] 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.

[0268] 166. A recombinant polypeptide according to any one of embodiments 157 to 165, comprising, from N-terminus to C-terminus, an scFv, a Fab, and a chimeric constant domain. 167. A recombinant polypeptide according to any one of embodiments 110 to 166, wherein the recombinant polypeptide is an antibody (optionally a multispecific antibody) or forms part of an antibody (optionally a multispecific antibody).

[0269] 168. A recombinant polypeptide according to any one of embodiments 110 to 167, wherein the recombinant polypeptide is a monovalent antibody or forms part of a monovalent antibody. 169. A recombinant polypeptide according to any one of embodiments 110 to 167, wherein the recombinant polypeptide is or forms part of a bivalent antibody.

[0270] 170. The recombinant polypeptide according to embodiment 169, wherein the recombinant polypeptide is or forms part of a bispecific antibody. 171. A recombinant polypeptide according to any one of embodiments 110 to 167, wherein the recombinant polypeptide is or forms part of a trivalent antibody.

[0271] 172. The recombinant polypeptide according to embodiment 171, wherein the recombinant polypeptide is or forms part of a bispecific antibody. 173. The recombinant polypeptide according to embodiment 171, wherein the recombinant polypeptide is or forms part of a trispecific antibody.

[0272] 174. A recombinant polypeptide according to any one of embodiments 110 to 167, wherein the recombinant polypeptide is or forms part of a tetravalent antibody. 175. The recombinant polypeptide according to embodiment 174, wherein the recombinant polypeptide is or forms part of a bispecific antibody.

[0273] 176. The recombinant polypeptide according to embodiment 174, wherein the recombinant polypeptide is or forms part of a trispecific antibody. 177. A recombinant polypeptide according to any one of embodiments 110 to 157, wherein the recombinant polypeptide is a fusion protein or forms part of a fusion protein.

[0274] 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% at an antibody or fusion protein concentration of at least 10 nM.

[0275] 179. A recombinant polypeptide according to any one of embodiments 167 to 178, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 10% at an antibody or fusion protein concentration of at least 10 nM.

[0276] 180. A recombinant polypeptide according to any one of embodiments 167 to 179, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 5% at an antibody or fusion protein concentration of at least 10 nM.

[0277] 181. A recombinant polypeptide according to any one of embodiments 167 to 180, wherein the antibody or fusion protein exhibits a cytotoxic activity of less than 4% at an antibody or fusion protein concentration of at least 10 nM.

[0278] 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% at an antibody or fusion protein concentration of at least 10 nM.

[0279] 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% at an antibody or fusion protein concentration of at least 10 nM.

[0280] 184. A recombinant polypeptide according to 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.

[0281] 185. A recombinant polypeptide according to 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.

[0282] 186. A recombinant polypeptide according to any one of embodiments 167 to 185, wherein the antibody or fusion protein shows increased expression in an expression system compared to an antibody comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0283] 187. The recombinant polypeptide according to embodiment 186, wherein the expression system is a Chinese Hamster Ovary (CHO) stable expression system. 188. The recombinant polypeptide according to embodiment 186 or embodiment 187, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 5%.

[0284] 189. A recombinant polypeptide according to any one of embodiments 186 to 188, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 10%.

[0285] 190. A recombinant polypeptide according to any one of embodiments 186 to 189, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 15%.

[0286] 191. A recombinant polypeptide according to any one of embodiments 186 to 190, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 20%.

[0287] 192. A recombinant polypeptide according to any one of embodiments 186 to 191, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 25%.

[0288] 193. A recombinant polypeptide according to any one of embodiments 186 to 192, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 30%.

[0289] 194. A recombinant polypeptide according to any one of embodiments 186 to 193, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 40%.

[0290] 195. A recombinant polypeptide according to any one of embodiments 186 to 194, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 50%.

[0291] 196. A recombinant polypeptide according to any one of embodiments 186 to 195, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 60%.

[0292] 197. A recombinant polypeptide according to any one of embodiments 186 to 196, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 70%.

[0293] 198. A recombinant polypeptide according to any one of embodiments 186 to 197, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 80%.

[0294] 199. A recombinant polypeptide according to any one of embodiments 186 to 198, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 90%.

[0295] 200. A recombinant polypeptide according to any one of embodiments 186 to 199, wherein the increase compared to an antibody or fusion protein comprising a wild-type IgG1 constant domain is at least 100%.

[0296] 201. The recombinant polypeptide of any one of embodiments 110 to 200, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 5-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0297] 202. The recombinant polypeptide of any one of embodiments 110 to 201, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 10-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0298] 203. The recombinant polypeptide of any one of embodiments 110 to 202, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 50-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0299] 204. The recombinant polypeptide of any one of embodiments 110 to 203, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 100-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0300] 205. The recombinant polypeptide of any one of embodiments 110 to 204, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 500-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0301] 206. The recombinant polypeptide of any one of embodiments 110 to 205, wherein the recombinant polypeptide exhibits binding to a human Fc receptor (FcR) that is at least 1,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0302] 207. The recombinant polypeptide of any one of embodiments 110 to 206, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 5,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0303] 208. The recombinant polypeptide according to any one of embodiments 110 to 207, wherein the recombinant polypeptide exhibits binding to human Fc receptor (FcR) that is at least 10,000-fold reduced compared to a polypeptide comprising a wild-type IgG1 constant domain comprising an IgG1 hinge, an IgG1 CH2, and an IgG1 CH3.

[0304] 209. A recombinant polypeptide according to any one of embodiments 201 to 208, wherein the FcR is FcRγ1. 210. A recombinant polypeptide according to any one of embodiments 201 to 208, wherein the FcR is FcRγ2A.

[0305] 211. The recombinant polypeptide according to any one of embodiments 201-208, wherein the FcR is FcRγ2B. 212. The recombinant polypeptide according to any one of embodiments 201-208, wherein the FcR is FcRγ3A.

[0306] 213. The recombinant polypeptide according to any one of embodiments 201-208, wherein the FcR is FcRγ3B. 214. A composition comprising a recombinant polypeptide according to any one of embodiments 110 to 213.

[0307] 215. The composition according to embodiment 214, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharma- ceutically acceptable carriers. 216. A nucleic acid or a plurality of nucleic acids encoding a recombinant polypeptide according to any one of embodiments 110 to 215.

[0308] 217. A host cell engineered to express a recombinant polypeptide according to any one of embodiments 110 to 213 or a nucleic acid(s) according to embodiment 216. 218. A method for producing a recombinant polypeptide comprising a chimeric constant domain, comprising culturing a host cell according to claim 109 or claim 217 under conditions in which the recombinant polypeptide is expressed.

[0309] 219. The method of claim 218, further comprising recovering and optionally purifying the recombinant polypeptide. Group B: Numbered Embodiments 1. A method for increasing the production of a fusion protein that contains, from the N-terminus to the C-terminus, (a) a chimeric immunoglobulin hinge, (i) the human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) at positions 216 to 225 (EU numbering); (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) at positions 226 to 229 (EU numbering); (iii) a chimeric immunoglobulin hinge comprising the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) at positions 230 to 238 (EU numbering); (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 239 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from positions 341 to 447 (EU numbering) of human IgG1, and optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitutions are 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.

[0310] 2. The method of embodiment 1, wherein the method increases expression relative to expression of a variant fusion protein comprising a control constant domain. 3. The method of embodiment 2, wherein the control constant domain is a constant domain of SEQ ID NO: 1 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0311] 4. The method of embodiment 2, wherein the control constant domain is a constant domain of SEQ ID NO: 9 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0312] 5. The method of embodiment 2, wherein the control constant domain is a constant domain of SEQ ID NO: 10 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0313] 6. The method of any one of embodiments 2-5, wherein the increase in expression compared to the variant fusion protein is at least 10%. 7. The method of any one of embodiments 2-5, wherein the increase in expression compared to the variant fusion protein is at least 15%.

[0314] 8. The method of any one of embodiments 2 to 5, wherein the increase in expression compared to the variant fusion protein is at least 20%. 9. The method of any one of embodiments 2 to 5, wherein the increase in expression compared to the variant fusion protein is at least 30%.

[0315] 10. The method of any one of embodiments 2 to 5, wherein the increase in expression compared to the variant fusion protein is at least 40%. 11. The method of any one of embodiments 2 to 5, wherein the increase in expression compared to the variant fusion protein is at least 50%.

[0316] 12. A method for increasing the activity of a fusion protein comprising, from the N-terminus to the C-terminus, (a) a chimeric immunoglobulin hinge, (i) the human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) at positions 216 to 225 (EU numbering); (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) at positions 226 to 229 (EU numbering); (iii) a chimeric immunoglobulin hinge comprising the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) at positions 230 to 238 (EU numbering); (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 239 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from positions 341 to 447 (EU numbering) of human IgG1, and optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitutions are 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.

[0317] 13. The method of embodiment 6, wherein the method increases expression relative to expression of a variant fusion protein comprising a control constant domain. 14. The method of embodiment 13, wherein the control constant domain is a constant domain of SEQ ID NO: 1 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0318] 15. The method of embodiment 13, wherein the control constant domain is a constant domain of SEQ ID NO: 9 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0319] 16. The method of embodiment 13, wherein the control constant domain is a constant domain of SEQ ID NO: 10 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0320] 17. The method of any one of embodiments 13 to 16, wherein the increase in activity compared to the variant fusion protein is at least 10%. 18. The method of any one of embodiments 13 to 16, wherein the increase in activity compared to the variant fusion protein is at least 15%.

[0321] 19. The method of any one of embodiments 13 to 16, wherein the increase in activity compared to the variant fusion protein is at least 20%. 20. The method of any one of embodiments 13 to 16, wherein the increase in activity compared to the variant fusion protein is at least 30%.

[0322] 21. The method of any one of embodiments 13 to 16, wherein the increase in activity compared to the variant fusion protein is at least 40%. 22. The method of any one of embodiments 13-16, wherein the increase in activity compared to the variant fusion protein is at least 50%.

[0323] 23. A method for increasing the expression and activity of a fusion protein comprising, from the N-terminus to the C-terminus, (a) a chimeric immunoglobulin hinge, (i) the human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) at positions 216 to 225 (EU numbering); (ii) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) at positions 226 to 229 (EU numbering); (iii) a chimeric immunoglobulin hinge comprising the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) at positions 230 to 238 (EU numbering); (b) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 239 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (c) a CH3 domain comprising a human IgG1 CH3 domain amino acid sequence from positions 341 to 447 (EU numbering) of human IgG1, and optionally comprising one or more CH3 domain amino acid substitutions, optionally wherein the substitutions are 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.

[0324] 24. The method of embodiment 23, wherein the method increases expression and activity compared to expression of a variant fusion protein comprising a control constant domain. 25. The method of embodiment 24, wherein the control constant domain is a constant domain of SEQ ID NO: 1 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0325] 26. The method of embodiment 24, wherein the control constant domain is a constant domain of SEQ ID NO: 9 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0326] 27. The method of embodiment 24, wherein the control constant domain is a constant domain of SEQ ID NO: 10 with or without mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations.

[0327] 28. (a) at least a 10% increase in expression compared to the variant fusion protein; (b) The method of any one of embodiments 24 to 27, wherein the increase in activity compared to the variant fusion protein is at least 10%.

[0328] 29. The method of embodiment 28, wherein the increase in expression compared to the variant fusion polypeptide is at least 15%. 30. The method of embodiment 28, wherein the increase in expression compared to the variant fusion polypeptide is at least 20%.

[0329] 31. The method of embodiment 28, wherein the increase in expression compared to the variant fusion polypeptide is at least 30%. 32. The method of embodiment 28, wherein the increase in expression compared to the variant fusion polypeptide is at least 40%.

[0330] 33. The method of embodiment 28, wherein the increase in expression compared to the variant fusion polypeptide is at least 50%. 34. The method of any one of embodiments 28 to 33, wherein the increase in activity compared to the variant fusion polypeptide is at least 15%.

[0331] 35. The method of any one of embodiments 28 to 33, wherein the increase in activity compared to the variant fusion polypeptide is at least 20%. 36. The method of any one of embodiments 28 to 33, wherein the increase in activity compared to the variant fusion polypeptide is at least 30%.

[0332] 37. The method of any one of embodiments 28 to 33, wherein the increase in activity compared to the variant fusion polypeptide is at least 40%. 38. The method of any one of embodiments 28 to 33, wherein the increase in activity compared to the variant fusion polypeptide is at least 50%.

[0333] 39. The method of any one of embodiments 1 to 38, wherein optionally one or more CH2 domain amino acid substitutions reduce binding of the chimeric constant domain to an Fc receptor and / or reduce effector function.

[0334] 40. The method of any one of embodiments 1-39, wherein the optional one or more CH3 domain amino acid substitutions provide for heterodimeric association of the chimeric constant domain with another constant domain.

[0335] 41. The method of any one of embodiments 1 to 40, wherein optionally one or more CH3 domain amino acid substitutions provide for selective purification of the fusion protein. 42. The method of any one of embodiments 1 to 41, wherein the fusion protein is an antigen-binding protein.

[0336] 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 or consists of DKKV (SEQ ID NO: 31) or DKRV (SEQ ID NO: 32).

[0337] 44. The method of any one of embodiments 1 to 43, wherein the fusion protein comprises at least one target binding domain. 45. The method of any one of embodiments 1 to 44, wherein the fusion protein comprises at least two target binding domains.

[0338] 46. ​​The method of any one of embodiments 1 to 45, wherein the fusion protein comprises at least three target binding domains. 47. The method of any one of embodiments 1 to 46, wherein the fusion protein comprises at least one Fab.

[0339] 48. The method of any one of embodiments 1 to 47, wherein the fusion protein comprises at least one scFv. 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.

[0340] 50. The method of any one of embodiments 1 to 49, wherein the fusion protein comprises two Fabs and one scFv. 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.

[0341] 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. 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.

[0342] 54. The method of any one of embodiments 1 to 53, wherein the fusion protein comprises, from N-terminus to C-terminus, an scFv, a Fab, and a chimeric constant domain. 55. The method according to any one of the preceding embodiments, wherein the fusion protein is part of or forms part of an antibody (optionally a multispecific antibody).

[0343] 56. The method of 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, and optionally each chimeric constant domain is independently selected from a constant domain having an amino acid sequence of any one of SEQ ID NOs: 3, 15, 16, 17, 18, 19, 20, 21, 22.

[0344] 57. The method of embodiment 56, wherein the fusion protein is a homodimer. 58. The method of embodiment 56, wherein the fusion protein is a heterodimer. 59. The method of embodiment 58, wherein one of the chimeric constant domains comprises a knob substitution and the other chimeric constant domain comprises a hole substitution.

[0345] 60. The method of embodiment 58 or embodiment 59, wherein the chimeric constant domain comprises a star mutation. 61. The method of any one of embodiments 1 to 60, wherein the fusion protein comprises two polypeptide chains.

[0346] 62. The method of any one of embodiments 1 to 60, wherein the fusion protein comprises three polypeptide chains. 63. The method of any one of embodiments 1-60, wherein the fusion protein comprises four polypeptide chains.

[0347] 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 in which the fusion protein is expressed.

[0348] 65. The method of embodiment 64, wherein the host cell is a mammalian host cell. 66. The method according to embodiment 64 or embodiment 65, further comprising recovering and, optionally, purifying the expressed fusion protein.

[0349] 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.

[0350] 68. The method of any one of the preceding embodiments, further comprising introducing into the host cell a nucleic acid molecule or a plurality of nucleic acid molecules encoding the fusion protein prior to said expression step.

[0351] 69. The method of embodiment 64, wherein the nucleic acid molecule or molecules are contained in one or more host cell expression vectors. 70. A population of fusion proteins produced by the method of any one of embodiments 1 to 69.

[0352] 71. The population of embodiment 70, comprising at least 10,000 fusion proteins. 72. A population according to embodiment 70 or embodiment 71, characterized by a higher activity compared to a population of fusion proteins comprising a control constant domain comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:9, or SEQ ID NO:10 with or without a mutation enabling heterodimerization or purification, e.g., a knob-in-hole mutation and / or a star mutation.

[0353] Group C: Numbered Embodiments 1. A fusion protein comprising a first polypeptide chain associated with a second polypeptide chain, (a) the first polypeptide chain comprises a first chimeric constant domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1, wherein the chimeric constant domain comprises: (i) having a PVA-absent sequence at amino acids 233 to 236 as defined by EU numbering; (ii) has one or more amino acid substitutions that promote heterodimerization with a second polypeptide chain; (b) the second polypeptide chain comprises a second chimeric constant domain comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1, provided that the chimeric constant domain comprises: (i) having a PVA-absent sequence at amino acids 233 to 236 as defined by EU numbering; (ii) a fusion protein having one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain.

[0354] 2. A fusion protein comprising a first polypeptide chain associated with a second polypeptide chain, (a) the first polypeptide chain comprises a first chimeric constant domain, the first chimeric constant domain having, from N-terminus to C-terminus: (i) a chimeric immunoglobulin hinge, (1) the human IgG1 upper hinge amino acid sequence EPKSCDKTHT (SEQ ID NO: 24) at positions 216 to 225 (EU numbering); (2) the human IgG1 core hinge amino acid sequence CPPC (SEQ ID NO: 25) at positions 226 to 229 (EU numbering); (3) a chimeric immunoglobulin hinge comprising the human IgG2 lower hinge amino acid sequence PCPAPPVAGP (SEQ ID NO: 42) at positions 230 to 238 (EU numbering); (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 237 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (iii) a CH3 domain comprising the amino acid sequence of a human IgG1 CH3 domain at positions 341 to 447 (EU numbering) of human IgG1; (iv) one or more amino acid substitutions that promote heterodimerization with a second polypeptide chain; and (v) optionally, one or more amino acid substitutions to reduce effector function and / or facilitate purification; (b) the second polypeptide chain comprises a second chimeric constant domain, the second chimeric constant domain having, from the N-terminus to the C-terminus: (i) a chimeric immunoglobulin hinge comprising the human IgG1 upper hinge amino acid sequence EPKSCDKTHTCP (SEQ ID NO: 43) at positions 216 to 227 (EU numbering) of human IgG1 and the human IgG2 lower hinge amino acid sequence PCPAPPVA (SEQ ID NO: 44) at positions 228 to 236 (EU numbering) of human IgG2; (ii) a CH2 domain comprising a human IgG1 CH2 domain amino acid sequence from positions 237 to 340 (EU numbering) of human IgG1, and optionally comprising one or more CH2 domain amino acid substitutions, optionally where the CH2 has one or more of the amino acid substitutions H268Q, K274Q, Y296F, A327G, A330S, and P331S (EU numbering), which do not occur simultaneously; and (iii) a CH3 domain comprising the amino acid sequence of a human IgG1 CH3 domain at positions 341 to 447 (EU numbering) of human IgG1; (iv) one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain; and (c) optionally, one or more amino acid substitutions to reduce effector function and / or facilitate purification.

[0355] 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 to the amino acid sequence of SEQ ID NO:1.

[0356] 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 to the amino acid sequence of SEQ ID NO:1.

[0357] 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 to the amino acid sequence of SEQ ID NO:1.

[0358] 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 to the amino acid sequence of SEQ ID NO:1.

[0359] 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 to the amino acid sequence of SEQ ID NO:1.

[0360] 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 the second chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 16.

[0361] 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 the second chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 16.

[0362] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 18.

[0363] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 18.

[0364] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 20.

[0365] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 22.

[0366] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 20.

[0367] 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 the first chimeric constant domain comprises the amino acid sequence of SEQ ID NO: 22.

[0368] 16. The fusion protein according to any one of embodiments 1 to 15, wherein the fusion protein is an antigen-binding protein comprising at least one target-binding domain. 17. A fusion protein according to any one of the preceding embodiments, wherein the fusion protein is or forms part of an antibody (optionally a multispecific antibody).

[0369] 18. A fusion protein according to embodiment 16 or embodiment 17, comprising at least two target binding domains. 19. The fusion protein of embodiment 18, wherein the fusion protein is or forms part of a bivalent antibody.

[0370] 20. A fusion protein according to any one of embodiments 16 to 18, comprising at least three target binding domains. 21. The fusion protein of embodiment 20, wherein the fusion protein is or forms part of a trivalent antibody.

[0371] 22. A fusion protein according to any one of embodiments 16 to 21, wherein at least one target binding domain comprises a Fab. 23. A fusion protein according to any one of embodiments 16 to 21, wherein at least one target binding domain comprises an scFv.

[0372] 24. A fusion protein according to any one of embodiments 16 to 21, comprising a Fab as the first target binding domain and an scFv as the second target binding domain. 25. The fusion protein of embodiment 20 or embodiment 21, comprising a Fab as the first target binding domain, a scFv as the second target binding domain, and a Fab as the third target binding domain.

[0373] 26. A polypeptide comprising a third polypeptide chain and a fourth polypeptide chain, (a) a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH1), a CH1 domain, and a first constant domain; (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; (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; (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;

[0374] 27. The fusion protein of embodiment 26, wherein VH1 and VH2 are identical. 28. The fusion protein of embodiment 26, wherein VH1 and VH2 are different. 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.

[0375] 30. The fusion protein of embodiment 28, wherein the first Fab domain and the second Fab domain bind to different target molecules. 31. A polypeptide comprising a third polypeptide chain and a fourth polypeptide chain, (a) a first polypeptide chain comprising, 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; (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; (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; (d) the fusion protein of embodiment 20 or embodiment 21, wherein the fourth polypeptide chain comprises a first light chain comprising a second light chain variable region (VL2) associated with a VH2 to form a second Fab domain.

[0376] 32. A polypeptide comprising a third polypeptide chain and a fourth polypeptide chain, (a) a first polypeptide chain comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH1), a CH1 domain, and a first constant domain; (b) the second polypeptide chain comprises, from N-terminus to C-terminus, an 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; (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; (d) the fusion protein of embodiment 20 or embodiment 21, wherein the fourth polypeptide chain comprises a first light chain comprising a second light chain variable region (VL2) associated with a VH2 to form a second Fab domain.

[0377] 33. The fusion protein of 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 in the following order: VH3-optional linker-VL3.

[0378] 34. The fusion protein of 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 in the following order: VL3-optional linker-VH3.

[0379] 35. A fusion protein according to any one of embodiments 31 to 34, wherein the scFv binds to a different target molecule than the first Fab domain and / or the second Fab domain. 36. The following: (a) the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering, within the first and second chimeric constant domains; and / or (b) the fusion protein according to any one of embodiments 1 to 35, which exhibits at least 10% increased expression in an expression system compared to a variant protein comprising a first and a second chimeric constant domain having the amino acid sequence of SEQ ID NO: 10 with or without a mutation enabling heterodimerization or purification, e.g., a knob-in-hole mutation and / or a star mutation.

[0380] 37. The fusion protein according to embodiment 36, wherein the expression system is a Chinese Hamster Ovary (CHO) expression system, or a HEK293 or derivative (e.g., Expi293F) expression system.

[0381] 38. The fusion protein according to embodiment 36 or embodiment 37, wherein the increase in expression is at least 20% or at least 30%. 39. Below: (a) the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering, within the first and second chimeric constant domains; and / or (b) the fusion protein of any one of embodiments 1 to 38, exhibiting at least 5-fold reduced binding to a human Fc receptor (FcR) compared to a variant protein comprising a first and a second chimeric constant domain having the amino acid sequence of SEQ ID NO: 9 with or without a heterodimerization or purification enabling mutation, e.g., a knob-in-hole mutation and / or a star mutation.

[0382] 40. The fusion protein of embodiment 39, wherein the reduction in binding is at least 10-fold, or at least 20-fold. 41. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ1.

[0383] 42. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ2A. 43. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ2B.

[0384] 44. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ3A. 45. The fusion protein of embodiment 39 or embodiment 40, wherein the FcR is FcRγ3B.

[0385] 46. ​​A composition comprising a fusion protein according to any one of embodiments 1 to 45. 47. The composition according to embodiment 46, wherein the composition is a pharmaceutical composition comprising one or more excipients and / or pharma- ceutically acceptable carriers.

[0386] 48. A nucleic acid or a plurality of nucleic acids encoding a fusion protein according to any one of embodiments 1 to 45. 49. A host cell engineered to express a fusion protein according to any one of embodiments 1 to 45.

[0387] 50. A host cell comprising one or more expression vectors encoding a fusion protein according to any one of embodiments 1 to 45. 51. A host cell according to embodiment 49 or embodiment 50, which is a CHO cell, or a HEK293 cell or a derivative (eg Expi293F) cell.

[0388] 52. A method for producing a fusion protein according to any one of embodiments 1 to 45, comprising culturing a host cell according to any one of embodiments 49 to 51, and optionally recovering and / or purifying the expressed protein.

[0389] 53. A population of fusion proteins according to any one of embodiments 1 to 45. 54. A population of fusion proteins according to embodiment 53, produced by a method according to any one of embodiments 52.

[0390] 55. The population described in embodiment 53 or embodiment 54, comprising at least 10,000 fusion proteins. Group D: Numbered embodiments 1. A recombinant protein comprising a constant domain, the constant domain comprising: (a) the amino acid sequence of SEQ ID NO:3; (b) the amino acid sequence of SEQ ID NO: 15; (c) the amino acid sequence of SEQ ID NO: 16; (d) the amino acid sequence of SEQ ID NO: 17; (e) the amino acid sequence of SEQ ID NO: 18; (f) the amino acid sequence of SEQ ID NO: 19; (g) the amino acid sequence of SEQ ID NO: 20; (h) the amino acid sequence of SEQ ID NO: 21; (i) the amino acid sequence of SEQ ID NO: 22; (j) a recombinant protein having an amino acid sequence having at least 97%, 98%, 99%, or 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, except that the Fc domain (i) has a PVA-absent sequence at amino acids 233-236 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).

[0391] 2. The recombinant protein of embodiment 1, which is a multimer. 3. The recombinant protein of embodiment 2, comprising two polypeptide chains. 4. The recombinant protein of embodiment 2, comprising three polypeptide chains.

[0392] 5. The recombinant protein of embodiment 2, comprising four polypeptide chains. 6. A recombinant protein according to any one of embodiments 1 to 5, comprising an Fc homodimer.

[0393] 7. The recombinant protein of embodiment 6, comprising two constant domains, each having the amino acid sequence of SEQ ID NO:3. 8. The recombinant protein of embodiment 6, comprising two identical 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, with the proviso that the Fc domain has a PVA-absent sequence at amino acids 233-236.

[0394] 9. The recombinant protein of embodiment 6, comprising two identical 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, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, with the proviso that the Fc domain has a PVA-absent sequence at amino acids 233-236.

[0395] 10. The recombinant protein of embodiment 6, comprising two identical constant domains, each having an amino acid sequence 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, with the proviso that the Fc domain has a PVA-absent sequence at amino acids 233-236.

[0396] 11. The recombinant protein of embodiment 6, comprising two identical constant domains, each having an amino acid sequence having 100% sequence identity with SEQ ID NO:3. 12. A recombinant protein according to any one of embodiments 1 to 5, comprising an Fc heterodimer.

[0397] 13. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 15 and a second constant domain having an amino acid sequence of SEQ ID NO: 16.

[0398] 14. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 15 and a second constant domain having an amino acid sequence of SEQ ID NO: 18.

[0399] 15. The recombinant protein of 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.

[0400] 16. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 17 and a second constant domain having an amino acid sequence of SEQ ID NO: 18.

[0401] 17. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 19 and a second constant domain having an amino acid sequence of SEQ ID NO: 20.

[0402] 18. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 19 and a second constant domain having an amino acid sequence of SEQ ID NO: 22.

[0403] 19. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 21 and a second constant domain having an amino acid sequence of SEQ ID NO: 20.

[0404] 20. The recombinant protein of embodiment 12, comprising a first constant domain having an amino acid sequence of SEQ ID NO: 21 and a second constant domain having an amino acid sequence of SEQ ID NO: 22.

[0405] 21. The recombinant protein of 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, with the proviso that the Fc domain (i) has a PVA-absent sequence at amino acids 233-236 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).

[0406] 22. The recombinant protein of 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, with the proviso that the Fc domain (i) has a PVA-absent sequence at amino acids 233-236 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).

[0407] 23. The recombinant protein of 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, with the proviso that the Fc domain (i) has a PVA-absent sequence at amino acids 233-236 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).

[0408] 24. The recombinant protein of embodiment 12, comprising two different constant domains, each 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 PVA-absent sequence at amino acids 233-236 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).

[0409] 25. A nucleic acid or a plurality of nucleic acids encoding a protein according to any one of embodiments 1 to 24. 26. A host cell comprising: (a) engineered to express a recombinant protein according to any one of embodiments 1 to 24; and / or (b) A host cell comprising one or more expression vectors encoding a recombinant protein according to any one of embodiments 1 to 24.

[0410] 27. The host cell according to embodiment 26, which is a CHO cell, or a HEK293 cell or a derivative (e.g. Expi293F) cell. 28. A method for producing a recombinant protein according to any one of embodiments 1 to 24, comprising culturing a 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.

[0411] 29. A method for increasing production of a polypeptide, comprising expressing the polypeptide as a protein as defined in any one of embodiments 1 to 24. 30. The method according to embodiment 29, comprising culturing a host cell according to any one of embodiments 25 to 27 so that the protein is expressed, and optionally recovering and / or purifying the expressed protein.

[0412] 31. The method of embodiment 29 or embodiment 30, wherein the method increases expression compared to expression of a variant protein comprising a control constant domain or pair of constant domains. 32. The method of embodiment 31, wherein the control constant domain or pair of constant domains has the sequence ELLG (SEQ ID NO: 23) at amino acids 233 to 236 as defined by EU numbering.

[0413] 33. The method of embodiment 31, wherein the control constant domain, or pair of constant domains, has the amino acid sequence of SEQ ID NO: 1 with or without one or more mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations and / or mutations that introduce disulfide bridges (e.g., S354C or E357C).

[0414] 34. The method of embodiment 31, wherein the control constant domain, or pair of constant domains, has the amino acid sequence of SEQ ID NO: 9 with or without one or more mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations and / or mutations that introduce disulfide bridges (e.g., S354C or E357C).

[0415] 35. The method of embodiment 31, wherein the control constant domain, or pair of constant domains, has the amino acid sequence of SEQ ID NO: 10 with or without one or more mutations that allow heterodimerization or purification, such as knob-in-hole mutations and / or star mutations and / or mutations that introduce disulfide bridges (e.g., S354C or E357C).

[0416] 36. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 10%. 37. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 15%.

[0417] 38. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 20%. 39. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 30%.

[0418] 40. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 40%. 41. The method of any one of embodiments 31 to 35, wherein the increase in expression and / or activity compared to the variant protein is at least 50%.

[0419] 42. A population of proteins according to any one of embodiments 1 to 24. 43. A population of proteins according to embodiment 42, produced by a method according to any one of embodiments 28 to 41.

[0420] 44. The population of embodiment 42, comprising at least 10,000 proteins. 45. A population according to any one of embodiments 42 to 44, characterized by a higher activity compared to a population of proteins comprising a control constant domain or pair of constant domains as defined in any one of embodiments 32 to 35.

[0421] 46. ​​The population according to any one of embodiments 42 to 45, in the form of a pharmaceutical composition comprising the population of proteins and one or more excipients and / or pharma- ceutically acceptable carriers. EXAMPLES

[0422] 8. Working Example 8.1. Materials and Methods 8.1.1. Experimental constructs Antibody constructs were generated containing the IgG1 PVA domain and control constant domains shown below. The components of the test and control constructs are listed in Table 2 below.

[0423] [Table 2-1]

[0424] [Table 2-2]

[0425] [Table 2-3]

[0426] [Table 2-4]

[0427] The test and control constructs include various bispecific and trispecific binding molecules, as shown in Table 3 below, which provides a description of the various control and test constructs utilized throughout the studies described herein. Certain bispecific antibodies comprised three antigen-binding sites (of which the first site binds to FGFR1c, the second site binds to the GH1 domain of KLB, and the third site binds to the GH2 domain of KLB), an IgG Fc domain (e.g., IgG1 PVA, IgG1 N180G (N297G according to EU numbering), IgG4, or IgG4s), and linkers of different 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 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.

[0428] [Table 3-1]

[0429] [Table 3-2]

[0430] [Table 3-3]

[0431] [Table 3-4]

[0432] Vector Construction DNA fragments encoding the anti-KLB GH1 Fab, anti-KLB GH2 Fab, anti-KLB GH2 scFv, and anti-FGFR1c Fab domains, various amino acid linkers, and various IgG hinge and Fc domains were synthesized by Integrated DNA Technologies, Inc. (San Diego, California) or Geneart / Thermo Fisher Scientific (Regensburg, Germany).

[0433] Mammalian expression vectors for the individual polypeptide chains were generated by one of the following approaches: using the NEBuilder HiFi DNA Assembly Kit (New England BioLabs Inc.); by restriction digestion followed by ligation according to standard molecular cloning protocols provided by New England BioLabs Inc.; or by DNA synthesis and cloning in ready-to-use constructs into the pcDNA3.4Topo Expression System (Life Technologies). DNA was transfected as a single plasmid or as heavy and light chain pairs according to the manufacturer's protocol. 50 ml of cell culture supernatant was harvested and processed for purification via HiTrap™ Protein G HP, HiTrap Protein A FF, or MabSelect SuRe pcc columns (Cytiva).

[0434] Certain constructs were expressed in Expi293F™ cells by transient transfection (Thermo Fisher Scientific). Proteins in Expi293F supernatants were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with either HiTrap™ Protein G HP or MabSelect SuRe pcc columns (Cytiva). After a single step elution, antibodies were neutralized and 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 HiPrep26 / 60 Sephacryl S-200 column was used.

[0435] 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 above. 8.1.3. Kinetics of Fc Receptor Binding by Biacore Briefly, surface plasmon resonance (SPR) experiments were performed at 25°C on a Biacore T200 instrument using carboxymethyl dextran coated (CM-5) chips. Mouse monoclonal anti-pentahistidine antibodies (GE Healthcare) were immobilized onto 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 the anti-pentahistidine amine coupled CM-5 chip and antibody stock solutions were injected over the captured proteins at 50μl / min for 2 minutes and serially diluted (6uM-24.7nM). mAb binding responses were monitored and steady-state binding equilibria were calculated for low affinity receptors. Data were processed using Scrubber 2.0 curve fitting software to determine kinetic association (ka) and dissociation (kd) rate constants by fitting to a 1:1 binding model. The binding dissociation equilibrium constants (KD) and dissociation half-lives (t1 / 2) were calculated from the kinetic rate constants as follows: 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.

[0436] 8.1.4. Enzyme-Linked Immunosorbent Assay (ELISA) Wells of a microtiter plate were coated with 6x-His tag (SEQ ID NO: 47) monoclonal antibody (4E3D10H2 / E3) (Thermo scientific) at 4 μg / ml in 100 μl of PBS (18 h, 4° C.) 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. Meanwhile, antibodies were added at 6.0×10 -06The antibodies were diluted in a 1:5 ratio from the starting concentration of 100 μM. The diluted antibodies (100 ul) were then added to the wells and incubated for 1 hour at room temperature. Peroxidase-conjugated goat anti-human IgG, F(ab')2 detection antibody 100 ul / well (1:5000 in blocking buffer) was added for 1 hour at room temperature and the reaction was visualized by adding 100 μl of peroxidase substrate (KPL-TMB) for 30 minutes. The reaction was stopped with 100 μl of TMB stop buffer and absorbance was measured at 450 nm using an ELISA plate reader (Envision, PerkinElmer). The plate was washed three times with wash buffer (PBS, pH 7.4, containing 0.05% (v / v) Tween 20) after each step.

[0437] 8.1.5. Surrogate ADCC Assays 8.1.5.1.Target cells HEK293 / hFGFR1c / hKLB / hCD20: HEK293 cells in which endogenous FGFR1 had been ablated by CRISPR-Cas9 were engineered to constitutively express full-length human CD20 (hCD20, amino acids M1-P297 in accession number NP_690605.1), FGFR1c (hFGFR1c, amino acids M1-R731 in accession number NP_075594), and KLB (hKLB, amino acids M1-S1044 in accession number NP_783864.1). Cells were sorted for high expression of all receptors.

[0438] 8.1.5.2. Reporter cells Jurkat / NFAT-Luc / FcγR3a 176Val: Jurkat T cells were engineered to stably express a Nuclear Factor of Activated T cells (NFAT) luciferase reporter construct together with the high affinity human FcγR3a 176Val allotype receptor (amino acids M1–K254 of accession no. P08637 VAR_003960).

[0439] 8.1.5.3. Assay Setup Three days before the experiment, Jurkat reporter cells were plated at 1.25 × 10 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 and reporter cells were transferred into assay medium (RPMI + 10% FBS + P / S / G) and split at a 1:1 ratio (each cell type: 3 × 10 4 The cells were added at 1000μg / well (1:100 / well) to a 96-well white microtiter plate. Multispecific anti-FGFR1c / KLB antibodies and hIgG4 S108P isotype control antibodies were titrated in 7-point, 1:4 serial dilutions ranging from 73.2 pM to 300 nM final concentrations, with the last 8th point containing no antibody, in duplicate to the cells. The plates were incubated for 4.6 h at 37 °C / 5% CO2, after which an equal volume of ONE-Glo™ (Promega) reagent was added to lyse the cells and detect luciferase activity. Emitted light was captured in relative light units (RLU) on a multilabel plate reader Envision (PerkinElmer). Antibody EC50 values ​​were determined from a 4-parameter logistic equation (including background signal) on an 8-point dose-response curve using GraphPad Prism software. Maximum fold induction was calculated using the following equation: Fold induction = Maximum mean RLU / mean RLU within the tested dose range for each antibody (background signal = no antibody) 8.1.6. Stable Expression and Antibody Titers Recombinant proteins encoding different antibodies with various IgG subclasses were cloned into expression plasmids, transfected into CHO cells, and selected with 400 mg / L hygromycin for 12-14 days, after which stably transfected pools were isolated. Stable CHO cell pools grown in suspension in chemically defined protein-free medium were used to produce proteins for testing.

[0440] Protein was produced by inducing cell cultures with 0.5 mg / L doxycycline for 5 days and harvesting the conditioned medium. Protein titers were determined on an Octet instrument (ForteBio) using a protein A sensor against known standards of various concentrations.

[0441] 8.1.7. Luciferase Reporter Assay Antibodies containing different IgG hinge and Fc domains were tested for their agonist activity using HEK293.SREluc.hFGFR1c / hKLB cells that stably expressed human FGFR1c and KLB and a luciferase reporter gene under the control of a promoter containing serum response element (SRE). Recombinant human FGF21 with a 6xHis tag (SEQ ID NO: 47) was used as a positive control, and the maximum reporter activity obtained from FGF21 was defined as 100% activity. Cells were treated with 6xHis-FGF21 ("HHHHHH" disclosed as SEQ ID NO: 47) for 6 hours and then subjected to luciferase assay. The percentage activity induced by individual antibodies was normalized to the maximum activity by FGF21. A dose-response assay was performed to determine EC50. An anti-FelD1 isotype (hIgG4-S108P) control antibody was used as a negative control.

[0442] 8.1.8. Human Primary Adipocyte Signaling Assays 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. Cells were lysed using lysis buffer for the AlphaScreen™ SureFire™ ERK Assay Kit, which measures phospho-ERK in treated cell lysates (PerkinElmer, Shelton, CT). The SureFire™ ERK assay was performed according to the manufacturer's protocol. His-tagged human FGF21 and an isotype control human IgG4 antibody were tested as positive and negative controls, respectively. FGFR1c / KLB bispecific antibody was also included in the experiment.

[0443] 8.1.9. Flow Binding Assay BaF3 cells overexpressing target protein X were cultured at 1 × 10 6 Cells were resuspended in FACS wash buffer (PBS containing 1% FBS) at 1 × 10 cells / mL. Staining was performed at 1 × 10 cells / well. 5 The antibody was added at 1.3 × 10 -07 The antibodies were diluted in a 1:5 ratio from the starting concentration of 1000 mM. The diluted antibodies were then added to the wells containing the cells. The cells were stained for 30 min at 2-8 °C and washed twice with FACS wash buffer. AF647-conjugated goat anti-hFc Fab (Jackson Immuno Research, 109-607-008, 1:400) was added to the wells and the cells were incubated for 30 min at 2-8 °C. The cells were then washed to remove excess antibody and fixed in 2% paraformaldehyde for 30 min at 2-8 °C. After two washes, the stained cells were analyzed using a BD LSR Fortessa™ FACS instrument. The results were analyzed by FlowJo. Mononuclear cells were selected using an FSC / SSC gate.

[0444] 8.1.10. Negative staining electron microscopy (EM) Complexes of C-terminal myc-myc-6xHis tagged CD40 ectodomain (residues (res.) 20-193) with anti-CD40 antibodies containing different hinge sequences (IgG1, IgG1-PVA, and IgG2) were isolated by size-exclusion chromatography using a Superdex 200 increase 10 / 300 column. Purified samples at 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).

[0445] Negatively stained EM grids were inserted into a Glacios TEM (Thermo Fisher) and imaged with a CETA camera (Thermo Fisher). Automated data collection was performed at 73,000x nominal magnification using EPU. EM data were processed using RELION 4.0. Particles were first picked using a Laplacian of Gaussian algorithm to generate 2D templates, which were then used for template-based particle picking. Particle images were subjected to multiple rounds of 2D classification, and after each round particles belonging to class averages with distinct features of IgG were selected. The diffuse density of bound CD40 allowed the assignment of two Fab arms that otherwise lacked distinctive features from the Fc region. Class averages with distinguishable features of two Fabs, each bound to CD40, were selected for Fab-Fab angle determination. Fab-Fab angles were measured manually using the angle tool in ImageJ and are the angle between lines drawn through the long axes of the two CD40-bound Fab arms.

[0446] 8.2. Example 1: Design, cloning, and expression of IgG1 PVA Overview IgG1Fc and IgG4Fc have different Fcγ receptor binding capabilities and charge distributions, which provide options for optimal Fc functional engagement and different compatibility with antibody building blocks such as Fab, scFv, and alternative format antibody fusion proteins. The hinge regions of IgG1 and IgG4 also have different lengths and flexibility. IgG4 (S108P, or S228P, EU numbering) is utilized in multiple approved antibody products such as pembrolizumab, nivolumab, and ixekizumab, where reduced Fc effector function is required. Due to the preference of antibody building blocks (e.g., Fab, scFv) for certain immunoglobulin subclasses, alternative and natural sequence variants based on human IgG1Fc that demonstrate reduced Fc gamma receptor binding and reduced Fc receptor effector function, different from IgG4 (S108P), were sought.

[0447] Figure 4 shows an alignment of various IgG hinge / Fc variants with sequences between the various wild type and modified human IgG1 and IgG4 hinge regions, as well as a description of the CH2 and CH3 Fc regions used, from amino acids 226 to 447 (EU numbering). The hIgG1 PVA was designed to contain PVA mutations in the lower hinge region in an otherwise entirely IgG1 background (e.g., IgG1 upper hinge, CH2, and CH3 regions).

[0448] To test the properties of hIgG1 PVA, it was incorporated into alternative format antibodies having either a 2+1 N-scFv or a 2+1 N-Fab format (see, e.g., FIG. 5 for an illustration of the 2+1 N-scFv format; in the 2+1 N-Fab format, the N-terminal scFv domain is replaced with a Fab domain).

[0449] 8.2.2.Results Control and bispecific antibodies incorporating various IgG hinge and Fc domains were successfully expressed and purified.

[0450] When expressed in CHO cells, AF1 constructs in an IgG1 PVA backbone with various linker lengths between the scFv and Fab had higher antibody titers (measured as total antibody species) than constructs containing IgG4 S108P (Figure 5).

[0451] 8.3. Example 2: Fc gamma receptor binding kinetics Overview The binding affinity and signal to Fc gamma receptors of various antibodies with different hinge Fc regions were measured by Biacore as described in Section 8.1.3.

[0452] 8.3.1.1.Results The results are shown in Tables 4 and 5 below.

[0453] [Table 4]

[0454] [Table 5]

[0455] In Table 5, NB refers to no binding and WB refers to weak binding. IgG1 PVA has no binding signals to FcγR1, FcγR2b, FcγR3a (F176), or FcγR3b. The binding signals to FcγR2a (both R131 and H131) are low, but at significantly reduced levels (91 and 21 RU, respectively) compared to IgG1 and IgG4 S108P. IgG1 PVA has a KD of 7.2×10 -05 M had a weak to moderate binding signal (144 RU) to FcγR3a (V176), which was much weaker than the binding signals of IgG1 and IgG4 S108P (Tables 4 and 5).

[0456] 8.4. Example 3: ELISA Binding to Fc Gamma Receptors Overview The binding of FGFR1c / KLB bispecific antibodies containing various IgG hinge and Fc regions was assessed by ELISA as described in section 5.1.4.

[0457] 8.4.2.Results Binding curves showing the ability of control and test antibodies to bind to various Fc gamma receptors are plotted in Figures 6A-6G. Antibodies bearing wild-type IgG1 hinge and Fc domains demonstrated the highest binding to hFCRγ1. Binding of hFCRγ1 was significantly reduced with IgG1 PVA, which showed similar binding to IgG4s (Figure 6A). Binding with IgG1 N180G was similarly reduced. IgG4 S108P demonstrated slightly reduced binding to hFCRγ1 compared to wild-type IgG1. Similar trends were observed in the binding of hFCRγ3A(V158) and hFCRγ3A(F158) (Figures 6E, 6F). Negligible differences in binding were observed with hFCRγ2A(H131), hFCRγ2A(R131) (Figures 6B and 6C). However, IgG1 PVA bound less well than IgG4 S108P and slightly less well than IgG1 in hFCRγ2B (FIG. 6D), and less well than IgG1 in hFCRγ3B (FIG. 6G).

[0458] 8.5. Example 4: Antibody-Dependent Cellular Cytotoxicity Overview Using the surrogate antibody-dependent cellular cytotoxicity (ADCC) assay described in Section 8.1.5, the cytotoxic activity of IgG1 PVA was determined and compared to that of other IgG variants (e.g., IgG1 N180G and IgG4 S108P).

[0459] The ability of multispecific antibodies targeting hFGFR1c and hKLB to interact with FcγR3a, prominently expressed on NK cells, to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was measured in a surrogate bioassay using reporter cells and antibody-bound target cells. In this assay, engineered Jurkat T cells express the high affinity human FcγR3a 176Val allotype receptor (Jurkat / NFAT-Luc / hFcγR3a 176The reporter gene luciferase is expressed under the control of the transcription factor NFAT (NFAT-Luc) together with NFAT-Val. The target cells are HEK293 cells engineered to express human CD20 in combination with full-length human FGFR1c and human KLB. The reporter cells are incubated with the target cells, and engagement of FcγR3a through the Fc domain of human IgG1 antibody bound to the target cells leads to activation of the transcription factor NFAT in the reporter cells, driving expression of luciferase, which is then measured by a luminescent readout.

[0460] 8.5.2.Results Representative data from the ADCC assays are depicted in Figures 7 and 8. Only the alternative format antibodies with wild type IgG1, AF1d IgG1 and AF2d IgG1 showed a 1.9-fold (EC50=307pM) and 3.4-fold (EC50=1.04nM) induction of luciferase signal, respectively. None of the alternative format antibodies in 2+1 N-scFv or 2+1 N-Fab format with IgG1 PVA, IgG1 N180G or IgG4 S108P showed activity in the surrogate ADCC assay.

[0461] 8.6. Example 5: Molecular Activity Overview The activity of FGFR1c / KLB bispecific antibodies, including IgG1 PVA and controls, was tested using the luciferase reporter assay and human primary adipocyte signaling assay described in Sections 6.1.7 and 8.1.8.

[0462] 8.6.2.Results The activity of the alternative format antibodies in HEK.293SREluc.hFGFR1c / hKLB is shown in Figure 9 (2+1 N-scFv, AF1a, 1b) and Figure 10 (2+1 N-Fab, AF2a, 2b, 3a, 3b). The activity in human adipocytes is shown in Figure 11 (AF1a, 1b, 3a, 3b). In both HEK FGFR1c / KLB cells (Figure 9) and human adipocytes (Figure 11), the antibody with the 2+1 N-scFv format incorporating IgG1 PVA (AF1a) showed better agonist activity than the antibody with IgG4 S108P (AF1b). Antibody AF2a (2+1 N-Fab format in IgG1 PVA) produced a greater maximum activation in the reporter cell assay than AF2b (the same antibody with IgG4 S108P) (Figure 10).

[0463] 8.7. Example 6: Target Cell Binding Overview AF5-a and AF5-b, multispecific tetravalent antibodies with four antigen-binding domains that bind Protein X, were designed and produced as described in Sections 8.1.1 and 8.1.2, with AF5-a and AF5-b designed to contain the same set of antigen-binding arms linked to either a human IgG1 PVA or IgG4 S108P backbone, respectively. The binding properties of AF5-a, AF5-b, and a non-binding isotype control antibody were assessed using the flow binding assay described in Section 8.1.9.

[0464] 8.7.2.Results 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 scaffold was associated with higher efficacy than the IgG4 S108P scaffold.

[0465] 8.8. Example 7: Fab-Fab angles associated with three different hinge sequences Overview The Fab-fab angles of CD40-binding anti-CD40 IgG antibodies bound to different IgG hinge sequences were calculated as described in Section 8.1.10.

[0466] 8.8.2.Results Three IgG-CD40 complexes were evaluated: IgG1-CD40 complex, IgG1-PVA-CD40 complex, and IgG2-CD40 complex. For IgG1-CD40, IgG1-PVA-CD40, and IgG2-CD40, respectively, approximately 35%, 35%, and 11% of the particles in the 2D sorting had IgG-like features but could not be assigned a Fab-Fab angle due to ambiguity in Fab arm identification. For the 2D sorting runs shown in Figures 13C-E, only a small number of particles (i.e., less than 2%) were classified into the "junk" 2D class that lacked IgG-like features.

[0467] The Fab-Fab angles determined from the 2D class means and particle population percentages are listed in Table 6.

[0468] [Table 6-1]

[0469] [Table 6-2]

[0470] Without being bound by theory, it is believed that the IgG1-PVA-Fc domain results in increased target binding of the antibody preparation by increasing the percentage of molecules in the antibody preparation with a favorable Fab-Fab angle for target binding.

[0471] 9. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. A multispecific antibody comprising a first polypeptide chain associated with a second polypeptide chain, (a) The first polypeptide chain comprises a first chimeric constant domain comprising an amino acid sequence having at least 99% identity with any one of SEQ ID NOs: 3, 15, 16, 17, 18, 19, 20, 21, and 22, wherein the chimeric constant domain has a P-V-A-absent sequence at amino acids 233-236 as defined by EU numbering. (b) A multispecific antibody wherein the second polypeptide chain comprises a second chimeric 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, and SEQ ID NO: 22, wherein the chimeric constant domain has a P-V-A-absent sequence at amino acids 233-236 as defined by EU numbering.

2. The multispecific antibody according to claim 1, wherein the first chimeric constant domain comprises one or more amino acid substitutions that promote heterodimerization with the second polypeptide chain.

3. The multispecific antibody according to claim 2, 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).

4. The multispecific antibody according to claim 2, 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).

5. The multispecific antibody according to claim 1, 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.

6. The multispecific antibody according to claim 1, wherein the second chimeric constant domain comprises one or more amino acid substitutions that promote heterodimerization with the first polypeptide chain.

7. The multispecific antibody according to claim 6, 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).

8. The multispecific antibody according to claim 6, 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).

9. The multispecific antibody according to claim 1, 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.

10. The multispecific antibody according to claim 1, 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.

11. The multispecific antibody according to claim 1, 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.

12. The multispecific antibody according to claim 1, 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.

13. The multispecific antibody according to claim 1, 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.

14. The multispecific antibody according to claim 1, 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.

15. The multispecific antibody according to claim 1, 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.

16. The multispecific antibody according to claim 1, 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.

17. The multispecific antibody according to claim 1, 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.

18. The multispecific antibody according to claim 1, further comprising a first target-binding domain and a second target-binding domain.

19. The multispecific antibody according to claim 18, wherein the first target-binding domain binds to a first target molecule, and the second target-binding domain binds to a second target molecule.

20. The multispecific antibody according to claim 18, wherein the first target-binding domain and the second target-binding domain bind to the same target molecule.

21. The multispecific antibody according to claim 18, further comprising a third target-binding domain that binds to a target molecule different from the first target-binding domain and / or the second target-binding domain.

22. The multispecific antibody according to claim 1, wherein the multispecific antibody is a bispecific antibody.

23. The multispecific antibody according to claim 1, wherein the multispecific antibody is a tripspecific antibody.

24. The multispecific antibody according to claim 1, wherein the multispecific antibody is a trivalent antibody.

25. The multispecific antibody according to claim 1, wherein the multispecific antibody is a tetravalent antibody.

26. The multispecific antibody according to claim 1, wherein the multispecific antibody exhibits an increase in expression of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the expression system compared to an antibody containing a wild-type IgG1 constant domain including IgG1 hinge, IgG1 CH2, and IgG1 CH3.

27. The multispecific antibody according to claim 1, wherein the multispecific antibody exhibits binding to the human Fc receptor (FcR) at a rate 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 lower than that of a polypeptide comprising a wild-type IgG1 constant domain including IgG1 hinge, IgG1 CH2, and IgG1 CH3.

28. The multispecific antibody according to claim 27, wherein the FcR is FcRγ1, FcRγ2A, FcRγ2B, FcRγ3A, or FcRγ3B.

29. A composition comprising a multispecific antibody according to any one of claims 1 to 28.

30. The composition according to claim 29, wherein the composition is a pharmaceutical composition and further comprises one or more excipients and / or a pharmaceutically acceptable carrier.

31. A nucleic acid molecule or a plurality of nucleic acid molecules encoding a multispecific antibody according to any one of claims 1 to 28.

32. A host cell manipulated to express a multispecific antibody according to any one of claims 1 to 28.

33. A method for producing a multispecific antibody according to any one of claims 1 to 28, comprising culturing the host cells according to claim 32, and optionally recovering and / or purifying the expressed protein.

34. A method for increasing the production of a multispecific antibody, comprising expressing the multispecific antibody as the multispecific antibody described in any one of claims 1 to 28.

35. The method according to claim 34, comprising culturing the host cells according to claim 32 so that a protein is expressed, and recovering the expressed protein.

36. The method according to claim 34, wherein the method increases the expression of a variant protein by at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the expression of a control pair of constant domains each containing the sequence E-L-L-G at amino acids 233-236 as defined by EU numbering.

37. A population of multispecific antibodies according to any one of claims 1 to 28, comprising, optionally, at least 10,000 multispecific antibodies.

38. A group of polyspecific antibodies according to claim 37, characterized by higher activity compared to a group of proteins containing a control pair of constant domains each containing the sequence E-L-L-G at amino acids 233-236 as defined by EU numbering.

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